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IONIZING RADIATIONS

AND

CELL METABOLISM

Ciba Foundation Symposia
General Volumes:
Visceral Circulation _____
Mammalian Germ Cells _ _ _ _

Preservation and Transplantation of Normal
Tissues _______

Hypertension: Humoral and Neurogenic
Factors _______

Leukaemia Research _____

Chemistry and Biology of Pteridines -

Experimental Tuberculosis _ _ _ _

Porphyrin Biosynthesis and Metabolism -

Histamine _______

Extrasensory Perception _ _ _ _

Bone Structure and Metabolism _ _ -

Paper Electrophoresis _____

The Nature of Viruses _____

Chemistry and Biology of Purines - _ -

A leaflet giving fuller details of these volumes, also of the

Ciba Foundation Colloquia on Endocrinology and Colloquia

on Ageing, is available from the Publishers.

est

CIBA FOUNDATION SYMPOSIUM

IONIZING RADIATIONS

AND

CELL METABOLISM

Editors for the Ciba Foundation

G. E. W. WOLSTENHOLME, O.B.E., M.A., M.B., B.Gh.

and
CECILIA M. O'CONNOR, B.Sc.

With 48 Illustrations

LITTLE, BROWN AND COMPANY

BOSTON

THE CIBA FOUNDATION

for the Promotion of International Co-operation in Medical and Chemical Research

41 Portland Place, London, W.l.

Trustees :

The Right Hon. Lord Adrian, O.M., F.R.S.

The Right Hon. Lord Beveridge, K.C.B., F.B.A.

Sir Russell Brain, Bt.

The Hon. Sir George Lloyd-Jacob

Mr. Raymond Needham, Q.C.

Director, and Secretary to the Executive Council:
Dr. G. E. W. Wolstenholme, O.B.E.

Assistant to the Director :
Dr. H. N. H. Genese

Assistant Secretary :
Miss N. Bland

Librarian :
Miss Joan Etherington

Editorial Assistants :

Miss C. M. O'Connor, B.Sc.

Miss E. C. P. Millar, A.H-W.C.

This book may not be reproduced by
any means, in whole or in part, with-
out the permission of the Publishers

Published in London by

J. & A. Churchill Ltd.

104 Gloucester Place, W.l

First published 1956
Printed in Great Britain

PREFACE

The Ciba Foundation, London, is an educational and
scientific charity founded by a Trust Deed made in 1947.
Its distinguished Trustees, who are wholly responsible for its
administration, are The Rt. Hon. Lord Adrian, O.M., F.R.S.;
The Rt. Hon. Lord Beveridge, K.C.B., F.B.A.; Sir Russell
Brain, Bt. ; The Hon. Sir George Lloyd-Jacob, and Mr.
Raymond Needham, Q.C. The financial support is provided by
the world-wide chemical and pharmaceutical firm which has
its headquarters in Basle, Switzerland.

The Ciba Foundation forms an international centre where
workers active in medical and chemical research are encour-
aged to meet informally to exchange ideas and information.
It was opened by Sir Henry Dale, O.M., F.R.S., in June 1949.

In the first seven years, in addition to many part-day dis-
cussions, there have been 40 small international symposia,
each lasting two to four days and attended by from twenty-
five to thirty outstanding workers from many countries. Other
symposia are planned at the rate of five or six a year.

The informality and intimacy of these meetings have per-
mitted discussion of current and incomplete research and
stimulated lively speculation and argument. They have also
been the occasion for reference to much published and un-
published work throughout the world.

The reader will probably be well aware that there have been
many conferences, national, international and inter-disciplin-
ary, in recent years on the effects and hazards of radiation.
This is partly due to rapid progress and expansion in this field
of research, and partly to a quickening interest in the signifi-
cance of the work shown by other scientists and by laymen.
Most of these conferences have been on a fairly large scale,
valuable for exchanges of information but usually affording
little opportunity, except privately, for thorough discussion.

vi Preface

Dr. A. Hollaender and Professor A. Haddow made these
points when approaching the Director late in 1954 with a
request that a symposium on the Influence of Ionizing
Radiations on Cell Metabolism should be included in the Ciba
Foundation's programme. The Director readily agreed, sub-
ject to receiving their expert advice on its organization, in
which they were later most helpfully joined by Professor
J. A. V. Butler and Dr. L. H. Gray.

The symposium, which was realized in March 1956, and
which was held under the skilful and kindly chairmanship of
Professor Haddow, is amply recorded in this book. The
Editors hope that their intervention has to some extent made
for easier reading, but that the reader will be able to enjoy, as
if he were a participant, the efforts made by the contributors
on this friendly occasion to bring forward new information and
to come to an understanding of each other's aims, methods,
problems and interpretations.

CONTENTS

PAGE

Chairman's opening remarks

by A. Haddow ........ 1

Cytoplasmic and nuclear structure in relation to metabolic
activities

by J. Brachet ........ 3

Discussion: Bracket, Butler, Davidson, Gray, Holmes,

Howard, Roller, Popjak ...... 20

The effects of ionizing radiations on enzymes in vitro

by W. M. Dale 25

Discussion: Alexander, Butler, Dale, Forssberg, Popjak 34

The activity of enzymes and coenzymes in irradiated
tissues

by Antoinette Pirie ...... 38

Discussion: Alexander, Alper, Van Bekkum, Butler,

Gray, Lajtha, Latarjet, Pirie ..... 56

Effects of X-rays and radiomimetic agents on nucleic
acids and nucleoproteins

by J. A. V. Butler ....... 59

Discussion: Alexander, Alper, Butler, Forssberg, Gray,
Haddow, de Hevesy, Hollaender, Roller, Lajtha,
Latarjet, Mitchell, Spiegelman .... 70

Oxidative phosphorylation in some radiosensitive tissues
after irradiation

by D. W. VAN Bekkum . " . . . . .77

Discussion: Van Bekkum, Brachet, de Hevesy, Holmes,

Howard, Roller, Laser, Loutit, Mitchell, Popjak . 89

The effects of extraneous agents on cell metabolism

by H. A. Rrebs ....... 92

Discussion: Alexander, Brachet, Cohn, Dale, Haddow,

Rrebs, Lajtha, Zamecnik . . . . . .103

vii

72024

viii Contents

PAGE

The influence of oxygen on radiation effects

by H. Laser ........ 106

Discussion: AllPer, Gray, Laser, Latarjet, Stapleton . 116

The influence of chemical pre- and post- treatments on
radiosensitivity of bacteria, and their significance for
higher organisms

by A. HoLLAENDER and G. E. Stapleton . . .120

Discussion: Alper, Van Bekkum, Gale, Gray, Haddow,

HOLLAENDER, LaSER, LaTARJET, POPJAK, SpIEGELMAN,

Stapleton, Stocken . . . . . . .135

Postirradiation treatment of mice and rats

by D. W. H. Barnes and J. F. Loutit . . . 140

Discussion: Alexander, Van Bekkum, Butler, Haddow,

HOLLAENDER, LaJTHA, LaTARJET, LOUTIT, SpIEGELMAN,

Stapleton, Stocken . . . . . . .153

Studies on the mechanism of protein synthesis

by P. C. Zamecnik ....... 161

Discussion: Bracket, Holmes, Pirie, Popjak, Spiegelman,

Work, Zamecnik . . . . . . . .169

Nucleic acids and amino acid incorporation

by E. F. Gale 174

Discussion: Bracket, Butler, Cokn, Gale, Pirie, Spiegel-
man .......... 183

Protein synthesis in protoplasts

by S. Spiegelman . . . . . . .185

Discussion: Alexander, Bracket, Cokn, Davidson, Gale,

Krebs, Lajtka, Spiegelman . . . . .193

Influence of radiation on DNA metabolism

by Alma Howard ....... 196

Discussion: Alexander, Alper, Van Bekkum, Bracket,
Davidson, Gale, Gray, de Hevesy, Hollaender, Holmes,
Howard, Lajtka, Latarjet, Spiegelman, Swanson . 206

Contents ix

PAGE

The influence of radiation on the metabolism of ascites
tumour cells

by A. FoRSSBERG ....... 212

Discussion : Forssberg, Howard, Krebs, Lajtha, Latarjet,

POPJAK ......... 222

Influence of radiation on metabolism of regenerating rat
liver

by Barbara E. Holmes ...... 225

Discussion: Gray, de Hevesy, Holmes, Howard, Roller,

Lajtha ......... 2.36

The induction of chromosomal aberrations by ionizing
radiation and chemical mutagens

by C. P. SwANSON and B. Kihlman .... 239

Discussion: Alper, Van Bekkum, Bracket, Haddow,
Hollaender, Holmes, Howard, Roller, Pirie, Spiegel-
man, SWANSON ........ 251

Primary sites of energy deposition associated with radio-
biological lesions

by L. H. Gray 255

Discussion: Alexander, Butler, Gray, Hollaender,

Roller, Lajtha, Latarjet, Swans on .... 270

Effects of radiation and peroxides on viral and bacterial
functions linked to DNA specificity

by R. Latarjet ....... 275

Discussion: Alexander, Alper, Butler, Dale, Gray,

Latarjet, Popjak, Spiegelman ..... 297

General Discussion : Alexander, Butler, Dale, Haddow, de
Hevesy, Hollaender, Holmes, Howard, Laser, Latar-
jet, Mitchell, Popjak ...... 300

Chairman's closing remarks

by A. Haddow ........ 307

List of those participating in or attending the Symposium on

"The Influence of Ionizing Radiations on Cell Metabolism"

held at the Ciba Foundation, 6th-9th March, 1956

P. Alexander .
TiKVAH Alper .

D. W. VAN Bekkum

J. Brachet

J. A. V. Butler

D. W. COHN

W. M. Dale

J. N. Davidson
A. Forssberg
E. F. Gale

L. H. Gray

A. Haddow
G. DE Hevesy

A. HOLLAENDER

Barbara E. Holmes
Alma Howard .

P. Koller
H. A. Krebs
L. G. Lajtha

H. Laser .
R. Latarjet

Chester Beatty Research Inst., London

Experimental Radiopathology Research Unit,
Hammersmith Hospital, London

Medical Biological Laboratory of the National
Defence Research Council T.N.O., Rijswijk

Laboratoire de Morphologic Animale, Univer-
sity Libre de Bruxelles

Chester Beatty Research Inst., London

Oak Ridge National Laboratory, Tennessee,
and Chemical Laboratory, University of
Cambridge

Dept. of Biochemistry^ Christie Hospital, and
Holt Radium Institute, Manchester

Dept. of Biochemistry, University of Glasgow

Inst, of Radiophysics, Stockholm

School of Biochemistry, University of Cam-
bridge

British Empire Cancer Campaign Research
Unit in Radiobiology, Mount Vernon
Hospital, Northwood

Chester Beatty Research Inst., London

Inst, of Organic Chemistry and Biochemistry,
University of Stockholm

Biology Division, Oak Ridge National Labora-
tory, Tennessee

Dept. of Radiotherapeutics, University of
Cambridge

British Empire Cancer Campaign Research
Unit in" Radiobiology, Mount Vernon
Hospital, Northwood

Chester Beatty Research Inst., London

Dept. of Biochemistry, University of Oxford

Dept. of Radiotherapy, The Churchill Hos-
pital, Oxford

Molteno Inst., University of Cambridge

Laboratoire Pasteur de I'lnstitut du Radium,
Paris

J. F. LOUTIT

J. S. Mitchell .
Antoinette Pirie

G. POPJAK .

S. Spiegelman .
G. E. Stapleton

L. A. Stocken .

C. P. SWANSON .

T. S. Work
P. C. Zamecnik

List of Participants

. Medical Research Council Radiobiological
Research Unit, Harwell

. Dept. of Radiotherapeutics, University of
Cambridge

Nuffield Laboratory of Ophthalmology, Uni-
versity of Oxford

Experimental Radiopathology Research Unit,
Hammersmith Hospital, London

Dept. of Bacteriology, University of Illinois

Biology Division, Oak Ridge National Labora-
tory, Tennessee

Dept. of Biochemistry, University of Oxford

Dept. of Biology, The Johns Hopkins Univer-
sity, Baltimore

National Inst, for Medical Research, Mill Hill,
London

Massachusetts General Hospital, Boston

O^^C/4^

CHAIRMAN'S OPENING REMARKS

A. Haddow

A GREAT deal of work has been carried out on the elucidation
of the changes in gross cellular structure produced by ionizing
radiations, on the histopathology of radiation damage, and on
the cytological and genetical effects. Yet what of the bio-
chemical changes, the metabolic changes we have to consider?
To quote Dubois and Petersen's review (1954, Annu. Rev.
Nuclear Sci., 4, 351), although research on the biochemical
effects of ionizing radiations has yielded a vast amount of
information, no satisfactory explanation of the exact mechan-
ism by which tissue damage is inflicted has yet been obtained.
Research on the biochemical mechanisms has been under way
for a relatively short period of time. A considerable amount
of research on the subject during recent years was of necessity
exploratory in nature. Many approaches to the problem of
mechanism have been employed. A large number of the earlier
studies dealt with in vitro systems. The information obtained
from such studies has been valuable in indicating the chemical
linkages and groups which are the most susceptible to altera-
tion by ionizing radiations. However, attempts to apply in
vitro findings with ionizing radiations to intact cells have been
generally disappointing. Biologists have therefore turned their
attention to the more difficult task of attempting to define
radiation damage in terms of interference with biochemical
systems, through research on irradiated animals and micro-
organisms. My colleague J. A. V. Butler has pointed out that
the basic puzzle of radiobiology, one which has been stressed
especially by L. H. Gray, is still unsolved — namely that com-
paratively small doses of radiation produce marked biological
changes, although in general rather large doses are required
to produce easily observable chemical changes. In Butler's

RAD. 1 2

2 A. Haddow

words, the passage of radiation through hving tissues obviously
initiates a long chain of events. We have the primary ioniza-
tions, the chemical consequences, and the biological events
which follow. Although the physical nature of the primary
actions has been well worked out, and the chemical con-
sequences have been established, at least in numerous simple
cases, the link between the chemical changes and the biological
consequences is almost completely unknown. Discussing the
radical initiated polymerizations of unsaturated substances,
Butler points out that the radical merely acts as a catalytic
agent in that it stimulates processes which can occur spon-
taneously. This recalls a recent impression that the chemical
carcinogens may simply expedite processes which occur
spontaneously at much lower rates. Again to quote Butler,
we are at the moment in the position of a man who tries to
elucidate the mechanism of a telephone exchange by throwing
bricks into it and observing some of the results.

Our subject is at an elementary stage, yet it is always
dangerous to say what will not happen in science. Even
Lord Rutherford at one time thought little of the prospects
of the release of atomic energy. From the study of the influence
of ionizing radiations on cell metabolism may, however, flow
the most profound consequences for the theory of ageing, for
the theory of carcinogenesis, and for the theory of heredity.
J. J. Thomson once said that if he were to start life again he
would take up the study of biology, this being, as he thought,
at the same stage as physics when he started his early career.
Our own subject is the ideal region in which physics, chemistry
and biology meet.

CYTOPLASMIC AND NUCLEAR STRUCTURE IN
RELATION TO METABOLIC ACTIVITIES

J. Brachet

University of Brussels

Among the numerous theories which have been proposed
with a view to explaining the functions of the nucleus in the
life of the cell, several have now been definitely rejected. This
is true in particular for the hypothesis of Loeb (1899) who
considered the nucleus as the prime centre of cellular oxida-
tions, for we have now shown, both for amoebae and for the
unicellular alga Acetahularia mediterranean that removal of
the nucleus does not appreciably reduce the rate of cellular
oxidations, even after a considerable length of time (Brachet,
1955a). It is also now well known that isolated nuclei have
an extremely low oxygen consumption and lack most of the
oxidative enzymes, this being true for amphibian egg nuclei
obtained by microdissection (Brachet, 1939) and for nuclei
of liver homogenates prepared by differential centrifugation
(cf. recent review articles by Bounce, 1955; Allfrey, Daly and
Mirsky, 1955). Extensive research on liver homogenates has
shown, in addition, that mitochondria are the primary,
though not exclusive, site of the energy-generating reactions
of the cell (oxidative phosphorylations). This work has been
ably summarized in recent reviews by de Duve and his co-
workers (de Duve and Berthet, 1954) and by Hogeboom and
Schneider (1955). An interesting exception, as yet uncon-
firmed, has been reported by Rubinstein and Denstedt (1954):
bird erythrocytes lack mitochondria and contain oxidative
enzymes (cytochrome oxidase and succinic dehydrogenase) in
their nuclei.

The fact remains, however, that the metabolism of enucle-
ated cytoplasm is never entirely normal. In the case of

4 J. Bracket

amoebae, removal of the nucleus leads to considerable dis-
turbances of phosphorylation. 32p.incorporation into non-
nucleated halves slows down almost immediately (Mazia and
Hirshfield, 1950), while their ATP content undergoes an
increase in aerobiosis which probably reflects a block in the
utilization of the phosphate-bond energy of ATP (Brachet,
1955a). Under anaerobic conditions, on the other hand, non-
nucleated cytoplasm shows a markedly reduced ability for
keeping ATP in phosphorylated form (Brachet, 1955a). More-
over, the general metaboUc disturbance of non-nucleated cyto-
plasm is also revealed in other biochemical systems. As we
have shown (1955a), the utilization of lipid and carbohydrate
reserve products is considerably reduced in non-nucleated
halves of amoebae.

These metabolic injuries can be accounted for, as we have
already suggested (Brachet, 1955a), by assuming that the cell
nucleus is involved in the synthesis of nucleotide coenzymes,
which are essential for glycolysis and cellular oxidations.
This hypothesis is in agreement with most recent findings.
Hogeboom and Schneider (1952) have shown that, in the liver,
the complete enzyme system for the synthesis of diphos-
phopyridine nucleotide (DPN) from ATP and nicotinamide
nucleotide is located in the nuclei. In the starfish oocyte,
as shown by our co-worker E. Baltus (1954), the same enzyme
system is concentrated in the nucleoli, which are fifty times
more active than entire oocytes in this respect. If one of
the biochemical functions of the cell nucleus consists in the
production of DPN-like nucleotide coenzymes, enucleation
should result in a rapid loss of these coenzymes from the
cytoplasm and Baltus (1956) has found that this is indeed the
case: the DPN content of fasted amoebae drops much faster
in the non-nucleated than in the nucleated halves.

Certain conclusions can be drawn from these various results.
At first it appears that the presence of the nucleus is by no
means essential to keep up the normal rate of cellular oxida-
tions and that those cytoplasmic granules which are specially
active in cellular oxidations, in particular mitochondria, are

Cytoplasmic & Nuclear Structure & Metabolism 5

largely independent of the nucleus. The latter, however, does
exert an indirect control by regulating these oxidation pro-
cesses through the synthesis of the nucleotide coenzymes. It
appears probable that these coenzymes are protected from
hydrolytic enzymes when bound to the mitochondria, in
which case removal of the nucleus can have little effect on
bound DPN and cannot interfere strongly with cellular
oxidations. On the contrary, free coenzymes, those not bound
to mitochondria, would appear to be left unprotected against
hydrolysis and this should result in a rapid drop of glycolysis
with an incomplete utilization of the stored glycogen after
removal of the nucleus. Thus non-nucleated cytoplasm,
I with its low content of free DPN and the resulting deficient
glycolysis, should no longer keep up its normal ATP content
in anaerobic conditions.

Such a direct action of the nucleus might be postulated not
only for the synthesis of DPN, but also for that of the other
nucleotide coenzymes (triphosphopyridine nucleotide, flavine-
adenine dinucleotide, coenzyme A, etc.). The experiments to
prove it have yet to be done but it remains an attractive
hypothesis, in view of the extremely important part taken by
the nucleus in the metabolism of a polynucleotide, ribonucleic
acid (RNA). We already know from ^^P experiments by
Marshak (1948), Marshak and Calvet (1949), Jeener and
Szafarz (1950) and Barnum and Huseby (1950), that nuclear
RNA shows a much higher specific activity than cytoplasmic
RNA. Studies Vvdth other radioactive precursors such as
orotic acid (Hurlbert and Potter, 1952), glycine (Bergstrand
et al., 1948), formate (Payne et al., 1952; Smellie et al., 1953)
have confirmed these results. In all cases, incorporation by
nuclear RNA was very high, higher in fact than that by any
cytoplasmic fraction.

There has been much debate as to whether, as suggested
by Jeener and Szafarz (1950), nuclear RNA is a precursor of
cytoplasmic RNA. Recent mathematical work by Barnum,
Huseby and Vermund (1953), as well as measurements show-
ing that nuclear and cytoplasmic RNA's have different

6 J. Bracket

molecular compositions (Crosbie, Smellie and Davidson, 1953;
Elson, Trent and Chargaff, 1955) give little probability to the
idea of nuclear RNA being the sole precursor of cytoplasmic
RNA. On the other hand, Goldstein and Plant (1955) recently
succeeded in grafting, in normal and in non-nucleated amoebae,
nuclei which had been labelled with ^^P. These experiments
strongly suggest that nuclear RNA can give rise to cytoplasmic
RNA, but they do not demonstrate that nuclear RNA is the
sole precursor of cytoplasmic RNA, nor do they prove that
nuclear RNA is not degraded prior to its conversion into
cytoplasmic RNA. It appears rather as if both forms of RNA
are synthesized independently, though at a faster rate in the
nucleus than in the cytoplasm. We shall see later that major
differences are also found in the fate of RNA in various
enucleated organisms.

Let us next consider another aspect of the role of the nucleus
in the life of the cell, the possible relations of the nucleus with
protein synthesis. As early as 1881, Verworn had suggested a
control by the nucleus of the cell's anabolism, making this
hypothesis in order to explain the usual incapacity of non-
nucleated cytoplasm to regenerate. Caspersson (1941, 1950)
has taken up this old hypothesis of Verworn and extended it.
On cytochemical grounds he has postulated that the nucleus
plays a fundamental role in protein synthesis, a suggestion
we shall now consider in the light of recent experimental
results from a number of laboratories.

The observation that cells in which an active protein
synthesis goes on have a particularly large nucleolus with a
correspondingly high content of RNA, has led Caspersson
(1941) to propose that the nucleus, and especially the nucle-
olus, is a key factor in protein synthesis. Simultaneously with
Caspersson (1941) but working independently, we proposed
the hypothesis that RNA plays a direct role in protein
synthesis (Brachet, 1941). This was suggested by the excep-
tionally high RNA content of all cells actively synthesizing
proteins. The hypothesis found further support in the results
of Hultin (1950) and of Borsook and co-workers (1952), who

Cytoplasmic & Nuclear Structure & Metabolism 7

found that microsomes (the smallest cytoplasmic particulates,
which have also the highest RNA content) are most active in
the incorporation of radioactive amino acid into proteins.
More recently, Gale and Folkes (1954, 1955) have found, in
bacteria lysed by ultrasonics, that protein synthesis will
only take place if RNA is left intact. Indeed this process is
brought to a stop if the nucleic acid fraction is extracted
by various means. In our laboratory also (Brachet, 1954,
1955a and b), it has been shown that ribonuclease, by specific-
ally attacking or binding the RNA of normal, living cells
(onion roots, amoebae, star-fish or amphibian eggs, etc.), has
a powerful inhibitory action on the incorporation of amino
acids into proteins, on the growth of the cell and on its overall
protein synthesis.

It is now a generally accepted fact, as pointed out by
Borsook (1955), Gale (1955) and Mirsky (1955), that nucleic
acids are directly and fundamentally involved in protein
synthesis. This is clear at least in the case of RNA (Gale and
Folkes, 1954, 1955; Brachet, 1954, 1955 a and b), but appears
less evident for DNA; some experiments of Allfrey (1954) and
AUfrey and Mirsky (1955) do indicate that desoxyribonuclease
inhibits amino acid incorporation into the proteins of isolated
thymus nuclei; the inhibition, however, is not so strong as
that by ribonuclease for the whole cell although ribonuclease
does not inhibit amino acid incorporation into the proteins of
isolated nuclei.

In view of the high nucleic acid content of cell nuclei and
because of the now well-established importance of these
compounds in protein synthesis, Caspersson's idea (1941,
1950) of a particularly important function of the nucleus in
protein synthesis has been brought into focus again and several
laboratories have initiated experiments on this problem. A
simple method, used chiefly by Mirsky and co-workers (Daly,
Allfrey and Mirsky, 1952) and by Davidson and co-workers
(Crosbie, Smellie and Davidson, 1953; Smellie, Mclndoe and
Davidson, 1953), consists in injecting a radioactive amino
acid into a living animal and then determining the specific

8 J. Bracket

radioactivity of the various constituents of its liver cells
(nuclei, mitochondria, microsomes and supernatant as obtained
by differential centrifugation). This technique is open to some
criticism. Results of the Mir sky group show that the methods
of preparation of isolated nuclei used entail serious losses of
some nuclear proteins. In this manner histones have been
shown to incorporate amino acids only slowly, while the rest
of the nuclear proteins do not differ much in activity from the
whole of cytoplasmic proteins. But it must always be kept
in mind that the current preparation processes may well
extract from the nuclei some proteins of considerable meta-
bolic importance.

In the light of these objections, we have taken up a dif-
ferent aspect of the same problem, one which appears more
worthwhile from a biologist's standpoint. Together with a
group of co-workers, we have investigated protein meta-
bolism in nucleated and non-nucleated halves of unicellular
organisms. We have deliberately selected two widely separ-
ated species : the amoeba (Amoeba proteus), the non-nucleated
halves of which cannot regenerate, and the giant unicellular
alga, Acetabularia mediterranean in which the non-nucleated
stems remain capable of extensive regeneration, as shown by
the classical work of Hammerhng (1934, 1953). We shall next
consider the results obtained in both cases.

If one cuts an amoeba into half, the non-nucleated frag-
ment soon rounds up and stops feeding. The nucleated half
keeps behaving normally and, if fed living micro-organisms, it
can resume growth and divide. Since the biochemical changes
in both halves should be studied under comparable conditions,
both fragments must be kept fasting in the course of the
experiment. Under these conditions the non-nucleated halves
remain alive for 10-15 days and the nucleated fragments for
about 3 weeks.

Our experiments have led us to the following conclusions.
As already pointed out, the oxygen consumption of non-
nucleated halves remains unaffected, but their ATP content
rises aerobically. Under anaerobic conditions, however,

Cytoplasmic & Nuclear Structure & Metabolism 9

anucleate cytoplasm cannot keep up its normal ATP content
for long. We already know that these non-nucleated fragments
rapidly lose the ability to utilize stored glycogen (Brachet,
1955a). Similarly they stop incorporating ^^P (Mazia and
Hirshfield, 1950) and cannot maintain a normal DPN content
(Baltus, 1956). This leaves no doubt that the loss of the nucleus
leads to serious defects of the metabolism of carbohydrates
and phosphorylated compounds. A logical consequence of
such an inhibition of the energy-providing mechanisms of
the cell would be a severe disturbance of protein synthesis,
since it is well established that the synthesis of proteins or
even of simple peptide bonds requires energy from the high
energy phosphate bonds of ATP (Borsook, 1950; Siekevitz,
1952; etc.).

With respect to protein synthesis, removal of the nucleus
brings into play an additional unfavourable factor in the
amoeba: we have already mentioned that RNA is also in-
volved in this synthesis. Removal of the nucleus, as shown
in 1951 by Linet and Brachet and, in confirmation, by
James (1954), leads to an immediate and marked fall in the
RNA content of cytoplasm (this can reach 70 per cent within
ten days). Such a drop in cytoplasmic RNA is, of course, in
agreement with the more recent autoradiograph experiments
of Goldstein and Plant (1955), showing that at least some of
the cytoplasmic RNA in amoebae is of nuclear origin. It is
not surprising that total protein content decreases faster in
non-nucleated than in nucleated halves (Linet and Brachet,
1951).

Given the conditions of our experiments (complete fasting),
one could not, of course, expect net protein synthesis to occur.
A process very near to it could be followed, however: the
incorporation of tagged amino acids into proteins. In a
recent paper, Mazia and Prescott (1955) have shown that
removal of the nucleus leads, within as little as 2 or 3 hours,
to a drastic decrease in the uptake of radioactive methionine
by the proteins of non-nucleated amoebae. The N/A ratio
(N = nucleated half; A = anucleate half) is already increased

10 J. Bracket

to a value of 6 after a few hours, and it reaches the value of
20 at the end of 2-3 days. We must bear in mind, however,
that methionine uptake by the proteins of non-nueleated
halves never drops to zero. Such uptake is strongly dimin-
ished, but never abolished by removal of the nucleus.

More recently, in experiments done with our co-worker
Mrs. A. Ficq, in which radioactive phenylalanine was used as
a precursor and located in the cell by autoradiography, we
have essentially confirmed Mazia and Prescott's results. The
differences we observed are less striking, however, since our
N/A ratios are in the neighbourhood of 2 (instead of 6-20) from
1 to 6 days after removal of the nucleus in the amoeba and in
the neighbourhood of 5 after 10 days. This is taken to mean
that, in the amoeba, removal of the nucleus does not immedi-
ately stop protein metabolism in the cytoplasm. As a matter
of fact, the amino acid uptake by proteins of the non-nucleated
halves begins markedly to decrease only when the RNA
content of the non-nucleated cytoplasm is already much
diminished (Linet and Brachet, 1951).

Thus we come to the notion that, in the case of the amoeba
at least, the nucleus cannot be the exclusive centre of protein
synthesis. Amino acid incorporation into proteins is main-
tained at a non-negligible rate in non-nucleated fragments as
long as the RNA content of the latter remains essentially
unchanged. The same experiments, on the other hand, show
very clearly that the nucleus exerts a control on protein
metabolism in the cytoplasm. This gives rise to another
problem: are all the cytoplasmic proteins equally dependent
on the nucleus?

This was investigated, still using amoebae, by following in
the course of time the changes of various enzymes (hence of
as many distinct proteins) in both types of fragments. The
results were essentially as follows (Brachet, 1955a). In the
amoeba, the removal of the nucleus results in widely different
effects in the case of different enzymes. Some enzymes, such
as protease, amylase and enolase, remain practically un-
changed after removal of the nucleus ; others, dipeptidase for

Cytoplasmic & Nuclear Structure & Metabolism 11

instance, show a very slow decrease in the non-nucleated
halves; a third group, including esterase and acid phos-
phatase, have practically disappeared from non-nucleated
cytoplasm after a few days. This establishes without any
doubt that different enzymes are to different extents under
nuclear control and that this postulated "control" from the
nucleus is much more complex than was expected at first.

It is still too early to state definitely why the various
enzymes we studied behave so differently after removal of the
nucleus; a likely explanation, though lacking formal proof as
yet, might be the different cytological localizations of these
enzymes; as shown by Holter and Lovtrup (1949), in the
amoeba, amylase and protease are bound to large mito-
chondrion-like particles. This would imply that mitochondria
are, by and large, independent of the nucleus. This is,
moreover, in perfect agreement with the finding, reported
above, that removal of the nucleus has little effect on the rate
of cellular oxidations. According to Holter and Pollock (1952),
dipeptidase is found in solution in the hyaloplasm; it is there-
fore not surprising that it should behave, after removal of the
nucleus, like the whole of the proteins of the organism.
Finally, acid phosphatase and esterase both show a striking
decrease in activity in non-nucleated cytoplasm, just like
RNA; it is not unlikely, therefore, that we are here dealing
with microsome-bound enzymes. If this proves to be the
case, it would mean that these small cytoplasmic granules are
under much closer nuclear control than the mitochondria and
the soluble proteins of the hyaloplasm.

In summary, removal of the nucleus in the amoeba is
followed essentially by a drastic decrease of DPN, RNA,
acid phosphatase and esterase, g. marked fall in the incor-
poration of amino acids into proteins, a loss of the ability to
retain phosphorylated ATP under anaerobic conditions, and
a slow decrease of the total protein and dipeptidase content.
On the other hand, removal of the nucleus hardly changes
oxygen consumption, aerobic ATP, protease, amylase and
enolase.

12 J. Bracket

Parallel studies done on the unicellular alga Acetabularia
mediterranea, an organism which can regenerate to a sizeable
extent when deprived of its nucleus (Hammerling, 1934), have
yielded quite different results. As we have shown recently
(Brachet, Chantrenne and Vanderhaeghe, 1955), Acetabularia
mediterranea behaves much like the amoeba as far as respira-
tion is concerned. In both, removal of the nucleus has no
measurable effect on cellular oxidations, showing that the
latter are not under direct nuclear control. Indeed, the reverse
appears to be true to some extent in Acetabularia. The
morphology and chemical composition of the nucleus are
influenced by the cytoplasm. If energy production in the
cytoplasm is diminished or blocked by dinitrophenol or an-
aerobiosis, the nucleolus soon changes its shape, losing in the
process some of its high content of RNA. Such a nucleolar
reaction has been observed before by Stich (1951) as a result
of merely placing the algae in the dark.

We have, however, noted a difference between Amoeba and
Acetabularia for phosphorus metabolism. While it is true
that ^^P incorporation decreases in non-nucleated Aceta-
bularia stems, this effect does not become noticeable until
after a long time (Brachet, Chantrenne and Vanderhaeghe,
1955), usually several weeks. Fragments separated for only
a few days show no significant differences in this respect
(Hammerling and Stich, 1954).

It is, however, with respect to RNA and protein metabolism
that Acetabularia shows most difference from what has been
reported for Amoeba. We have recently been able to show
(Brachet, Chantrenne and Vanderhaeghe, 1955) that the non-
nucleated stem of Acetabularia retains for several weeks the
ability to incorporate radioactive ^^COg into proteins (in the
light) and orotic acid into RNA. These anabolic processes
continue at a normal rate for fifteen days in non-nucleated
cytoplasm. Together with regenerative potency, they then
begin to diminish. Even after three months without a nucleus,
fragments will still be capable of a noticeable uptake of radio-
active precursors into RNA and proteins.

Cytoplasmic & Nuclear Structure & Metabolism 13

Even more striking is the fact that non-nucleated cyto-
plasm can actually effect a net synthesis of proteins and RNA.
Indeed, during the first days after halving, this simultaneous
synthesis of RNA and proteins is even more rapid in the non-
nucleated than in the nucleated half. Perhaps this is due to
the fact that the nucleus competes with the cytoplasm for
ribonucleoprotein precursors. If the nucleus utilizes these
precursors at a higher rate than does the cytoplasm (we shall
later see that it could very well be so), the acceleration of net
RNA and protein synthesis with removal of the nucleus is
easy to understand.

The fact that net protein and RNA synthesis is possible in
the absence of the nucleus has interesting implications: for
instance, it is clear that, in contradiction to one of the theories
we have reviewed above, cytoplasmic RNA does not neces-
sarily originate in the nucleus. Furthermore, if RNA is
organized under the influence of DNA as has been suggested
by Gale and Folkes (1954), it is obvious that such a mechanism
must be a remote one. In Acetahularia, RNA synthesis is
certainly possible in the absence of DNA and the experiments
show that simple ideas such as "DNA makes RNA, and RNA
makes protein" are the result of an oversimplification of the
facts.

It remains, however, that this RNA and protein synthesis
in the absence of the nucleus does not go on indefinitely : the
process graduaUy slows down after 10 days or so. This shows
again that the nucleus does exert some control on protein
synthesis in the cytoplasm, but this control is remote and
indirect, not immediate as might have been expected.

The chemical nature of the nuclear control exerted by the
nucleus on protein synthesis is still unknown. It might be
that, as in Goldstein and Plant's (1955) experiments with
amoebae, part of the cytoplasmic RNA originates from the
nucleus in Acetahularia also and protein synthesis might come
to a standstill when this RNA of nuclear origin has been
exhausted. It would be important to know whether the
RNA, which is so quickly synthesized by the non-nucleated

14 J. Bracket

Acetabular ia stems, has the chemical composition of the nuclear
RNA or that of the cytoplasmic RNA, provided the two differ
in this respect; for the essential fact remains that the non-
nucleated Acetabularia mediterranea is capable of forming a
specific mediterranea regenerate in the absence of a nuclear
production of either RNA or DNA. It is unlikely that the
morphogenetic substance produced by the Acetabularia
nucleus is DNA, since we have been unable to detect the
presence of DNA in the non-nucleated stems with a sensitive
isotope dilution method.

The observed differences, with respect to protein and RNA
synthesis in non-nucleated cytoplasm, between Amoeba and
Acetabularia are not altogether unexpected if we consider
that the non-nucleated stem of an Acetabularia retains, as we
have seen for ourselves, a perfectly normal photosynthetic
activity. Thus the energy requirements for nucleoprotein
synthesis are still met with in a non-nucleated piece of
Acetabularia, but not in a non-nucleated Amoeba half.

We must finally point out the very clear correlation which is
found in both Amoeba and Acetabularia between the fate of
RNA and that of the proteins. In the former, removal of the
nucleus is followed by a rapid loss of RNA and a marked
decrease in protein metabolism. In Acetabularia, on the con-
trary, both processes are accelerated in a parallel manner.
Those are by no means special cases, since what we have just
said for Acetabularia applies also to reticulocytes. These are
immature red cells which have lost their nucleus, but still
retain a nearly normal amount of RNA in the cytoplasm.
They still have the power of incorporating tagged amino acids
into their proteins, an activity which is completely lacking
in mature erythrocytes. The latter have almost completely
lost their RNA (Borsook et al., 1952; Koritz and Chantrenne,
1954; Holloway and Ripley, 1952). The same reticulocytes,
in spite of the loss of their nucleus, can also incorporate
radioactive glycine in their RNA, as shown recently by Kruh
and Borsook (1955); these anucleate cells can even synthesize
haemoglobin (Nizet and Lambert, 1953) and various enzymes

Cytoplasmic & Nuclear Structure & Metabolism 15

(Koritz and Chantrenne, 1954). This ability of reticulocytes
to synthesize proteins and to incorporate radioactive precur-
sors into their proteins and RNA, decreases as they lose their
basophilia, i.e. their RNA, in the course of the maturation
process. This correlation between the drop in basophilia and
the decrease in glycine uptake into proteins is particularly
obvious in autoradiographic observations by Gavosto and
Rechenmann (1954) : their technique allowed both processes to
be followed simultaneouslv.

These results on reticulocytes are in full agreement with the
data from Acetabularia: removal or spontaneous elimination
of the nucleus does not necessarily lead to a rapid block of
protein synthesis. As a matter of fact, the results of the
experiment are chiefly dependent on the effects on cytoplasmic
RNA brought about by removal of the nucleus. If cytoplasmic
RNA is quickly broken down in the absence of the nucleus,
as happens in Amoeba, protein metabolism is immediately
affected. If, on the other hand, the non-nucleated cytoplasm
keeps its normal RNA content for a long time (as in Aceta-
bularia and reticulocytes), it can still synthesize proteins, at
least for a while.

Similar instances could no doubt be found in the case
of eggs deprived of their nucleus: unfortunately the data
gathered in this field so far [by Malkin (1954) on sea urchin
eggs and by Tiedemann and Tiedemann (1954) on Triton
eggs], lack a sufficiently complete analysis. We can at least
state, for both the sea urchin and the newt, that the non-
nucleated half is no less potent than the nucleated fragment
with respect to incorporating radioactive precursors into
proteins and RNA. It is a fact, however, that in unfertilized
eggs, as used in the above experiments, the net synthesis of
proteins and RNA is likely to be negligible. It follows that
those results of Malkin (1954) and Tiedemann and Tiedemann
(1954) should probably be taken as meaning that the turnover
of RNA and proteins remains at its normal level in non-
nucleated egg cytoplasm.

From the data available so far, we can now draw a general

16 J. Bracket

conclusion: the nucleus exerts, at the most, only a remote
and delayed control on the synthesis of cytoplasmic proteins.
It does not necessarily follow that the actual role of the
nucleus is negligible in the synthesis of proteins in the whole,
intact cells. We have already recalled the results from the
laboratories of Mirsky (Daly, Allfrey and Mirsky, 1952) and
of Davidson (Crosbie, Smellie and Davidson, 1953; Smellie,
Mclndoe and Davidson, 1953) in which it was established
that some nuclear proteins take up marked amino acids at a
rate comparable to that of the whole of cytoplasmic proteins.
We have pointed out on this occasion that the methods used
in isolating these nuclei may well involve the loss of soluble
proteins which might be very active metabolically.

This criticism is motivated by some recent autoradio-
graphic observations. When amino acid incorporation into
proteins is followed by this method, a much higher activity
is found in the nucleus than in the cytoplasm. This has been
shown for various materials: growing oocytes (Ficq, 1953),
amphibian eggs in the course of their development (Ficq,
1954; Sirlin, 1955) and mammalian liver (Ficq and Errera,
1955; Moyson, 1955). This higher activity of the nuclei
becomes much less obvious when the liver sections are
extracted by dilute citric acid, as used for the isolation of
nuclei. It appears possible, therefore, that this acid extracts
some metabolically active proteins from the nuclei. This
might be shown conclusively by a study of radioactive
amino acid incorporation into nuclei isolated in non-aqueous
media.

These autoradiographic studies have not been carried out to
a sufficient extent to allow general conclusions to be drawn
from them. It is by no means certain that the nuclei of all
cells are more active in this manner than the cytoplasm : that
has only been shown, so far, for cells with a high rate of
protein synthesis (oocytes, livers) and for actively dividing
cells (embryos in the course of development).

The autoradiographic experiments of Ficq (1953, 1955) on
starfish oocytes are more informative. In this material, it

Cytoplasmic & Nuclear Structure & Metabolism 17

is the nucleolus which has the most rapid and considerable
uptake of the tagged precursors (adenine, phenylalanine) of
RNA and proteins. This is most marked in young, actively
growing oocytes in which considerable nucleoprotein synthesis
is going on. In such oocytes, the activity of the nucleolus can
be a hundred times that of the other constituents of the cell
(nuclear sap, cytoplasm). Similar results for RNA have also
been published by Taylor (1953) and by Stich and Hammerling
(1953), using ^sp as a precursor: the former using auto-
radiographic methods, the latter measuring the activity
in the giant nucleolus of Acetabularia, isolated by micro-
dissection.

We can therefore conclude that the nucleus, especially the
nucleolus, is the site of a particularly active protein and RNA
metabolism ; this metabolism, however, can remain unaffected
for a long time in non-nucleated cytoplasm; net synthesis of
proteins and RNA can even take place in such cytoplasm.
In all known cases, a very strict parallel has been found
between the fate of RNA and that of protein anabolism.
Such a parallel lends support to the hypothesis put forth by
Caspersson (1941) and ourselves (1941) that RNA is directly,
involved in protein synthesis.

Summary

It has now been conclusively shown that the nucleus is
not a prime centre of cellular oxidations. It seems, neverthe-
less, that the nucleus plays a fundamental role in the synthesis
of nucleotides and of ribonucleic acid (RNA) and it may be
that the nucleus, and especially the nucleolus, is directly con-
cerned in the synthesis of nucle6tide coenzymes.

It is certain that the nucleus plays an important role in
protein synthesis, although in the unicellular alga Acetabularia
protein synthesis can go on for long periods without a nucleus.
Indeed, non-nucleated fragments of Acetabularia are able to
synthesize RNA and proteins for some fifteen days. The rate
of these syntheses decreases afterwards, showing that the

18 J. Bracket

nucleus exerts a very real, but remote, control on the pro-
duction of cytoplasmic proteins. These experiments also
demonstrate that cytoplasmic RNA cannot originate exclu-
sively from nuclear RNA.

Cases of closer control by the nucleus of protein synthesis
are shown by reticulocytes and especially by amoebae, in
which removal of the nucleus leads to a rapid decrease in the
incorporation of tagged amino acids into proteins and a drop
in RNA content of the cytoplasm. In the amoebae, removal
of the nucleus has different effects on various enzymes. It
appears that those enzymes bound to microsomes are parti-
cularly affected by the removal of the nucleus, while mito-
chondrial enzymes are especially independent of the presence
or absence of the nucleus.

Finally, autoradiographic experiments have shown that,
in cells in which a high mitotic activity or an active protein
synthesis goes on, the nucleus is more active than the cyto-
plasm in incorporating tagged amino acids into its proteins.

Acknowledgement

The author wishes to thank Dr. P. Couillard, who kindly translated
from the French text.

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DISCUSSION

Gray: Prof. Brachet, do you know the actual time-scale in which
DPN disappears after removal of the nucleus ?

Brachet: Yes, there is a fall of about 25 per cent in the DPN content
of Amoeba within 24 hours. It is a fast phenomenon. We have not done
it yet in Acetabularia.

Gray: Does DPN disappear both under aerobic and anaerobic
conditions ?

Brachet: Our observations were made under aerobic conditions. It
will be difficult to test the effects of anaerobic conditions because, in
order to measure the DPN content of Amoeba, using a very sensitive
method, we have to cut about 4-5,000 amoebae for each experiment.
What we would really like to know is whether the distribution of DPN
would be altered in non-nucleated cytoplasm: there is a possibility that
part of the DPN may be linked to mitochondria. Perhaps only the free
DPN is attacked, while the DPN bound to the mitochondria is more or
less preserved. It may be that this loss of DPN, especially if it is soluble
DPN, is one of the causes of the poor utilization of carbohydrate
reserves.

Davidson: I should like to raise one point in connection with nuclear
RNA : my colleagues and I believe that nuclear RNA is heterogeneous,
that at least two types of RNA, not just a single RNA, are present in
the nucleus. If we isolate cell nuclei by methods which avoid the use of
aqueous media, we can obtain perfectly clean cell nuclei from which

Discussion 21

we can extract both protein and RNA by means of dilute citric acid.
If we use labelled nuclei isolated from an animal that has received radio-
active phosphorus beforehand, then the RNA which is extracted from
the isolated nuclei has a lower specific activity than the RNA which
remains. We do not yet know whether there is any difference in base
ratios, but there certainly is a difference in specific activity, and more-
over, both the easily extractable and the non-extractable RNA have
specific activities which differ from those of any of the RNA's of the
cell cytoplasmic fraction. We have done the same sort of thing with
nuclei which have been isolated in sucrose-CaClg media. Whether we
label the animals with radioactive phosphorus or radioactive carbon
in the form of i*C-formate, when the nuclei are treated with dilute
citric acid or dilute phosphate buffer, an RNA can be extracted which
has a different specific activity from that of the RNA which remains,
and a different specific activity from that of any of the cytoplasmic
fractions. So here we have soine fairly substantial evidence that the
RNA of the nucleus is heterogeneous.

One might, of course, argue that the easily extractable RNA is simply
cytoplasmic RNA which is adhering to the nuclei. Against this is the
fact that nuclei prepared by the methods which we use are very clean
indeed according to microscopic examination. We have had them
examined microscopically by critics who were doing their best to find
flaws in our technique and they failed to do so, so that we do believe
that the RNA which is extracted is essentially nuclear RNA.

The possibility of nuclear RNA being the precursor of cytoplasmic
RNA is extremely interesting, but as Prof. Brachet said, the evidence
for a direct move of RNA from nucleus to cytoplasm is not good at the
moment.

Brachet: The localization of these two RNA's in the nuclei is not
known. If you study it by means of interference microscopy — I suppose
there is enough RNA which can be removed by the treatment with
citric acid — you might find out where it is located. There may be one
difficulty in this work on analysis of bases, i.e. it is difficult to get homo-
geneous RNA fractions, even from the tissues; so that it may be that
in the case of nuclei which have already been isolated, for instance by
extraction with citric acid, one may not get the true specific activity.
It may be a biochemical artifact to a certain extent.

Butler : WTiat is the evidence for the presence of RNA in chromatin ?

Brachet: As far as I know, there are only two pieces of evidence for
RNA in chromatin: one is that with cytochemical methods one finds
that there are differences in the staining ability of the nuclei from
different tissues. As a rule, the nuclei of tissues which do not synthesize
proteins stain almost green with methyl green-pyronin. In the case of
liver, pancreas, etc., there is a lilac or violet colour of the nuclei. If you
treat the sections with ribonuclease, you will find that the nuclei now
stain completely green. The red colour of the chromatin disappears as
well as the red colour of the cytoplasm and the nucleolus. Furthermore,
Mirsky and Ris, in their work on isolated chromosomes, obtained some
RNA in the threads.

22 Discussion

Davidson: Are you keeping in mind that Bounce (1955, In The Nucleic
Acids, ed. E. Chargaff and J. N. Davidson, Vol. II, p. 147) found that
in isolated rat liver the nucleic acid was mainly DNA, not RNA? So
where can the nuclear RNA be in the rat but in the nucleolus, unless it
be in the chromatin?

Bracket: I wonder how much of Bounce's material consisted of
nucleoli.

Butler: If you isolate the chromatin threads it is quite difficult in
group preparations to detect any RNA. I don't happen to remember
the figure of Mirsky.

Bracket: It came to about 10 per cent of the BNA content.

Butler: I should say we do not find that in our analyses of thymus
BNA.

Bracket: Thymus w^ould be rather different from liver, because
thymus nuclei stain almost completely green with methyl green-
pyronin.

Butler: BNA from normal rat livers is also effectively free from

RNA.

Holmes: Br. Jacobson (Jacobson, W., and Webb, M. (1952), Exp. Cell
Res., 3, 153) showed by staining methods that, as prophase begins,
ribonucleoprotein is added to the outside of the chromosome. This
remains during metaphase and anaphase, but in late anaphase ribonucleo-
protein appears to be shed from the chromosomes into the cytoplasm
between the two groups of chromosomes. By late telophase this ribonu-
cleoprotein has disappeared from the nucleus. The amount found must
depend a little on whether the tissue is a dividing or a resting tissue,
and on the state of the nuclei.

Bracket: I have had an experience similar to that of Br. Jacobson.
There certainly are changes in the staining ability of the chromosomes
during the mitotic cycle, but it is very difficult to know exactly what is
happening unless one makes quantitative estimations. The shape of
the chromosomes changes so much that it becomes extremely difficult
to decide whether or not there is an increase of a substance. This
dilution effect is dangerous in cytochemical work.

Popjak: Since BPN synthesis is confined to the nucleus, it is not
surprising that the enucleated part of the amoeba eventually runs down,
because one of the primary acceptors of electrons from the various sub-
strates is gradually eliminated, and that is why vital functions cannot
proceed. In that connection, therefore, I wonder to what extent we can
ascribe a function to nuclear BNA or nuclear RNA on the one hand
and to the running down of BPN on the other, in the changes of meta-
bolism of cytoplasm.

With regard to the experiments on the transfer of labelled nuclei,
where the labelling was with ^^p, is there any other evidence that the
label that subsequently appears in the cytoplasm is in fact associated
with RNA?

Bracket: With regard to the second point, it was done by an auto-
radiograph method. With such a method, it is likely that many soluble
phosphorus-containing substances are lost. Since, after using fixative,

Discussion 23

staining, etc., Goldstein and Plant found that all of the label could be
removed both from the nucleolus and the cytoplasm by ribonuclease, it
seems that what has been marked really is RNA. But I do not think
that the experiment shows more than the fact that cytoplasmic RNA
is labelled under these conditions. We cannot be sure that the nuclear
RNA has gone directly into the cytoplasm, because it is quite possible
that intermediary biochemical stages break down this RNA. Goldstein
and Plant say that RNA does not go the other way round, i.e. from the
cytoplasm to the nucleus. But, to be really sure, a normal nucleus
should be introduced into very strongly labelled cytoplasm, and this
experiment has not been done.

As regards Dr. Popjak's first question, I believe that quite a number
of changes occur in a non-nucleated Amoeba. Why they ultimately die
off, I do not know, but this is a slow, progressive process; the overall
lifespan is always somewhat lower without the nucleus than with it and
the same applies to Acetabularia. The very first changes which occur
are unfortunately not known to us: when you cut an Amoeba in half,
you can identify the enucleated half 15 minutes later, because it has
rounded up ; there is something going on very quickly in the membrane
which is not yet understood.

Hoivard: Prof. Brachet, what would happen if you added the nucleus
of one species of Acetabularia to the non-nucleated half of a different
species ? Would it grow a hat of the species from which the nucleus came ?

Brachet: I don't think the experiment has been done. The only thing
that has been done by Hammerling is the transfer of a nucleus between
two species and what then happens is this: if you cut Acetabularia
mediterranea just before the hat is formed, you will get a small, but
typical Acetabularia mediterranea hat. If you graft a nucleated half of
another species — for instance Acetabularia crenulata — in the stem of
Acetabularia mediterranea before the formation of the hat, then you get
hybrid hats. The purpose of the hat is the reproduction of the alga.
This hybrid hat is never fertile; it can be replaced by a second hat,
which will now be a typical crenulata hat.

These experiments of Hammerling show that there are morphogenetic
substances produced under the influence of the nucleus ; whether they
originate from the nucleus or are due to nucleocytoplasmic interactions,
we do not know.

Roller: You mentioned that in Acetabularia when you cut off the hat,
the enucleated stem will develop another hat of the same kind as the
original. This shows that protein synthesis in the enucleated part is
still under genetic control. It would be interesting to see how far
genetic control remains in operation. By removing the regenerated hat
from the enucleated Acetabularia and repeating the process it might be
possible to distinguish morphogenetic substances which are derived
from the nucleus from those which are produced in the enucleated part.

Brachet: I cannot answer that question because we have not done the
experiment. I believe that, probably very soon after the hat has been
formed, you will no longer be able to form a hat. We have carried out
experiments where we have tried to find out how long the non-nucleated

24 Discussion

half is capable of regenerating a hat. This experiment is very simple :
you cut a number of algae, take the non-nucleated halves. Light is
needed to provide energy for the regeneration. You put some of the
algae immediately in the light, you keep another batch for one week,
another for two weeks or three-four weeks in the dark before exposing
them to the light. AVhen you put a batch of non-nucleated algae
immediately in the light, you may get about 70 per cent hats. If you
leave them one or two weeks in the dark, you still get 70 per cent hats ;
but if you leave them three weeks in the dark, you get only 25 per cent
hats, and after four weeks you have none. There is something which
dies off in the dark as well as in the light. The time when the percentage
of the hats decreases (i.e. two weeks) is the same when net protein
synthesis and incorporation of ^^COg in the proteins also decrease in the
light.

THE EFFECTS OF IONIZING RADIATIONS
ON ENZYMES IN VITRO

W. M. Dale

Department of Biochemistry, Christie Hospital and Holt Radium, Institute,

Manchester

One important task for the biologist is to correlate the
radiation effects obtained in experiments in vitro with those
in living matter. Since enzymes are essential constituents of
cells, this short survey of radiation effects on enzymes in vitro
is meant to form a background against which the action of
radiation in vivo should be viewed, and I hope that the follow-
ing papers and their discussion will open new ways of approach
to decide what part enzymes may play in the mode of action
of radiation in living matter. As you will presently see, we
shall have to consider not only radiation effects on enzymes
themselves but also radiation effects on non-enzymic sub-
stances in their relation to enzymes.

Although enzymes are not, in their response to radiation,
fundamentally different from other substances capable of
reacting with radicals, their inactivation may have more far-
reaching biological consequences because of their catalytic
properties and the fact that they are present in cells in only
small amounts. It has been shown that they are subject to the
indirect action of radiation in aqueous solution (Dale, 1940),
i.e. via radicals, as well as to direct action in the dry state
(Lea, et al., 1944).

When solutions of a crystalline enzyme are irradiated the
number of molecules inactivated to a given proportion of re-
maining activity by a given dose is constant and independent
of the initial concentration. In consequence the percentage
destruction in a dilute solution is greater than in a concen-
trated solution, and therefore a dilute solution would appear to
be radiosensitive and a concentrated one radioresistant. Thus

W. M. Dale

radiation doses of the order of 100 r can cause appreciable
percentage destruction in a dilute solution (Dale, 1940).

One will have to consider in the discussion whether this
dilution effect can be operative in the inhomogeneous interior
of a cell. Fig. 1, which refers to the enzyme carboxypeptidase,
shows that the efficiency of the radiation decreases only in
extremely dilute solutions. This is usually interpreted as
being due to the fact that the distance between solute
molecules is so great that part of the radicals recombine
before they have a chance of reacting with solute.

O 15

10
5

10"

»-4

10^

10"" 10 10

ENZYME CONC.ing./ml.
Fig. 1. Yield: concentration relationship for carboxypeptidase.

From a certain concentration onwards, however, the yield
remains constant over a wide range of concentrations (Dale,
Gray and Meredith, 1949). Other enzymes, e.g. trypsin
(McDonald, 1954&, 1955) and chymotrypsin (McDonald and
Moore, 1955a) show some increase in the ionic yield when this
concentration is increased. The cause of this is not quite
certain though it may be connected with a lower probability
of elimination of radicals by the enzyme molecule.

An interesting occurrence of two different ionic yields has
been found by Aronson, Mee and Smith (1955), working with
a-chymotrypsin, which has an esterase and protease function.

Ionizing Radiations and Enzymes

The ionic yield of the esterase activity was three times greater
than that of protease activity. A possible explanation given
was that there are two active centres in the molecule.

The initial ionic yields for various enzymes generally lie
between 1 and 0-1. An example of a very low yield is catalase
which is of the order of 10"^, though there is, according to
Forssberg (1946), some dose-rate dependence. A low ionic

INDIRECT

<
o

cc:

>IRECT|
I S 10 IS

g. ENZYME PER lOOg. SOLUTION

Fig. 2. Relative contribution of "Indirect" and

"Direct" action to the total effect of X-rays on car-

boxypeptidase in solution (Dale, 1947. Reproduced by

permission of Brit. J. Radiol.).

yield of this powerful enzyme may be of importance to its
survival and its consequent availability for decomposing
HgOo formed by radiation.

Returning to Fig. 1, it will be seen that the constancy of the
yield extends to a concentration of 15 per cent, at which the
enzyme is no longer soluble. It will be useful to demonstrate
how much of the radiation effect has to be assigned to the
direct and to the indirect actions respectively, at the various
concentrations.

Fig. 2 demonstrates the respective contributions to the
observed effect, at various concentrations, of both the direct

28 W. M. Dale

and the indirect actions. Even at a concentration of 20 per
cent weight for volume the indirect action is predominant.
This presentation apphes to enzymes which have a concen-
tration-independent yield of the order of 0-18. If the yields
are lower, i.e. if bigger doses are required to achieve the same
effect, the chances of the direct action grow correspondingly.

We have now to consider the situation when an enzyme
solution is irradiated whilst the solution contains a second
solute. Then the available radicals will be shared by both
solutes according to their respective concentrations and
reactivity, and as a result they will mutually reduce the radi-
ation effect. In other words, the presence of a second solute
"protects" the first against the effects of radiation. This
protection effect is responsible for an exponential curve when
the activity of an irradiated enzyme solution is plotted against
radiation dose. As soon as irradiation has started there are
two types of solute molecules present, active and inactive,
the inactive ones still reacting with radicals, i.e. sharing radi-
cals, though this reaction is not scored. The opposite effect,
namely that the presence of a second solute leads to an increase
of radiation effects, i.e. sensitization, has so far not been
observed with enzymes, though it does occur with other sub-
stances. This applies in particular to the presence of oxygen
as a second solute, which in many biological radiation re-
sponses causes an increase of radiation effects via the HO 2
radicals and HgOg. No such increase has been observed with
carboxypeptidase (Dale, Gray and Meredith, 1949), ribo-
nuclease (Colhnson, Dainton and Holmes, 1950) and trypsin
(McDonald, 19546, 1955). Carboxypeptidase (Dale, Gray and
Meredith, 1949) and ribonuclease (Colhnson, Dainton and
Homes, 1950) are stable towards HgOg and are inactivated
by the — OH radical. Trypsin is also inactivated by the — OH
radical but is reversibly inhibited by HoOg independent of the
time of contact, while irradiation has an irreversible effect
(McDonald and Moore, 19556).

The protection effect has been shown to operate quite
generally. In particular it was shown in the case of D-amino

Ionizing Radiations and Enzymes 29

acid oxidase (Dale, 1942), that the two components making
up the enzyme, namely the flavineadenine dinucleotide and
the specific protein, when irradiated together lost in terms of
oxygen uptake about 60 per cent of their activity, this loss
being due to the inactivation of the protein only while the
dinucleotide was protected. When irradiated singly, and then
joined, the loss was about 90 per cent.

It is important to be aware of possible differences in the
response to radiation by non-enzymic substances which may
be present as protecting co-solutes, e.g. as substrates for
enzymic action or as products of metabolism. The protection
effect offers a method by which the effect of radiation on
non-enzymic substances can be measured when they are
co-solutes in an enzyme solution. The enzymic activity is
then the reference against which the effect of radiation on the
co-solute is measured. Such measurements make it possible
to detect radiation effects when ordinary analytical methods
applied to the non-enzymic substance itself would sometimes
fail because of the smallness of the effect.

The results of such experiments are as follows (Dale, 1947;
Dale, Davies and Meredith, 1949): for large molecules the
protective power is roughly proportional to the molecular
weight and no specificity is found. However, if one considers
small molecules, of which a special atomic group forms the
greater part, very marked specificities appear, which, even if
they do occur in big molecules, would get lost in the over-
whelming excess of other atomic groups of average reactivity
with radicals. The outstanding examples of such a specific
effect is given by a comparison of the protective effects of urea
and thiourea. Whereas urea is hardly protective at all, the
substitution of O by S in thiourea causes a 10,000-fold increase
in protective power.

Without going into details of the specificity of the pro-
tection effect, I should like to stress that sulphur-containing
compounds, and sulphur itself, play a special role, and that
generally the remainder of the molecule has an effect on the
protective power of any particular atomic group.

W. M. Dale

In Table I is listed the protective power of various sulphur-
containing compounds, when radiation took place with carb-
oxypeptidase as the indicator. Qp is the protective power
per iig. of protective substance, and Qg is the protective power
of such amounts of protector as contain one [ig. of sulphur in
each case. One can, therefore, estimate how the non-sulphur
residue in any one compound affects the protective power of
one [jLg. of sulphur contained in it, taking the colloidal sulphur
as reference. Elemental sulphur is about as protective as
thiourea and sodium thiosulphate, but the introduction of

Table I

The Protective Power of Various Sulphur-
containing Compounds with Carboxypepti-
dase as the indicator

Thiourea
Dimethylthiourea
Colloidal sulphur
Sodium thiosulphate

110

130

110

118

two methyl groups into thiourea causes a considerable de-
crease in the protective power of the sulphur.

Because of its possible bearing on cell constituents, I should
not omit to mention that the straightforward sharing mechan-
ism of radicals between two solutes is not always valid. If it
were, the protective power per unit weight of protector would
be constant, whereas in certain cases it declines appreciably
with increasing concentration of protector. This declining
protective power may be of significance from the biological
point of view when the effect of protective substances within
the interior of cells is considered. The phenomenon, which we
called the "changing quotient", is shown in Fig. 3, in which
the log of the protective power Q is plotted on the ordinate,
and the concentration of the protector on the abcissa. In
these experiments carboxypeptidase was used as the indicator.

Ionizing Radiations and Enzymes

The diminishing efficiency of the protective substance
when its concentration is increased can be explained best by-
assuming that a protector molecule, after reaction with a
radical, may possibly be in a metastable state, or may have
formed another organic radical and thus be able to "hand on"
the effect of the first collision with a radical to the indicator

CHANCING quotient' FOR CP
CURVES ARE THEORETICAL, POINTS EXPT

A 0 DIMETHYLUREA WITH CP 3<^7*/
C B ° GLUCOSE WITH CR SO^V*^

C.R 90 Wm^

LOCO

»'

CONC. IN

lO-"

lO"

Fig. 3. Curves showing "Changing quotient" for carboxypeptidase. Curves

are theoretical, points experimental (Dale, Davies and Meredith, 1949.

Reproduced by permission of Fhil. Trans.).

molecule. Protector molecules reacting in the described
manner would fail to fulfil their function as protectors.

Barron and co-workers (Barron and Dickman, 1949;
Barron et al., 1949) assume that the principal point of attack
by oxidizing radicals is the SH group in enzymes in which
SH is essential for enzymic activity. Inactivation could be
prevented by blocking the SH groups with mercaptide-
forming reagents, and lost activity restored by adding gluta-
thione to reduce the disulphide, provided that the X-ray
doses were not so high as to lead to denaturation of the

32 W. M. Dale

enzyme protein. When phosphoglyceraldehyde dehydro-
genase was inactivated by 100 r, complete reactivation by
glutathione was possible, but after 200 r only 62 per cent
reactivation occurred. Thus the differentiation between
action on SH groups and denaturation of protein lies within
rather narrow limits and and does not seem fully justified.

Closely linked with the question of denaturation of protein
is the observation of after-effects. Continued inactivation
after cessation of irradiation has been found with pepsin
(Anderson, 1954), and with trypsin (McDonald, 1954a). Some
modification of the enzyme molecule has been produced which
makes the molecule more sensitive to thermal denaturation,
similar to the action of radiation on the albumin examined by
Fricke (1952).

Effect of oL-radiation. With regard to the effect of radiation
of different ionization density, only one extensive study on the
effect of a-radiation as compared with X-radiation, on carb-
oxypeptidase, has been carried out (Dale, Gray and Meredith,
1949). The result was that the efficiency of a-rays was shown
to be only one twentieth that of X-rays, and this low efficiency
could be accounted for by the S-radiation which accompanies
the a-radiation, and is similar in ion density to X-radiation.

We have so far dealt with enzymes and co-solutes in solu-
tion. There is, however, evidence that there are many
enzymes firmly bound to cell structure. Not very much is
known about these from in vitro experiments. Mazia and
Blumenthal (1950) made an attempt to investigate a mono-
molecular film of pepsin-albumin on the surface of water,
and exposed it to radiation. This difficult experiment is open
to some criticism since the substrate, which was not in great
excess of the enzyme, was also exposed to radiation. However,
they reported an inactivation of a thousand enzyme mole-
cules per ionization under the circumstances of the experi-
ment. More model experiments with phase-bound enzymes
would be valuable.

In conclusion of this survey of in vitro experiments with
enzymes it may be useful to stress the main features of the

Ionizing Radiations and Enzymes 33

mode of action of radiation which will have to be considered
when attempting to correlate the results with the structural
organization and the metabolic activities of cells.

(1) Can one assume that the dilution effect may operate
inside a cell? In other words, are there intermicellar spaces
through which enzyme molecules diffuse from storage depots
when called upon by metabolic requirements of the cell?

(2) Are these intermicellar spaces in the inhomogeneous
cell structure filled with high concentrations of protective
substances?

(3) Is there a spatial separation between substrates and
surface-bound enzymes which may involve an action of radia-
tion on substrates in transit?

(4) Should one consider not a depletion of stored enzymes
but rather a decrease of the functional fraction of enzymes
which, by slowing down reaction velocities and possibly also
by the initiation of non-enzymic chain reactions, disorganizes
the delicate sequence of metabolic steps?

(5) Can one expect from an analysis of tissue extracts or
homogenates irradiated as such, or made from irradiated
tissues, any answer to the question of the participation of
enzymes in biological radiation effects?

Answers to all these questions will depend on the degree of
knowledge of the internal organization of cells at the sub-
microscopic level, and I hope that the discussion will clarify
some of the issues raised.

REFERENCES

Anderson, R. S. (1954). Brit. J. Radiol., 27, 56.

Aronson, D., Mee, L., and Smith, C. L. (1955). IV Int. Conf. Radiobiol.

Edinburgh : Oliver & Boyd.
Barron, E. S. G., and Dickman, S. (1949). J. gen. Physiol., 32, 595.
Barron, E. S. G., Dickman, S., Muntz, J. A., and Singer, T. P. (1949).

J. gen. Physiol., 32, 537.
CoLLiNSON, E., Dainton, F. S., and Holmes, B. (1950). Nature, Lond.,

165, 266.
Dale, W. M. (1940). Biochem. J., 34, 1367.
Dale, W. M. (1942). Biochem. J., 36, 80.
Dale, W. M. (1947). Brit. J. Radiol., SuppL, 1, 46.

BAD. 3

34 W. M. Dale

Dale, W. M., Davies, J. V., and Meredith, W. J. (1949). Brit. J.
Cancer, 3, 31.

Dale, W. M., Gray, L. H., and Meredith, W. J. (1949). Phil. Trans.,
242 A, 33.

Forssberg, a. (1946). Acta radiol., Stockh., 27, 281.

Fricke, H. (1952). J. phys. Chem., 56, 789.

Lea, D., Smith, K. M., Holmes, B., and Markham, R. (1944). Parasito-
logy, 36, 110.

Mazia, D., and Blumenthal, G. J. (1950). J. cell. comp. Physiol., 35,
171.

McDonald, M. R. (1954a). Brit. J. Radiol., 27, 62.

McDonald, M. R. (19546). J. gen. Physiol., 38, 93.

McDonald, M. R. (1955). J. gen. Physiol., 38, 581.

McDonald, M. R., and Moore, E. C. (1955a). Radiation Res., 3, 38.

McDonald, M. R., and Moore, E. C. (19556). Radiation Res., 2, 426.

DISCUSSION

Alexander: I think we are extremely fortunate that in the days when
the indirect effect was not well understood Dr. Dale happened to choose
the particular enzyme systems which he has described, because this
enabled him to put the indirect effect on the sound basis which we now
take for granted. If he had used other proteins or, as criteria, changes
other than inactivation of enzymes, then the effect of concentration and
the relation with dose would have been much more complicated. When
solutions of protein — we have studied serum albumin and lysozyme —
are irradiated in dilute solutions, aggregation occurs and units of very
much larger molecular weight are formed. There is no simple relation-
ship between the dose or protein concentration and size of the aggregates
formed. This often exceeds several million and with higher doses the
whole material becomes quite insoluble. All these molecules which form
part of the very large aggregate have, in a sense, been removed from
the bulk solution, but are not necessarily enzymatically inactive. De-
pending on the method of test, very strange dose-relationships between
radiation effect and /or concentration may be observed. Changes in
ionic strength can alter critically the aggregation phenomena and thus
influence the radiation effect in a way which cannot be explained from
simple consideration of indirect action.

The second point which I want to make is that for these aggregation
phenomena direct action is remarkably efficient, i.e. if we irradiate
serum albumin dry, and then dissolve it up and measure the amount of
change which has occurred, we find that direct action is remarkably
great. Six electron volts (or a G value of 18) is sufficient for the dis-
appearance of a protein molecule and its shift into an aggregate; if we
were choosing this as a criterion, we would find on Dr. Dale's histogram,
giving the proportion of direct and indirect effect at different concentra-
tions {see page 27), that the direct effect would be the most important
at the local concentrations of proteins encountered in cells.

My last point concerns the attack on the tyrosine in proteins. Wlien

Discussion 35

serum albumin has been irradiated in dilute solution, the u.v. absorption
peak at 2,800 increases and this has been interpreted by Barron and
others as a reaction by the free radicals with the tyrosine residues in the
protein molecule. In reality this increase is due to aggregation and not
due to a change in the actual light-absorbing groups. On irradiation the
protein molecules form aggregates which scatter more light. The amount
of light scattered varies inversely as to the fourth power of the wave-
length, and a solution which does not appear cloudy in visible light may
scatter a great deal at 2,8QP A. We proved that the increase in absorp-
tion at the 2,800 peak is entirely due to aggregate formation, by measur-
ing the amount of light scattered in a special instrument. In the case of
lysozyme which contains much tryptophan, the position is slightly
different: one does first of all get a decrease in the absorption peak and
this is due to the destruction of the tr^T)tophan. With higher doses the
absorption goes up, but this increase is due to aggregation.

Dale: I should like to answer the first part. I have no experience of
the second part, which is actually a communication of your experiments
rather than a question. I think it would be rather unfortunate to choose
this criterion of aggregation, because it is a very common experience
with all colloids that the particle size increases on standing. I should
like to ask whether you have examined these solutions after irradiation
has been finished, and whether there are after-effects of aggregation
or not.

Alexander: Not after the first 30 minutes, which was the shortest
period in which we were able to look at it after irradiation.

Dale: When you precipitate colloids with various precipitins, with
various salts, you find continuous aggregation leading eventually to
flocculation. You must have had a similar phenomenon because you
mentioned the effect of addition of salts. I don't know whether you
varied valency of the salts using divalent ions and trivalent ions and so
on and whether you had a negative or a positive colloid, but the very
fact, as you say, that the enzymatic activity is not necessarily changed
does not bring this phenomenon within these experiments, because what
we measured is the effect on the activity of the enzyme rather than
on aggregation. I think the aggregations are rather non-specific changes
which with bigger doses probably also point to denaturation and I think
it does not affect activity measurements with radiation, which are
strictly quantitatively what you would expect, that doubling the dose
or making the dose 100 times as great has 100 times greater effect, apart
from the region where you have wide separation of solute molecules
with recombination of radicals.

Forssberg: When speaking about colloids and irradiation it may be
relevant to recall that J. A. Crowther and others, some twenty years
ago, studied the changes produced by very small doses on, for example,
colloidal gold and graphite, but also on proteins. It would seem that
irradiation causes cyclic changes both in particle size and in charge.
These changes sometimes proceed even after the irradiation is finished,
which implies that they are a function of the time of assay. It is not
known whether similar effects occur in vivo.

36 Discussion

Alexander: I think we should differentiate between lyophobic colloids,
which are essentially unstable colloids which will aggregate in time, and
solutions of macromolecules with which one deals in serum albumin
which is stable. The reason why I mentioned the physical changes in
proteins produced by radiation is that they can play a part in removing
the enzyme from its sphere of action and this may be as serious to the
cell as true inactivation. Aggregation induced by radiation is very
dependent on the conditions of irradiation and may contribute to the
variation in the radiosensitivity of cells with changing conditions.

Dale: This is a useful suggestion.

Popjak: I would like to raise a question about our general way of
thinking about effects of radiation on enzymes. I suppose the reason
why most people are looking for inactivation of enzymes really springs
from the overall effects observed, i.e. that the radiation eventually kills
an animal or a cell. Now, are we right in assuming that radiation will
necessarily inactivate an enzyme? The biological effects that are ob-
served are observed with relatively small doses; how far is one justified
in concluding from the irradiation in vitro with very large doses of a
crystalline enzyme, divorced from its substrates and all its other com-
panions, that the same sort of phenomenon is operating in the cell?
When an enzyme is inside the cell it is working fairly fast, and there is
a continuous movement of electrons and protons in and around the
molecule probably forming some kind of resonating system. I wonder
whether we might not by irradiation change enzyme specificity, change
rates of reactions, and whether it might not be worth while directing
some work towards that end rather than watching the enzyme in-
activation, and whether more information as to biological effects might
not be obtained in this way.

Dale: This is just what I had in mind when I put those questions at
the end of my presentation. My point of view is that it is quite possible
that a very minute functional part is changed while it is in transit and
that small changes may upset the proper sequence of events, changes
which may be so small that they are not analytically detectable, but
whilst the reaction is going on in the cell it may be of much greater
significance than the depletion of absolute amounts of enzyme. If, for
instance, in the enzymic reaction the functional part is slowed down it
cannot provide precursors for another reaction at the right time, so
that the integration of various interdependent reactions is destroyed
and, in my opinion, any attempt at trying to find a difference in enzyme
content of irradiated tissues or cells or disintegrated cells is, from the
start, completely futile because you only catch the total amount of
enzyme which does not matter at all. We are quite agreed that radiation
can only deal with a minute fraction of the enzyme present, but it all
depends on whether this minute fraction in the circumstances in which
it works in the cell is not relevant to the effect and, of course, from the
practical point of view it is very difficult to find experimental conditions
for checking what you suggested.

Butler: I would like to support Popjak's view on these grounds: you
have two classes of enzymes, those that are present in the cell in quite

Discussion

large quantities in which case, under any reasonable circumstances, the
percentage of inactivation is quite small, whether your reaction is
direct or indirect. The other case is the possibility of enzymes being
present in very small amount; as Mcllwain has shown (1946, Nature,
Lond., 158, 898), the possibility exists of enzymes being present only
to the extent of a few molecules per cell. But the situation is also
difficult there because the chance of a unique molecule of an enzyme
being inactivated is correspondingly small. So that in either case it seems
to me that the possibility of an enzymic explanation of metabolic effects
is not a very favourable one.

THE ACTIVITY OF ENZYMES AND
COENZYMES IN IRRADIATED TISSUES

Antoinette Pirie

Nuffield Laboratory of Ophthalmology, University of Oxford

The biochemical effects of radiation have been recently
reviewed by Ord and Stocken (1953), DuBois and Petersen
(1954), Errera (1955) and by Bacq and Alexander (1955) and
these valuable reviews form the basis from which any survey
of this jungle of a subject must be made. They make a general
survey of the jungle unnecessary and I propose to define the
problem as best I can and then to describe in some detail
those pieces of experimental work which provide evidence for
an initial effect of radiation on enzymes in the cell. The
effect of radiation on DNA, RNA, protein and phosphorus
metabolism is being considered by other speakers and will
not be touched upon.

If we are looking for an effect of radiation on cell enzymes,
what characteristics should we demand? A change in enzyme
activity found after radiation of the whole animal could be a
direct or indirect effect, i.e. reaction with the ionizing radiation
or particle itself or with the radicals formed in the medium.
It could also be a secondary effect resulting from structural or
chemical changes in other molecules which repercuss on the
activity of the particular enzyme we are studying. It seems
probable that such secondary effects on enzymes will become
more marked with time unless recovery sets in. Both direct
and indirect effects will be instantaneous and may be detect-
able as soon as it is possible to test ; but they may or may not
be maximal at this time. An enzyme, slightly damaged by
direct or indirect action, may continue to deca}^ Thus, for
example, Anderson (1954) found that pepsin, and McDonald .
(1954) found that trypsin gradually lose further activity dur-

Enzymes in Irradiated Tissues 39

ing the 24 hours following X-radiation. Kleczkowski (1954)
has found that if chymotrypsin is irradiated with ultraviolet
light and then kept at 2°, no further activity is lost, but if
kept at 37° for 48 hours the irradiated sample loses more
activity than a parallel control sample. Irradiation has made
the enzyme abnormally susceptible to body temperature. We
cannot therefore draw any hard and fast line between the
cause of enzyme change detected as soon after radiation as we
are able to make the estimation, and enzyme change that
develops gradually.

The time that must elapse between irradiation and bio-
chemical testing is necessarily long compared to the time
taken in metabolic reactions; a 10-second illumination of
green algae in the presence of carbon dioxide is sufficient
for the synthesis of a very large number of compounds
(Bassham et al., 1954). The time that elapses between irradi-
ation and metabolic examination is ample for a long
sequence of changes to interpose between direct and indirect
effects of radiation and the change we measure.

It does, however, seem worth while to concentrate on what
Errera (1955) has termed initial effects. The fact that few, if
any, such effects have been found may be an indication of our
ignorance of large parts of tissue metabolism, and the fact
that even the known parts have not yet been thoroughly
surveyed in relation to radiation. But it could also be ex-
plained if the only effect of radiation is to upset the molecules
of DNA and to upset its synthesis. This may well be an
enzymic effect since one could expect that DNA molecules
may have enzymic activities and will be synthesized by
enzymic processes, but at least it restricts the area of investi-
gation. I do not think any evidence we have at present can
decide. Certainly radiation affects cells other than those
capable of division. Patt (1955) has pointed out that there are
many departures from the simple condition relating radio-
sensitivity to growth and differentiation. The metabolic
activity of the cell can determine radiosensitivity, e.g.
chilling lymphocytes to 2° either shortly before or shortly

40 Antoinette Pirie

after radiation will remove the protective effects of anoxia or
of cysteine treatment (Patt, 1955). A low temperature also
seems to make the glycolytic enzyme systems of tumour and
retina more sensitive.

One concept of the reason why it is difficult to find an
immediate change in enzymic activity is that radiation has a
greater effect on enzyme synthesis than on the enzymes them-
selves. The cell, therefore, may only gradually go down hill
as enzymes fail to be replaced; but in yeast and Escherichia
coli, which have been investigated, the ability to form
adaptive enzymes seems unimpaired immediately after a dose
of X-rays that has killed 90-99 per cent of the cells. Thus
Brandt, Foreman and Swenson (1951) find that yeast cultures
given 4,800 r, which kills 90 per cent of the cells, will still form
galactozymase. Spiegelman, Baron and Quastler (1951)
found both galactozymase and maltozymase formation un-
affected. Yanofsky (1953) found that Esch. coli could still form
lactase normally after a dose of 5,000-10,000 r and Billen and
Lichstein (1952) showed that hydrogenlyase formation by
Esch. coli was normal after 15,000 r, for 100 minutes, although
it then fell off.

Concerning the effect of radiation on enzyme formation in
animal tissues Ranch and Stenstrom (1952) found that
400-000 r of X-radiation restricted to the pancreas, in dogs
w^ith pancreatic fistulae, caused a lowered secretion of amylase,
trypsin and lipase when tests were made 12 hours later. The
volume and pH of the secretion remained unchanged but the
enzyme content fell. The effect was reversible but could be
reproduced with further irradiation. The glands showed no
histological damage. Since the secretion of enzymes was not
studied at once after radiation we cannot say that this is an
initial effect, but it is an interesting approach.

Therefore, as there is doubt in this last case, we can say
that irradiation with X-rays [ultraviolet does inhibit (Errera,
1955)] has no immediate effect on enzyme synthesis. Thus, a
bacterial cell can continue to maintain its integrity and to
synthesize some at least of its enzymes after an amount of

Enzymes in Irradiated Tissues 41

radiation that prevents cell division. This does not lend
support to the view that the gradual decay of enzymes after
irradiation of animals is due to failure of synthesis, but the
evidence is far too scanty to be taken as refuting such a view.

It seems useless to attempt to catalogue all the work that
has been done which shows delayed effects of radiation on cell
enzymes. This includes my own work on lens metabolism.
It would appear to be more profitable to make a few bald
statements which can be amphfied, if necessary, in discussion.
This may lead to oversimplification but a detailed presenta-
tion leads only to confusion.

Within the boundaries of our ignorance it seems true to say
that lethal doses of radiation do not immediately change the
overall oxygen consumption of the animal or the major
respiratory enzymes. There is no change in oxygen consump-
tion of guinea pigs after an LD50 of X-rays (Smith, Budding-
ton and Greenan, 1952) or of the rat during the first four
days after a lethal dose (Mole, 1953). On the other hand, liver
dispersions examined 6 hours after whole body X-ray show
increased endogenous respiration (Kunkel and Phillips, 1952),
as do also bone marrow cells from the rabbit examined as
soon as possible after radiation (Altman, Richmond and
Solomon, 1951). These increases may indicate that there has
been a change in substrate concentration or another form of
enzymic activation. They are evidently insufficient to alter
the oxygen consumption of the whole animal. With Esch. coli,
Billen, Stapleton and Hollaender (1953) found that with one
strain a killing dose made no immediate difference to respira-
tion with glucose, succinate and pyruvate. With another
there was an immediate fall in oxygen uptake with pyruvate.

Cytochrome oxidase and succinic dehydrogenase, two major
respiratory enzymes of the mitrochondria, have been widely
investigated and in no case has any change been found as a
result of radiation, even hours or days later (Powell and
Pollard, 1955; Ashwell and Hickman, 1952; LeMay, 1951;
Hagen, 1955; Ryser, Aebi and Zuppinger, 1954; Thomson,
Tourtellottc and Carttar, 1952; Fischer, Coulter and Costello,

42 Antoinette Pirie

1953). Cytochrome c itself is not affected in Tlsch. coli (Hagen,
1955), nor is the uptake of radioactive ^^Fe into cytochrome b
of the liver (Bonnichsen and Hevesy, 1955).

The citric acid cycle accounts for a large part of the oxida-
tive metabolism of the tissues. The activity of the enzymes
of this system can be determined by determining the formation
of fluorocitric acid after injection of fluoroacetate. DuBois,
Cochran and DouU (1951) have found that a lethal dose of
X-rays to the rat inhibits fluorocitric acid accumulation in the
spleen, thymus and kidney within three hours. A dose of 100 r
inhibits synthesis temporarily in the spleen only. Now within
three hours these tissues will be already grossly altered struc-
turally and hence one cannot conclude that radiation has had
a direct or indirect effect on the enzymes concerned. That it is
a secondary effect is suggested by the fact that if the spleen
is exteriorized and it alone is irradiated (Table I) the fall in
citric acid synthesis does not occur (Petersen, Fitch and
DuBois, 1955).

Table I
Citrate Synthesis in Rat Spleen

24 hours after 800 r

Control

. -^ ^

To whole body To spleen only

Citric acid

y-g'ls-

1001

676 1002

DNAP

mg./lOO g.

124

55 98

Next, to consider coenzymes. These substances are
ubiquitous and function in many different metabolic processes
so that change in activity could have wide effects. Diphospho-
pyridine nucleotide coenzymes are thought to be synthesized
in the nucleus and change might indicate change of nuclear
function. No immediate change in their activity has, however,
been found. Eichel and Spirtes (1955) found no change in
the oxidized or reduced DPN content of rat liver 1 • 5 min.
after a lethal dose to the whole animal. Coenzyme A activity,
measured as the power of the animal to acetylate sulphanil-

Enzymes in Irradiated Tissues 43

amide (DuBois, Cotter and Petersen, 1955) or p-aminobenzoic
acid (Thomson and Mikuta, 1954) or to form hippuric acid
(Schrier, Altman and Hempelmann, 1954) was unimpaired.
It therefore appears that both coenzyme A and the enzymes
concerned with these acetylations function normally. The
level of coenzyme A and of nicotinic acid, which is some
measure of DPN and TPN, remained normal in the early
stages of X-ray cataract (van Heyningen, Pirie and Boag,
1954). The level of pyridoxin in the liver remains unaltered
(MacFarland et ah, 1950). Glutathione has not been found
to decrease in any tissue immediately after radiation (Bacq
and Alexander, 1955).

The glycolytic activity of tissues has not been extensively
studied. One particular investigation will be described later
but here one can say that where individual enzymes concerned
in glycolysis have been examined no change has been found
immediately after radiation. Thus aldolase, glyceraldehyde
phosphate dehydrogenase and lactic acid dehydrogenase of
lens, liver, kidney and spleen are not early affected (van
Heyningen, Pirie and Boag, 1954; DuBois and Petersen 1954).

Throughout, one has been expecting a fall in enzyme
activity; but results show that some enzyme processes are
immediately increased. Altman, Richmond and Solomon
(1951) showed that if the bone marrow was removed from
rabbits immediately after giving 800 r to the whole animal
and the synthesis of saturated and unsaturated fatty acids
from labelled acetate was measured in vitro the irradiated bone
marrow had 2-3 times the activity of the normal (Table II).
The oxygen uptake by the tissue was also greater. The actual

Table II

Effect of Radiation on the Synthesis of Fatty Acids and on
Respiration of the Bone Marrow of the Rabbit

.o/ ^«%«^!^« , O^ uptake

( % pre-radiation value) ^i Q^jg. wet wt.

Saturated Unsaturated in 3 hours
No radiation 100 100 300

0 hours 231 344 810

48 hours 108 — 280

44 Antoinette Pirie

time that elapsed between irradiation and the measurement of
synthesis is unfortunately not given. Later, the bone marrow
decayed. We know that coenzyme A, the coenzyme concerned
in fatty acid formation, is normal after radiation and it appears
that the enzymes are activated at once, either directly or by
change in substrate concentration, or change in permeability
of the mitochondria which are the seat of fatty acid synthesis.

This ties in with the work of Hevesy and Forssberg (1951)
who found that if mice given 2,000 r were then injected
immediately with ^^C-labelled glucose, and the exhaled CO 2
collected, starting 8 minutes later, the irradiated mice gave
ofP less CO 2 over the first hour than did the controls. There
was also increased ^*C in liver fats under these conditions.

The synthesis of haemoglobin also appears to be stimulated
immediately. Richmond, Altman and Solomon (1951) again
using the rabbit and a whole body dose of 800 r, found that
bone marrow and spleen dispersions taken immediately after
radiation incorporated ^*C -labelled glycine into haemin and
globin with greater rapidity than the normal. Forty-eight
hours after radiation synthesis had declined.

Similarly, Nizet, Lambert and Bacq (1954) found stimulation
of haemin synthesis in vitro by reticulocytes taken from a dog
30 minutes after a whole body dose of 500 r or by reticulocytes
irradiated in vitro. In three of four dogs tested it appeared
that plasma from the irradiated dog stimulated haemin forma-
tion in non-irradiated cells. This makes it appear that stimula-
ation of synthesis is not a direct or indirect effect on enzymes
of the red cell but a change in environment.

On the other hand, Bonnichsen and Hevesy (1955), who
point out that "Haemoglobin is one of the comparatively few
molecular constituents of the adult organism that is formed in
close connection with cell division", found decreased haemo-
globin formation in irradiated guinea pigs which were in-
jected with ^^Fe 6 hours after X-ray and killed 17 hours later.
This decrease in synthesis did not take place immediately
after radiation and it is suggested that the red blood corpuscles
of the marrow which are in an advanced stage of maturation

Enzymes in Irradiated Tissues 45

complete the synthesis of their haemoglobin after a dose of
radiation which will ultimately stop new haemoglobin forma-
tion entirely.

However interesting may be these results which show
immediate stimulation of fatty acid synthesis and of haemin
and globin formation, there is always the doubt that they are
direct or indirect effects of radiation since they have been
obtained with tissue preparations from animals which had
received whole body radiation. Therefore the effects might be
secondary and due to environmental change, i.e. change in
available substrates in the tissues. In vitro work with such
enzyme systems might give the answer.

To turn now to experiments where rather simpler conditions
have been used than irradiation of the whole body :

Formation of adrenal steroids

Ungar and co-workers (1955) have found that irradiation
of the perfused adrenal gland of the calf reduces the formation
of adrenal steroids. These glands, obtained from the slaughter-
house, are perfused and irradiated simultaneously for 2-3
hours, the dose being around 2,000 r of gamma-radiation from
^^Co. The blood, to which ACTH was added to stimulate
steroid formation, is passed through the gland only once and
therefore received only a small dose of radiation. At the end
of the perfusion the adrenal steroids in the blood were
isolated and estimated chromatographically. Production of
hydrocortisone, corticosterone and unidentified steroids was
markedly diminished.

Rosenfeld and co-workers (1955) further showed that if
various steroid precursors were added to the perfusing blood,
irradiation inhibited their conversion.

Table III shows that lip-, 17a- and 21-hydroxylations are
inhibited as well as oxidation of the A^-Sp-hydroxyl group to
to A^-3-ketone group. The percentage decrease in the conver-
sion products paralleled both in range and variability the
percentage decrease in corticoid output which was found in

Antoinette Pirie

Table III

Inhibition of Steroid Synthesis in Perfused Adrenal Gland by

Gamma- RADIATION

Dose 2,000-3,000 r

Substrate

Substance S

DOC

Progesterone

21-Desoxy-
cortisone

A^-Androstene-
3p-OH-17-one

Transformation
product

Hydrocortisone

Corticosterone

Hydrocortisone

Cortisone

lip-OH-
A*-androstene-
3 : 17-dione

Decreased

conversion

per cent

75, 34, 54
35, 55

37 (+7)

22, 19

48, 21

Specific

reaction

inhibited

1 1 P -hydroxy lation

1 1 [i-hydroxylation

11(3, 17a +

21 -hydroxylation

2 1 -hydroxylation

A5-3P-OH-
A*-3-ketone

the ACTH-stimulation studies. Radiation therefore appears
to inhibit many of the steroid-synthesizing enzymes and to
have httle specificity. The inhibition is apparent immediately
radiation ceases though it is true that the time of radiation is
considerable as is also the dose. However, other systems in
vitro are far more resistant to radiation than this. A point of
interest is that the enzyme which catalyses lip-hydroxyl-
ations is active in mitochondrial preparations (Brownie and
Grant, 1956) and the effect of radiation can be tested therefore
on this isolated enzyme that seems to be radiosensitive in the
tissue.

A further point is to try to relate the changes in the gland
in vitro with changes in vivo after radiation. Radiation of the
rat with 800 r causes loss of ascorbic acid and of cholesterol
from the adrenal within 1 hour (Bacq and Alexander, 1955).
Hochman, Bloch and Frankenthal (1953) found that 25-50 r
caused decrease in adrenal ascorbic acid tested 1 hour after
irradiation. The decrease, however, was not found to be dose
dependent; large doses still caused only about 26 per cent
decrease of adrenal ascorbic acid. Prof. Pincus tells me that
the ascorbic acid content of the adrenal glands used in his
perfusion work was extremely low and therefore no studies of
change during irradiation were made as it was felt these would

Enzymes in Irradiated Tissues 47

be meaningless. Excretion of steroids after radiation seems to
have been little studied. It is therefore impossible at present
to link the in vitro depression of steroid synthesis with in vivo
changes.

Effect of radiation on the retina

Crabtree (1936) found that if he gave long periods of
irradiation with radium — he does not specify the dose —
either to tumour tissue or to retinal tissue in vitro at 0°, the
anaerobic glycolysis was very much depressed whereas the
respiration of the tissue was unaffected. Tumour irradiated
at 37° showed no failure of anaerobic glycolysis. Retina could
not be irradiated at the higher temperature as the control
non-irradiated retina was unstable here.

Crabtree and Gray (1939) repeated this work using known
doses of X-rays, beta-rays or gamma-rays, keeping the time
of irradiation short and measuring the metabolism as quickly
as possible after radiation ended. They studied the retina of
the rat. Table IV shows that after a dose of 1,250 r given in

Table IV

Inhibition of Anaerobic Glycolysis of Rat Retina by

x-irradiation at 0-5°

Anaerobic glycolysis was measured 20 minutes after end of radiation

Time
radiation

Dose
r

of anaerobic

ru irtiiiuuiuti,

glycolysis

minutes

10 minutes

20 minutes

1250

130

15-3

2500

41 0

420

5000

61-8

62-5

10000

67-0

71-8

5 minutes at 0° the anaerobic glycolysis, when measured 20
minutes later, had fallen by 13 per cent. As the dose of radia-
tion given was increased the inhibition of glycolysis became
more marked. Fig. 1 shows that equal doses of p-radiation
and of X-radiation have the same effect. A criticism of this
work is that the figures given are percentage decreases and
if one calculates back from these percentages to the actual

Antoinette Pirie

lOO

.!?
'vi

8 8o

§ 60
'«»

«v 40
o

i^ 20

"^"H

■

■ (1 —

> I<

D 1

5 2

0 25 ' 30 35 40min.

Duration of Exposure
Percentage Residual Glycolysis after 20 minutes.

• p radiation at 250 E.S.U./c.c./min.
o X radiation at 250 E.S. U. /c.c./min.

Fig. 1. Equivalence of j3-rays and X-rays in depressing anaerobic glycolysis

of rat retina.

manometric readings that lie behind them then it seems dif-
ficult to place much confidence in the small change found after
the lowest dose of radiation in the first 10 minutes of experi-
ment. But this figure does not stand by itself. It forms part
of a series of results all of which show that radiation inhibits
anaerobic glycolysis when this is measured as soon as possible
after radiation ceases. Table V gives some results from an

Table V

Respiration and Glycolysis of Tissue After Irradiation at Low or

Body Temperature

Time

Tissue

of
irradi-
ation

Tr
hours

Temp.

— ■ ^

Tr Irradiated

Irradiated

J.Iv.S.

0-5

8-2

7-0 6-8

34-3

35-6

8-6

37-5

41-2

420

360

Retina

0-5

20-9 181

—

77- 1

4-5,9-4

0-5

19-4 17-3

68-4

25-6->'5-8

Enzymes in Irradiated Tissues 49

earlier paper where the actual Qq^ and Q^^ (anaerobic glyco-
lysis) are given and show the great difference in result accord-
ing to temperature. Therefore, I consider that we have here

a case where radiation a relatively large but not enormous

dose — inhibits an enzyme sequence in vitro. If the retina is
like the tumour used in Crabtree's earlier work it appears that
irradiation must be carried out at 0° for the effect to occur.

No work, as far as I know, has been published on the effect
of radiation on retinal metabolism in vivo. But that is not to
say that radiation has no effect. First of all, it is known that
low doses of X-rays produce a sensation of light — the X-ray
phosphene has been known since the last century — and there
is considerable evidence that X-rays act on visual purple, the
light-sensitive substance of retinal rod cells (Lipetz, 1955a
and b). This action of X-rays in stimulating the retina is not
known to have any relation to the inhibitory action of X-rays
on retinal glycolysis as the visual purple in rat retinas used
for metabolic experiments may have been largely bleached
although the retinas were prepared carefully in dim light.
Let us just use this as an indication that X-rays can stimulate
retinal tissue in vivo.

Evidence that X-rays have a very rapid damaging effect
on the retina in vivo has come from recent work by Cibis,
Noell and Eichel (1955) who have found that a dose of 2,000 r
given to the eye of a rabbit abolishes the 6-wave of the
electroretinogram within 10 minutes of the cessation of radia-
tion. This change may be reversible but with larger doses the
rod cells of the retina show degenerative changes within 3-5
hours, and over a period of days these cells disappear. The
changes produced by X-rays are strikingly similar to those
produced by injection of iodoacetic acid, that well known
inhibitor of the enzymes of glycolysis. Noell (1951) found
that non-lethal doses of iodoacetate caused immediate
reduction in amplitude of the electroretinogram measured in
the living animal and gradual histological decay of the rod
cells. Cone cells were relatively immune, as they are also to
X-rays.

50 Antoinette Pirie

Various considerations come to mind. First, is the rapid
diminution of the electroretinogram due to bleaching of the
visual purple by the X-rays? This seems unlikely since
Lipetz (1955&) using isolated retinas found little bleaching
until 10^-10^ r were given and then the picture was muddled
by heating effects. Calculations based on ratio of threshold
dose to bleaching dose for light and for X-rays also predict
that a very large dose of X-rays is necessary for bleaching.
Second, is the change in electroretinogram and the degenera-
tion of the rod cells due to vascular change in the choroid?
Cibis, Noell and Eichel (1955) state that vascular engorgement
occurs, but it is said that this type of retinal degeneration
would not be expected from such choroidal change.

With a dose of 1,400 r of X-rays we have noticed degenera-
tive changes in the outer limbs of rod cells of the rabbit eye
when the animal was killed some weeks or months later.
We have not examined many animals. Biegel (1955) using
radiation from the betatron failed to find more than minimal
changes after 3,600-4,500 r, in the rabbit retina. The results,
therefore, are a little variable.

In trying to assess the effects of radiation on the retina let
us return to the work of Crabtree and Gray. At 0° the
anaerobic glycolysis was inhibited. Now at 0° Terner, Eggleston
and Krebs (1950) have shown that retinal tissue is unable to
maintain osmotic control. It loses K and takes up Na. Low
temperatures have, in fact, a most remarkable effect on tissues.
It has long been known that they swell at this temperature,
and Conway, Geoghegan and McCormack (1955) find that
kidney and muscle tissue frozen in liquid O2, ground up and
then maintained at 0°, lose ATP and hexosephosphate and
increase their non-protein N. Hence, if one irradiates a tissue
at 0° not only is it in a state of metabolic arrest but it will be
in a state of metabolic decline. The change in K and Na in the
retina at 0° are reversible at 37° in the presence of glucose and
glutamate but not in their absence.

It seems possible that radiation could have a quite different
effect at low temperatures from that at normal — without

Enzymes in Irradiated Tissues 51

having recourse to an oxygen effect to explain this. This
problem needs a great deal more work but seems an example
of a rapid effect of X-rays on an enzyme system containing
at least 11 enzymes. No individual enzyme was studied, but
there is a certain specificity since the respiratory mechanism
is unaltered.

Auxin

A third instance where radiation is thought to have a
direct or indirect effect on a cytoplasmic enzyme is the in-
hibition of auxin synthesis in the plant by very low doses of
X-rays. Skoog (1935) first investigated the effect of radiation
on auxin and thought that auxin itself was inactivated rather
than that its synthesis was affected. Later, Gordon and
Weber (1955) have concluded that auxin is not particularly
sensitive to radiation, but that the synthesis is easily inhibited
(Weber and Gordon, 1951a, b and c; 1952a, b and c; Gordon,
1956). The effect was first described by Weber and Gordon
(1951a) in an Argonne National Laboratory report. Since
that time there have been many brief reports in ANL publica-
tions and Gordon gave a brief paper at Cambridge in 1955,
but, as far as I am aware, no details of the methods used and
the results obtained have ever been published and though one
can get some notion of the methods from others of Gordon's
papers not concerned with radiation, I have yet found it
difficult to assess the work. I feel a protest must be made
against this habit of publication of brief reports in laboratory
journals which are not available to most scientists. The
subject deserves more serious treatment.

Now, to summarize as critically as possible the ANL reports
on the inhibition of auxin synthesis by X-rays. Weber and
Gordon (1951a) find, first, that low doses of X-rays cause an
immediate drop in the auxin content of the young mung bean
plant; second (Weber and Gordon 19516), that shoots of the
mung bean infiltrated with tryptophan, and then irradiated,
form less auxin than similar non-irradiated shoots: third

52 Antoinette Pirie

(Weber and Gordon 1952a), extracts of irradiated mung bean
plants form less auxin from tryptophan than extracts from
normal plants. These changes were noticeable immediately
after radiation with low doses such as 10-50 r but synthesis
of auxin was not stopped completely even by very high doses.

The synthesis of auxin from tryptophan goes through a
series of steps. Weber and Gordon (1951c) found that im-
mediately after irradiation of the plant with 25-50 r there
was a rise in concentration of an aldehyde. Similarly, using
extracted enzymes from irradiated plants Weber and Gordon
(1952a) found increased formation of an aldehyde which is
considered to be indoleacetaldehyde. They therefore conclude
that it is the enzyme which converts indoleacetaldehyde to
indoleacetic acid or auxin that is specifically inhibited by
radiation.

If there is any doubt about this — and until details of the
work done between 1949 and 1955 have been published
there must be a doubt — I think it lies in the conclusion that
irradiation affects the conversion of indoleacetaldehyde to
indoleacetic acid. The reasons for uncertainty are these:
indoleacetaldehyde has not been proven to be an intermediary
in auxin synthesis. It has not been isolated from the plant and
has only once been synthesized and obtained pure. Gordon
has not, I think, been able to use indoleacetaldehyde in any of
his work. Assay of indoleacetaldehyde is achieved by conver-
sion to auxin either by infusions of soil or by enzymes from
leaves or by the aldehyde oxidase of milk, followed by bio-
logical assay of auxin formed. Since conversion is not com-
plete, the assay involves complicated calculations.

Brown, Henbest and Jones (1952) who synthesized indole-
acetaldehyde, tested the pure substance as an auxin or auxin
precursor in various biological tests. They found it to be not
more than 10 per cent as active as indoleacetic acid. Weber
and Gordon (1952&) have used neutral ether extracts of
cabbage as a crude source of indoleacetaldehyde in testing
the conversion of the aldehyde to auxin. But Jones and co-
workers (1952) have isolated indoleacetonitrile from neutral

Enzymes in Irradiated Tissues 53

extracts of cabbage and find that both the naturally occurring
and synthetic substances are as active as auxin itself in some
of the growth assay procedures, though not in others. On the
other hand Gordon and Nieva (1949a and b) in two full
papers have produced evidence that pineapple leaves, if
extracted with ether, yield an aldehyde in the neutral fraction
and this can be partly purified by reaction with dimedon or
bisulphite. The aldehyde, when regenerated from its bisul-
phite compound, will react with soil enzymes or leaf enzymes
to form auxin, but the conversion is not more than 10 per cent
after 24 hours.

This subject, therefore, is in a state of flux. That radiation
causes an immediate change in auxin formation seems
certain but there still seem to be doubts as to what is the
exact enzyme inhibited. Since this is such a fascinating
piece of work one looks forward with particular interest to
full publication.

What conclusions, if any, may be drawn from this partial
survey? Two things stand out in my mind: first, that we
cannot yet say in a single case that radiation directly or
indirectly damages an enzyme; second, that this present
position by no means rules out the possibility that enzymic
damage is important. The study of the effect of radiation on
enzymes is still in its infancy in spite of all the work that has
been done.

Gray has pointed out that the amount of energy from
radiation that is capable of preventing cell division is very
small indeed and that damage must either be to a key enzyme,
or be of a self-multiplying nature — or a break in continuity of
an important structure.

Inactivation of a key enzyme might be the cause of the
failure of DNA synthesis; break in continuity of a struc-
ture could be break up of DNA itself, or, for example, change
in structure of mitochondria. The self-duplicating form of
damage can be pictured as failure of enzyme synthesis.

Unfortunately we do not know what is the lifespan of
cellular enzymes or at what stage new synthesis takes place.

54 Antoinette Pirie

Perhaps enzymes are stable once formed, or perhaps they
wear out in use. Miller (1950) finds that depletion of protein
in the diet leads to loss of liver enzymes and replacement of
protein leads to their rapid restoration. Enzyme synthesis is
therefore possible in the non-dividing mammalian cell and
presumably takes place. In a number of experiments the
manifestation of radiation damage has depended on meta-
bolic activity and this could be imagined to be related to
wearing out and non-replacement of enzymes. In bacteria,
adaptive enzyme formation is not affected by doses that
prevent division but one wonders whether one can equate
bacteria that are extremely radioresistant with mammalian
cells that are radiosensitive. But at present we have no
evidence for failure of enzyme synthesis or failure of a key
enzyme after radiation of a mammalian cell.

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Enzymes in Irradiated Tissues 55

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56 Antoinette Pirie

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DISCUSSION

Latarjet: You quoted experiments of Nizet, Lambert and Bacq, who
found stimulation of liaemin synthesis by irradiated reticulocytes. The
stimulation of normal biological functions by radiations themselves or
by their chemical intermediates has always been surprising, and has
even been questioned. May I point out in connection with this that
Dr. Monier in my laboratory has treated pepsin with very small amounts
of an organic peroxide, and has found an increase, by a factor of 1 • 5, in
the enzymatic activity.

Van Bekkum: Dr. Pirie, you quoted data from Altman, Richmond and
Solomon in your Table II ; as far as I know, these experiments have not
been repeated by others so far and the evidence in support of an increase
in fatty acid synthesis is very meagre indeed, because each figure you
showed was derived from one rabbit only, and the authors did not
indicate the variation in the control rabbits.

Pirie: It was whole body irradiation, so they could not take one leg
and use the other as a control. Have you got similar reservations about
the data on increase in synthesis of haemin and globin?

Van Bekkum: I do not have reservations of the same kind but I have

Discussion 57

some reservations, because these data do not agree with what we know
about haemoglobin synthesis in vivo from other studies, for instance
those of Prof, de Hevesy.

Lajtha: In connection with haemoglobin synthesis, we have done
some irradiation of bone marrow in vitro and iti vivo. In our in vitro
studies we irradiated with doses of up to 5,000 r and then studied the
iron uptake with a high resolution autoradiography. We could detect
no increase or decrease in the uptake, neither in normoblasts nor in
erythrocytes. Jn vivo we gave up to 220 r. Bacq and Nizet gave 800 r
in vivo, and they gave huge doses, up to 100,000 r, in vitro.

Alexander: They gave those large doses in vitro because small doses
had no effect; they irradiated reticulocytes outside the dog and they
got no decisive effect until they reached 100,000 r.

Gray: They did get one effect at about 500 r. One gets the impression
that with the four dogs the results were rather variable. In one case an
effect was obtained at 500 r and in another none was observed at 10,000 r.

Alexander: I know the details of this work fairly well. The conclusion
which is based on limited data only is that plasma from an irradiated
dog was sufficient to stimulate unirradiated reticulocytes to greater
haemoglobin synthesis. The results about which I think there can be
no doubt are that the reticulocytes taken from dogs irradiated with
500 r synthesize haemin at a considerably faster rate than those which
had not been irradiated.

Lajtha: How long after irradiation did they take blood from the dog
to measure the reticulocyte stimulation?

Alexander: They took it as soon as they could.

With regard to the general problem of changes in enzyme activity
after irradiation of animals, Prof. Bacq and I reached the conclusion
a few years ago on reviewing the literature that there seem to be no
immediate decreases and in a few cases there was an increase in activity.
The effect of radiation may be to disturb the internal barriers of the cell
so as to allow enzymes to get access to sites from which they are normally
excluded (Bacq, Z. M., and Alexander, J. (1955). Fundamentals of
Radiobiology, p. 187). In this way one would find an increase in activity
if one looks immediately after irradiation. But after a time there will
be a loss in activity due to the mutual destruction of enzymes which
radiation had allowed to come together. An experiment is under way
at the moment to test this hypothesis. Errera found that the rigidity of
a nucleoprotein gel obtained by placing nuclei in water was decreased
by irradiation. The effect was greater when the intact cells were
irradiated than when the isolated nucleoprotein was irradiated. With
Prof. Bacq we have now done some experiments on spleen nuclei; if one
puts spleen nuclei into water they swell very much but do not go into
true solution, since on high speed centrifuging all the u.v.-absorbing
material (DNA-protein) goes to the bottom of the cell. On standing for
as little as 30 minutes at room temperature, but not at 0° C, this is no
longer the case and the u.v.-absorbing material is not spun down. It
looks as if an enzyme is liberated during the swelling of the nuclei in
water, which attacks the nucleoprotein gel and changes it from a gel

58 Discussion

to a sol. If the intact cells are irradiated and the nuclei isolated sub-
sequently, then the gel obtained is partially damaged. Irradiation of
the isolated nuclei with small doses does not have this effect on the
nucleoprotein gel. This may be interpreted as the release, on irradiation
of the cell, of an enzyme which is able to break down this nucleoprotein
gel, turning it into a sol.

Butler: The question which Dr. Pirie has been discussing is whether
in addition to effects on preformed enzymes there are effects on the
synthesis of enzymes, and one step in the synthetic process is, or may
be, the incorporation of amino acid. We have done a few experiments
in which we looked at the effects of irradiation on the incorporation of
amino acid in the proteins, actually of the rat liver. I know that other
experiments of that type have been done with rather inconsistent
results. It might be of interest to you in connection with the stimula-
tions you mentioned that we do find a stimulation of incorporation by
rather small doses of X-rays. It is not known what effect this has on
synthesis of proteins, but the radiation certainly has some effect on the
incorporation reaction.

Pirie: I think that the question of whether enzyme synthesis is upset
or not is one of the most interesting ones, and I wonder whether the
fact that in bacteria and yeast it is not affected is really relevant for the
very much more radiosensitive mammalian cell. It is difficult to get
data about enzyme lifespans in mammalian cells, but there certainly
are situations where enzymes are synthesized very rapidly. Miller found
that if he starved a rat, i.e. gave it a low protein diet, then there were
quite rapid changes in enzymes of the liver, and on replacing protein in
food the enzymes returned to normal activity in a few days. That is
a situation where enzyme synthesis is going on and could be studied.

Alper: Since the question of relative sensitivity of bacteria and
mammalian cells comes up quite often, I would like to point out here
that the sensitivity is not as different as is generally supposed. The
sort of doses which give, for instance, chromosome breaks in cells, are not
very far off the sort of doses with which you get long forms and much
increased lag. The doses which give killing effects in bacteria are not
really so different from those which kill mammalian cells. It is often
said that you cannot compare them, but I think this is not correct.

EFFECTS OF X-RAYS AND RADIOMIMETIG

AGENTS ON NUCLEIC ACIDS AND

NUCLEOPROTEINS

J. A. V. Butler

Chester Beatty Research Institute, Institute of Cancer Research,
Royal Cancer Hospital, London

I INTERPRET my fuHction as being to give an account of
present views on nucleic acid and nucleoprotein structure and
the chemical effects of irradiation, as a background for the
more specialized discussions which will follow on the actual
effect of ionizing radiations on metabolic processes in which
these substances are known or suspected to take part. The
discussion of DNA must begin with the structure proposed by
Crick and Watson (1953) which, although it may be subject
to modification in some minor respects, has proved adequate
so far to accommodate the known facts. In this structure, as
is well known, two complementary nucleotide chains are held
together by hydrogen bonds between the bases guanine and
cytosine and adenine and thymine. Numerous measurements
of molecular weight have given values of the order 6-8 X
10^. This implies a chain length of approximately 5 X 10* A
or 5 X 10"* cm. As determined by physical measurements,
the actual length of the particle is considerably less than this,
viz. 4-6 X 10^ A (Sadron, 1955). It follows that the particle,
although a fairly rigid structure, must be bent or coiled.
When studied by Shooter and Butler (1955) in the ultra-
centrifuge at low concentrations, a -very considerable range of
sedimentation constants was observed (often from S = 10 to
S = 40), so that there must be present a variety of fixed
shapes or sizes. Since one source (calf thymus) has given a
variety of products, we have to conclude that the product is
sensitive to the mode of preparation {e.g. by enzyme actions).

It must also be realized that the structure of DNA is not a

60 J. A. V. Butler

very stable one. It is disrupted by heat in water or salt solu-
tions, and is also sensitive to the action of dilute acids or
alkalies. Heating (e.g. at 100° for 15 minutes in water) causes
a great decrease in viscosity of solutions of DNA, with no
very marked change of sedimentation behaviour (Zamenhof,
Alexander and Leidy, 1954; Doty and Rice, 1955). There
have been conflicting interpretations of this, due mainly to
differences of conditions. However, there is no doubt that a
considerable amount of disruption of the hydrogen-bonded
structure occurs on heating, with a decrease in molecular
weight which depends partly on the specimen used and partly
on the concentration. On the basis of their results Dekker and
Schachman (1954) have suggested that the nucleotide thread
is interrupted at various points and that the DNA particle is
held together by hydrogen bonds between the overlapping
segments. It is, of course, difficult to establish whether such
interruptions are (1) originally present in the DNA; (2) pro-
duced during the preparation by the DNAse present in the
cells; (3) not originally present, but only caused by heat. The
drop in molecular weight (1/4 — 1/6) on heating was found by
Shooter, Pain and Butler (1956) to be much less in good speci-
mens than that found by Dekker and Schachman. It has also
been shown by Shooter and Butler (1956) that degradation
occurs at quite a rapid rate in the cell homogenates and even
in isolated (aqueous) nucleoprotein. The third possibility
would imply "weak points" in the nucleotide at which dis-
sociation by heat occurs, which might be the case if a few of the
PO4 bonds are triply esterified and thus easily hydrolysed.
No independent evidence of this has been obtained. However,
comparable heating in the solid state produces no degradation,
so that the eff'ect of heat may involve hydrolytic changes.

The effect of ionizing radiations on DNA has been studied
under a variety of circumstances. In aqueous solution (0-1
per cent) the characteristic high viscosity of DNA is greatly
reduced by comparatively small doses of radiation (see Fig.l),
about 8000 r being required to reduce the intrinsic viscosity
by one half (at 0-1 per cent). This is due mainly to a

Nucleic Acids and Nucleoproteins

decrease in particle size, but the relation between it and
molecular weight changes is not a simple one.

2 DOSEXlO"S

Fig. 1. Effect of 15 mcv electrons on solid and aqueous DNA. (Measurements
by R. H. Pain on materials irradiated by Prof. J. Rotblat.)

Chemical changes can also be observed in aqueous solutions
although large doses of radiation are required to produce
easily measured effects. Among the reactions which have

H2O

/ (OH
PO4/ ^

■'H)©

'N.

'C-

•'CH2-CH ChJKI-C .' N

N-C

\_ / ; ;\' /

^tJ® '"^^^ 'l^H, -'H)

Fig. 2. Some chemical effects of radicals on
DNA in aqueous solution.

been observed (Fig. 2) are (1) deamination of the bases;
(2) dehydroxylation ; (3) fission of the sugar-base linkage and
in some cases breakage of the pyrimidine ring; (4) oxidation
of the sugar moiety and (5) breakage of the nucleotide chains

J. A. V. Butler

and liberation of inorganic phosphate; (5) occurs normally
as a consequence of (4) (Scholes and Weiss, 1952; Butler and
Conway, 1953).

All these chemical effects are primarily radical reactions
and can be produced by — OH radicals formed in other ways.
The reactions of — H are not so well defined, except in so

NO IRRADIATION
I X 10 £ IN SOLUTION
2xl0^r IN SOLUTION
8x lO^r IN SOLID
|-2xl0^r SOLUTION

2 3 4

HOURS HYDROLYSIS

— p-

Fig. 3. Hydrolysis of DNA by heating with sulphuric acid
after irradiation (Butler and Simson, 1954).

far as it combines with oxygen to form the oxidizing radical
OgH. The liberation of inorganic phosphate varies with the
square of the dose, as is to be expected since two phosphate
ester bonds have to be broken in order to liberate PO4
(Butler and Conway, 1953). It is not easy to demonstrate
directly the breakage of the nucleotide chains. The effect of
single breakage is to liberate a terminal phosphate group, but
it can be shown that after irradiation free phosphate is more
quickly liberated on acid hydrolysis (Butler and Simson,
1954; see also Fig. 3).

Nucleic Acids and Nucleoproteins 63

It had been found that the dose required (in solution),
expressed in r units, to bring about a given amount of change
increases directly with the concentration (Cox et ah, 1955).
This is, of course, a characteristic of indirect action and will
be true if a constant fraction of the radicals is effective in
bringing about the observed change.

In dilute solutions about 100 ionizations are required per
molecule in the whole solution to reduce the viscosity by one
half, i.e. the process of degradation is comparatively inefficient.

When irradiated in the solid state, much greater doses are
required to bring about a given change, about 5 X 10^ r is
required to reduce the intrinsic viscosity by one half, i.e. the
factor for solid/dilute solution (0-1 per cent) is of the order of
100 for viscosity (Fig. 1). However, the actual efficiency per
ionization in producing a viscosity change is indeed greater
in the solid than in the solution, since as pointed out by
Alexander and Stacey (1955) less than 3 ionizations per
molecule produced in the solid do enough damage to reduce
the molecular weight by one half, and Fluke, Drew and
Pollard (1952) found that about one ionization will inactivate
Pneumococcus-tT2i\\sioTYmi\g principle. The greater sensitivity
per ionization produced in the solid state might be antici-
pated, as in dilute solution many of the ionizations give rise to
radicals at considerable distances from the DNA particles,
and will recombine with each other before reaching them.

This does not mean that the effect of radicals in vivo is
necessarily insignificant. The overall effect of irradiation is
greater in solution because ionizations occurring over a
considerable volume are effective by the radical mechanism.
When only a comparatively small amount of water is present,
the effect of ionizations in the water is relatively greater than
in dilute solutions and of the same order as that in an equal
volume of DNA; e.g. in 20 per cent DNA the sensitivity to a
given dose of radiation is 4 or 5 times that in solid DNA.

The effect of X-rays is also similar to that of heat in causing
a denaturation of the DNA. This is primarily a breakage of
hydrogen bonding between the threads. The result is shown

J. A. V. Butler

up in the fact that heat denaturation occurs more easily after
irradiation (Fig. 4). This effect occurs both in solution and
in the solid state, since it has also been found by Cox and
co-workers (1955), by titration curves, that X-ray treatment

loo

c?60

-I
I 50

s^ 40

©-

30
20
lO

20 30 40 50 60 70
TEMPERATURE

80 90 IOO°C

Fig. 4. Effect of irradiation on the temperature required to
denature DNA in 0 01 per cent aqueous solution.

0 Unirradiated DNA.

/\ Irradiated with 10^ r of 15 Mev electrons.

......... Irradiated with 4 X 10« r of 15 Mev electrons.

(Measurements by R. H. Pain on materials irradiated by Prof.

Rotblat.)

of solutions with doses of 8000 r causes a considerable break-
age of the hydrogen bonding. It is clear that this is a fairly
efficient process.

When we ask what biological effects are produced by these
changes we are in a much more difficult position. If DNA is

Nucleic Acids and Nucleoproteins 65

the genie substance it is probable that ehemical damage of
any kind will have some effect, but it is difficult to parti-
cularize as we do not know how the genetic information is
carried. If reduplication occurs by each base attracting its
complement as in the Crick and Watson scheme, it is clear
that loss of — NH2 or — OH groups by bases will prevent
their reduplication at this particular point. A break in a
nucleotide chain might make it impossible for the new particle
of DNA to be formed intact. However, it is not at present
certain that DNA duplication occurs in this way and there
are other possibilities in which some damage of the molecules
might be possible without impairing the genetic character.
It is noteworthy that Stent (1955) found that a considerable
amount of breakage of the nucleotide chains of phage could
occur without any loss of activity.

It is difficult to see how the mere breakage of hydrogen
bonds between the nucleotide chains could cause permanent
genie damage as the reduplication, at least on the Crick and
Watson scheme, involves the separation of the two fibres,
unless the particle can only take part in the duplicating
process if it is intact and complete. It would be expected
that a small degree of hydrogen bond breakage could easily
be made good. This might possibly happen in some "reacti-
vation" processes. However, attempts to reactivate trypsin
which had been inactivated to the extent of 50 per cent by
irradiation in the solid state by exposure to ultraviolet,
infrared and heating at 100° were unsuccessful (Butler and
Philhps, 1956, unpublished experiments). It might, however,
be possible to reverse slight degrees of damage.

It must also be remembered that the DNA is actually
present in somatic cells combined with histone. The histone
is known to be complex and to contain several substances of
different composition. However, since the total amount of the
bases present in the histone corresponds to at least 85 per
cent with the total amount of phosphate (Davison and Butler,
1956), it is reasonable to suppose that all or nearly all the
bases are attached to phosphate groups of DNA. We do not

RAD. 4

66 J. A. V. Butler

know what the function of this part of the nucleoprotein
system is.

The influence of this protein on radiation effects is not
easily assessed. It might be expected a priori that histone
would have a protective effect on DNA by mopping up
radicals, which would be prevented from reaching the DNA.
No accurate experiments on this have been made as it is
difficult to be sure that DNA has been recovered quantita-
tively from the nucleoprotein, and slow degradative changes
occur in the nucleoprotein on standing.

It may be useful to estimate the degree of damage in
the chromosome particle by different processes. If we take
the molecular weight of DNA as 6 X 10^, it can be estimated
that 10^ r will cause about 7 ionizations within each molecule,
and we have seen that 2-3 ionizations will cause a considera-
able amount of damage, which can be expressed as sufficient
to reduce the viscosity in aqueous solution by half. In the
hydrated state in the presence of water it may be expected
that this will be increased by several times at least, i.e. about
10^ r will cause the same degree of damage (expressed as 0*5
of the original viscosity). As there are 10^ particles in the
chromosome, this means that the probability of any one DNA
particle in the chromosome being damaged to this extent by
exposure to 1 r is of the order of 10~^.

It is estimated that (in mice) the probability of mutation
in any one locus is between 3 and 200 X 10"^ per r unit
(Russell, 1952). If we take 10"'^ or 10~^ as possible values, we
see that the probability of producing a specific mutation is
about 10"^ or 10"^ of that of damaging, to the extent mentioned,
the DNA particle which carried the gene in question. This
means that a considerable amount of damage can be done to
a DNA particle without affecting a specific gene carried by it.
This can be expressed in another way by saying that the locus
in question is not greater than the order of 10 ~^ or 10 ~^ of the
size of an average DNA particle. There are, of course, great
uncertainties in many of the quantities entering into this
calculation.

Nucleic Acids and Nucleoproteins 67

Let us turn now to the so-called radiomimetic agents
like the nitrogen mustards, which are capable of producing
(1) chromosome breaks, and (2) mutations. In vitro they com-
bine with DNA in various ways, especially with — NHg and
— OH. Combination with phosphate groups may also occur
but will in general be rather labile. The effect of combination
with the — NH2 and — OH groups is to break up the hydrogen-
bonded structure of DNA, which has been shown to be fol-
lowed by a slow degenerative breakage of nucleotide chains,
probably caused by hydrolysis of triply esterified phosphate (see
Butler, Gilbert and James, 1952). It might be asked if this
kind of reaction actually occurs in vivo, especially since there
are so many competing molecules such as proteins with which
reaction could occur.

It has been found that (1) when a nitrogen mustard (in this
case a radioactively labelled phenylalanine nitrogen mustard)
reacts with intact deoxyribonucleoprotein in aqueous medium,
reaction occurs with both the DNA part and the histone part
in the ratio of about 2 : 1 (Table I). This means that nitrogen

Table I

Reaction of a Nitrogen Mustard p-Di(2-Chloroethyl)amino-dl-i*C-
Phenylalanine with Deoxyribonucleoprotein Extracted from Various

Tissues.

(Experiments by J. A. V. Butler and A. R. Crathorn.)

Activity in Activity in

DNA Fraction Protein

Calf Thymus (dried preparation) 3-24 1-03

Rat Thymus (fresh wet preparation) 4-27 2-09

Spleen preparation 2-62 2-20

mustards can easily react with DNA even when the latter are
combined with histone; (2) when the same nitrogen mustard is
introduced into the rat, within a period of 1-3 hours (and
possibly shorter times) reaction has occurred with DNA and
RNA in all the cell fractions and in all the organs examined
(Table II). These substances therefore react very extensively
with nucleic acids in vivo.

J. A. V. Butler

Table II

Specific Radioactivities of Protein and Nucleic Acid Fractions from

Rats Administered with a ^''C-labelled Nitrogen Mustard p-Di(2-

Chloroethyl)amino-dl-Phenylalanine. (Experiments by P. Cohn.)

Tissue

Dose

Protein

RNA*

DNA*

Spleen

^ 10 mg./kg.:
rats killed

-. 0-017

0 004

0 010

Thymus 1 after one

^ or two days.

" 0021

0-007

0-010

Liver

(mitochondria)

^10 mg./kg. ^
twice at

0 083

0-037

Liver

(microsomes)

intervals

0 073

0-037

of 2 days:
killed on

Kidney

(supernatant)

^ 4th day. ^

0-33

0-20

* Purified by precipitation with ethanol.

In the absence of precise knowledge of the functions of these
substances, it is difficult to say what effects might be expected
to follow from these reactions. However, it is obvious that
reaction with DNA and RNA will not only interfere with their
ability to reproduce themselves but also with their metabolic
functions.

Table III

Comparison of Inhibitory Effect of Some Aromatic Nitrogen Mustards

ON THE "Exchange Reaction" in Staphylococcus awrej/s with the Inhibition

OF Growth of the Transplanted Walker Carcinoma.

(Experiments by G. D. Hunter and A. R. Crathorn.)

Inhibition of

Percentage inhibition

Formula

growth of

of exchange reaction

R = (C1-CH2CH2)2N-C6H4

Walker
carcinoma

with
phenylalanine

RCHgCOgH

+ + +

R(CH2)3C02H

+ + + +

R(CH2)4C02H

RO(CH2)3C02H

RO(CH2)4C02H

+ +

DL-R - CH2CH(NH2) - CO2H

+ + +

D-R • CH2 - CH(NH2) • CO2H

L-R • CH2CH(NH2)C02H

+ + + +

Nucleic Acids and Nucleoproteins 69

It has been found by my colleagues Dr. Hunter and Dr.
Crathorn, that with Staphylococcus aureus the effect of a
series of nitrogen mustards in inhibiting the incorporation of
an amino acid under conditions of starvation runs parallel to
their effects on tumour inhibition (Table III, Hunter and
Crathorn, 1956, unpublished experiments). There are thus
reasonable grounds for expecting that the reaction of the
nitrogen mustards with nucleic acids in the cell will have
metabolic consequences and will also affect the genie charac-
teristics.

Acknowledgement

I am indebted to my colleagues Drs. A. R. Crathorn, P. Cohn, G. D.
Hunter and D. M. Phillips for permitting me to quote unpublished
results.

REFERENCES

Alexander, P., and Stagey, K. A. (1955). IV Int. Conf. Radiobiol.,

Edinburgh : Oliver & Boyd.
Butler, J. A. V., and Conway, B. E. (1953). Proc. Roy. Soc, (B), 161,

562.
Butler, J. A. V., Gilbert, L., and James, D. W. F. (1952). J. chem.

Soc, 3268.
Butler, J. A. V., and Simson, P. (1954). Liege Radiobiology Sym-
posium, p. 46.
Cox, R. a., Overend, W. G., Peacocke, A. R., and Nelson, S. (1955).

Nature, Lond., 176, 919.
Crick, F. A. C, and Watson, J. D. (1953). Nature, Lond., 171, 737.
Davison, P. F., and Butler, J. A. V. (1956). Biochim. biophys. acta,

in press.
Dekker, C. a., and Schachman, H. K. (1954). Proc. nat. Acad. Sci.,

Wash., 40, 894.
Doty, P., and Rice, S. A. (1955). Biochim. biophys. acta, 16, 446.
Fluke, D., Drew, R., and Pollard, E. (1952). Proc. nat. Acad. Sci.,

Wash., 38, 180.
Russell, W. L. (1952). Cold Spr. Harb. Symp. quant. Biol., 16, 327.
Sadron, C. (1955). Ill Int. Congr. Biochim., p. 125.
ScHOLES, G., and Weiss, J. (1952). Exp. Cell Res., Suppl. 2, 219.
Shooter, K. V., and Butler, J. A. V. (1955). Nature, Lond, 175, 500.
Shooter, K. V., and Butler, J. A. V. (1956). Nature, Lond., 177, 1033.
Shooter, K. V., Pain, R. H., and Butler, J. A. V. (1956). Biochim.

biophys. acta, in press.
Stent, G. (1955). J. gen. Physiol., 38, 853.
Zamenhof, S., Alexander, H., and Leidy, G. (1954). J. exp. Med.,

98, 373.

70 Discussion

DISCUSSION

Roller: Why do you think that mutation may involve damage to the
gene molecule? Why not assume that mutation is caused by changing
the sequence of purine-pyrimidine bases in the DNA ?

Butler: You would have to break it up in order to change the sequence.
There is no easy chemical way of changing the sequence.

Haddow : You were thinking of cytology ?

Roller: Yes. Owing to the fact that mutation can be reversed and the
original gene structure reformed, the term "damage ", which is commonly
used by chemists and physicists, should be more clearly specified.

With regard to the question of hydrogen bonding, can we assume that
reduplication of the chromosome would involve the breaking of all the
hydrogen bonds in the DNA, which is a very high number? Are there
other possibilities to explain chromosome duplication and separation?

Spiegelman: I think that the doubled molecule has really been elimin-
ated by the Levinthal experiment which shows that you don't get
randomization of the ^^p making duplicates.

Butler: I heard that Mazia has the opposite results.

Spiegelman : I don't think the two types of experiments can be com-
pared. Mazia studied chromosomal multiplication and Levinthal the
duplication of viral DNA. Levinthal's results are consistent with the
obvious duplication mechanism deducible from the Watson-Crick
structure. He starts out with virus particles heavily labelled with ^^p
and examines the distribution of ^^p in the progeny particles produced.
An electron- sensitive photographic emulsion is used for the measurement
of the radioactivity of a single virus particle or a single DNA molecule.
He finds that 40 per cent of the DNA is contained in one large piece
which replicates to produce two particles, each containing half of the
original atoms. No further distribution of the original atoms takes
place with subsequent replication.

Alper: This whole experiment seems a bit mysterious if it is done with
phage, when it is considered to be fairly well established that the first
thing that happens when a phage gets inside a bacterium is that it just
breaks up completely.

Spiegelman: No, all that happens is that DNA goes in and leaves the
protein of the phage behind.

Alper: But it is not recognizable as phage chromosome for quite a
while, so that it is hard to see how things should come together, the
hot with the hot and the cold with the cold, so to speak.

Spiegelman: I don't understand why you believe that it falls apart
completely.

Alper: It depends on what one means by completely. You cannot
pick up phage recognizable as phage, or indeed any virus as virus, for
quite a long period.

Spiegelman: One cannot find infectious virus particles until the coats
and tails have been synthesized and put together, since these are neces-
sary for attachment and infection. However, the virus DNA is im-
mediately recognizable in such instances, e.g., T2 which contains 5-OH-
methylcytosine .

Discussion 71

Alper: Yes, the phage DNA, but I am talking about the actual
biological continuity of the phage particle as such. Now you are talking
about these threads presumably as carriers of the genetic material.
What is supposed to happen when phage gets into a bacterium is that
the genetic components come apart and they are somehow reconstituted.

Spiegelman: I don't think that is true.

Alper: I think it is.

Mitchell: I would like to make a suggestion about the therapeutic
action of radiation (see Mitchell, J. S. (1956), J. Colloid Sci., in press).
It is well known that a dose of say 2,250 r of gamma-rays produces
permanent healing of a typical small carcinoma of the skin in man. The
DNA content per nucleus in such a tumour was found by u.v. photo-
micrographic absorption methods to be 7-8 X 10'^^ g. The mean dia-
meter of the tumour cell nuclei was 6-86 microns. The number of ion
pairs within the material of the nuclei thus corresponds to a number of
DNA molecules of arithmetic mean molecular weight almost exactly
7 millions. This may suggest inhibition of reduplication, but that
hypothesis is not essential to the argument. While classical target
theory is obviously not the mechanism involved, one must think of a
macromolecular lesion of DNA or DNA-protein as the basis for the
therapeutic effect.

A possible experimental test is that one factor in radiosensitivity
would be the molecular weight of the DNA within the cell. I have
already started by methyl-green staining of sections cut in the same
block from radio-curable and radio-incurable tumours of the uterine
cervix. In the first pair of specimens there was very much less intense
methyl-green staining in the radio-incurable case. Aluch further work is
required.

Lajtha: I should like to ask Prof. Butler three questions, the first one
being whether he thinks that DNA in the cell may be more radioresis-
tant than DNA in solution. We labelled bone marrow cells in vitro with
^*C-adenine and then irradiated with 500 r and followed whether up to
48 hours there was any loss of labelled DNA; we found no loss after
500 r. We have repeated the experiment using 5,000 r, not with labelling
but just following the staining reaction of these cells with methyl
green-pyronin and Feulgen, and were unable to detect any significant
decrease in stainability. I don't tnink our technique is very sensitive, so
there may have been soine small losses not detectable, but certainly no
significant loss.

Secondly, how can one explain the differential radiosensitivity of the
incorporation of i*C-formate into DNA on the hydrogen bond breakage
theory ? Thirdly, what does Prof. Butler think is the mechanism of the
indirect radiation effects on bone marrow when the spleen is irradiated ?
Butler: As to the first question, that is really a type of chemical
experiment and you would picture it as similar to a test-tube experiment
in which one observed the liberation of adenine.
Lajtha: Yes.

Butler: I think it would require more than 5,000 r to produce any
observable effect.

72 Discussion

Lajtha: We get about 12| million i*C atoms incorporated into DNA!

Butler: You would require a fairly heavy dose in order to produce
detectable liberation of adenine. I think even in solution you require
fairly heavy doses, about 100,000 r ; 8,000 r will produce perhaps one
break in nucleotide chains, a very small fraction of the whole. With
regard to the other questions, these involve synthetic reactions and I
don't know what the synthetic reactions are in the mechanism of
synthesis of DNA. It is true that radiation has a marked effect on
spleen and thymus, and it has been reported that the DNA obtained
from the radiated spleen and thymus is relatively broken down. We
tried to repeat that, but we were not able to detect any difference in the
isolated DNA. What happens, of course, is that the radiation kills the
cells and you may get DNA which has been metabolically damaged by
enzymes from dying cells.

Forssberg: In vivo irradiation sometimes causes a marked degradation.
Bachmann and Harbers irradiated Walker carcinoma with 5,000 r and
isolated two DNA fractions according to Bendich, DNAi having a high
and DNAii a lower molecular weight. In the non-irradiated material
there was about three times as much high molecular DNA as low
molecular; but after irradiation with 5,000 r, within two hours the ratio
was about 0 • 2-0 • 3 ; so there is a very high grade depolymerization in this
carcinoma. Also the incorporation rates of isotopes are different in
these two fractions.

Gray: I would like to ask Prof. Butler for his observations on the
paper by Dr. Kaufmann and his group which seemed to me very inter-
esting as stressing the great sensitivity of the nucleoprotein as distinct
from DNA. They performed several experiments. In the first series of
experiments they formed a gel from calf thymus, irradiated the gel
with 1,000 r and then studied the fall in viscosity. This was much more
rapid than when they had high salt concentration present which dis-
sociated the nucleoprotein. In another experiment the effect of radiation
on a dilute solution of an artificial nucleoprotein obtained by mixing
calf thymus DNA with bovine albumin was compared with the effects of
radiation on dilute solutions of the DNA and the protein separately.
After exposure to 1,000 r the fall in viscosity was very much greater in
the former case, that is, when the nucleoprotein was irradiated. It is of
interest that the DNA used in these experiments was derived from calf
thymocytes and I wonder whether these experiments provide a clue to
the rather remarkable radiosensitivity of these cells.

Butler: This is rather reminiscent of Anderson's experiment at Oak
Ridge. The only thing that one can say about it is that you have there
a complex, a gel-like system of filaments which are bound together in
some way, and it certainly is true that very small doses of radiation
break down this structure. However, it is a very labile binding, because
it is affected even by shearing forces. What the mechanism of it is I don't
know; Dr. Hollaender may.

Hollaender: This nucleoprotein, which Anderson has prepared from
thymus, spleen, and other organs, responds readily to as little as 25
roentgens and is almost like water (Fig. 1). It behaves in many ways

Discussion

like polymer systems which have been studied previously. It can be
protected against irradiation by certain protective substances which will
be discussed later, and it can be studied quantitatively. In many ways
the preparation resembles the chromosome in the cell, at least as we
picture it from microdissection studies. It has, for example, pronounced
elasticity. It will break down in response to shearing stress, as Prof.
Butler pointed out, but the breakdown need not interfere with the
observation of radiation effect. Mr. Fisher, who is now working in our
laboratory, has devised a viscometer in which the viscosity may be
measured without previously subjecting the preparation to a shearing

VISCOSITY VS DOSE. RAT THYMUS IN « M NaCI

50 100 150 200 400 600

DOSE(r)

Fig. 1. (HoUaender). Effect of X-rays on viscosity
of crude rat thymus preparation (W. Fisher, un-
published).

field. With it he has been able to get very reproducible results. It
should be pointed out that sodium deoxyribonucleate is also sensitive to
mechanical stresses, and can be broken down during isolation, but it is
much more resistant than the nucleoprotein.

I would like to say that the nucleoprotein which Kaufmann has
isolated is in many ways much closer to what exists in the living cell
than the pure salt suspension which has been isolated. Unfortunately
these nucleoproteins cannot be dried, but it might be possible to reduce
them to a very low temperature where a direct effect could be observed.

Alper: What about the oxygen effect?

HoUaender: These are very viscous and the moment you bubble
oxygen through you break down the pattern in which they are organ-
ized. We have not learned how oxygen may be removed and so far it
cannot be removed; the moment you bubble nitrogen or any other gas

Discussion

through it or put a vacuum on so that you get the gas out, you break
the structure down.

Gray : If you remove the oxygen chemically then they are protected ?

Hollaender: Yes. In the curve shown in Fig. 1 the change in viscosity
with increasing irradiation was seen. Now if AET is added (Fig. 2), no
effect on viscosity is observed without irradiation (upper curve). With
250 r a very large decrease is observed without the AET (middle curve),
but with small concentrations a large protective effect is seen. With
900 r (lower curve) more AET is needed for maximal protection. Much
more complete protection is observed with lower doses of radiation.

CONCN. OF AET (mmoles/liter)

Fig. 2. (Hollaender). Protective effect of *S'-j3-amino-

ethyh'sothiouronium bromide HBr on crude rat

thymus preparation (W. Fisher, unpublished).

There is, however, a viscosity change which AET will not protect
against.

de Hevesy: Dr. Lajtha mentioned the effect of the irradiation of the
spleen. Is this effect not due to an interference with the formation of a
humoral substance and is the much greater effectiveness of the whole-
body radiation not partly due to the supression of the formation of this
substance in the whole organism?

Alexander: With regard to the irradiation of DNA in vitro, the resist-
ance of DNA to radiation, which has been stressed by Butler, is more
apparent than real. Dr. K. A. Stacey (1955, Int. Conf. TtadiohioL,
Cambridge, 1954, B.E.C.C. report, 32, 29) at our Institute irradiated
DNA from herring sperm in the dry state and then measured its mole-
cular weight by light scattering. From the change in molecular weight
the energy needed to break one polynucleotide bond can be determined.

Discussion

If the measurements are made in dilute salt solution, quite high doses
are required to decrease the apparent molecular weight. When un-
irradiated herring sperm DNA is dissolved in concentrated urea solution,
its molecular weight is halved, and is now only 3 million instead of 6 million
(Alexander and Stacey, (1955), Biochem. J., 60, 194). If the irradiated
DNA is measured in solution containing urea then relatively small doses
produce a decrease in molecular weight. A typical experiment is as
follows : a sample of DNA containing about 5 per cent of moisture is
irradiated with 2x10^ rad and its molecular weight measured dissolved

IRRADIATION

Fig. 1. (Alexander). A diagrammatic representation of
the effect of urea on irradiated DNA which shows how
breaks hidden in the dimeric structure (b) become ap-
parent in urea solution when all the hydrogen bonds
between the chains have been broken.

either in salt or in urea solution. In salt solution the average molecular
weight of the sample is hardly changed (6-5 x lOHo 6-0 x 10^), while
in urea solution the average molecular weight is more than halved.
We have interpreted this as follows : in urea solution the herring sperm
DNA molecule is dissociated and the two parts move freely; hence any
breaks introduced in any one chain will be revealed by molecular
weight measurements. When the material is measured in salt, the two
parts do not exist independently and two breaks have to occur in fairly
close juxtaposition in the two chains before a break can be detected by a
physicochemical measurement. Fig. 1 shows diagrammatically what we
believe to happen. Because of the dimer structure of DNA some of
these breaks are hidden and are only revealed when the structure is
opened out. With light scattering one can makf direct measurements of

76 Discussion

the molecular weight and determine the number of main chain bonds
broken, so that one can get quantitative values for the radiation change.
From a series of measurements we find that for every 10-15 electron
volts deposited in the nucleic acid (or a G value of more than 5) one
break occurs in the main chain if the DNA is irradiated in the solid
state. If the DNA is irradiated in solution the G value in the same
reaction is of the order of 0 • 1. In other words, direct action is about 50
times more effective than indirect action for depolymerizing DNA. In
the cell where the DNA is present at relatively high local concentration,
direct action will be much more important than indirect action as far as
depolymerization is concerned. This is relevant to the point made by
Prof. Mitchell (see p. 71) with regard to differences in the radiosensi-
tivity of tumours.

Latarjet: Experiments which were carried out on a transforming agent
of Pneumococcus by Dr. Ephrussi-Taylor and myself are not in agreement
with what Dr. Alexander has just said. The purified transforming agent,
which is DNA, is tested not by physicochemical properties but by its
biological specific activity, and its sensitivity to radiation remains the
same whether it has been treated with 5 m urea or not.

Haddow: I don't think that is necessarily an inconsistency.

Alexander: The answer may be that DNA's from different sources
don't all behave in the same way towards urea. Herring sperm DNA is
dissociated, DNA from thymus is not dissociated until it has received a
further treatment (Alexander, P., and Stacey, K. A. (1955), Nature^
Lond., 176, 162).

OXIDATIVE PHOSPHORYLATION IN SOME
RADIOSENSITIVE TISSUES AFTER
IRRADIATION

D. W. VAN Bekkum

Medical Biological Laboratory of the National Defence Research Council TNO,

Rijsivijk

Introduction

Although a considerable number of biochemical studies
on irradiated organisms and tissues has been made, informa-
tion concerning the primary biochemical lesion in irradiated
cells is still lacking. So far it seems that the investigation of
radiation effects in radiosensitive tissues has been more profit-
able than the study of radioresistant tissues. Unfortunately
our knowledge of the biochemistry of the former is limited
compared to that of some of the radioresistant tissues e.g. the
liver, the muscles and the brain.

In certain radiosensitive tissues massive cell death occurs
within a few hours after irradiation of the animal with a
lethal or sublethal dose and these events are bound to be
accompanied by a variety of biochemical changes. Therefore
the biochemical effects observed in these organs can only be
interpreted when the cytological effects are registered as well.
Biochemical changes that can be demonstrated before struc-
tural damage to the cells becomes apparent are of the greatest
interest, because these may be expected to be closely related
to or even identical with the primary biochemical lesion. This
does not infer however that structural alterations should neces-
sarily always be preceded by some enzymatic disturbance.

The nuclear functions of DNA synthesis and mitosis have
been found to be extremely sensitive to ionizing radiation.
However, the inhibition of these processes is not limited to
radiation and there is no convincing evidence as yet to justify

78 D. W. VAN Bekkum

identification of the disturbance of DNA synthesis with the
primary biochemical lesion. Since synthetic processes in the
nucleus are generally considered to be dependent on energy-
generating reactions which occur in the cytoplasm, it was
thought possible that the disturbance of nuclear functions
which occurs after irradiation might be the result of damage
to biochemical reaction systems outside the nucleus. The
cytoplasmic reaction chains by which energy is produced in a
transportable form, e.g. ATP, are chiefly anaerobic glycolysis
and the oxidations performed by way of the citric acid cycle.
In terms of the production of high energy phosphate bonds,
the latter are by far the most important.

The oxidative phosphorylations occur in the mitochondria
and these cytoplasmic structures have received relatively
little attention from radiobiologists. The older literature con-
tains some reports on morphological changes in mitochondria
which were observed shortly after irradiation of plant and
animal cells (Nadson and Rochlin, 1934; Hirsch, 1931;
Colwell, 1935). The changes described are presumably non-
specific but this applies to most of the radiation-induced
nuclear changes as well. Several of the authors claim that
the mitochondrial effects precede the morphological changes
which occur in the nuclei. On the other hand, Trowell (1952)
in a recent investigation of the effect of radiation on lympho-
cytes has reported only minor changes of the mitochondria.

In 1952 studies were initiated in this laboratory with the
object of collecting information on the effects of ionizing
radiation on the oxidative phosphorylations. In preliminary
experiments with mitochondria from various tissues, a
decreased phosphorylation was observed in rat spleen mito-
chondria after total body irradiation (van Bekkum et al., 1953).
Most of our subsequent work has been carried out with rat
spleen mitochondria but this effect has also been demon-
strated in mitochondria from mouse spleen and rat thymus.
A depression of oxidative phosphorylation in rat spleen
mitochondria after total body X-irradiation has also been
described by Potter and Bethel (1952) but their paper does not

Oxidative Phosphorylation in Irradiated Cells 79

include detailed results. Maxwell and Ashwell (1953) have
reported a decrease in oxidative phosphorylation of mouse
spleen mitochondria at 1-7 days after a lethal dose of total
body irradiation. The cellular composition of the spleen has
undergone radical changes at the time of their studies, so
that the results do not throw much light on the significance of
the disturbance with regard to the initial radiation effect.

Most of our own observations have been made 4 hours or
less after irradiation and considerable attention has been paid
to simultaneously occurring cytological effects. Part of the
results, which have already been published, will be reviewed
briefly below.

Methods

To enable the reader to evaluate our data, a few remarks
on the methods employed will be made. The animals — rats in
most of the experiments — were irradiated with penetrating
X-rays (H.V.L. : 1-8 mm. Cu, dose rate 45 r/min.) under
conditions of maximum backscatter. The mitochondria were
isolated by differential centrifugation of the homogenates in
0-25 M sucrose solution. Generally the mitochondria were
washed twice. The preparations thus obtained from spleen
and thymus contain a certain amount of impurities, part of
which consists of small nuclear fragments. With the various
homogenating techniques it has not been possible to disrupt
the majority of the cell membranes without simultaneously
breaking a significant number of nuclei. In view of the fact
that most of the cells of these tissues have only a narrow
brim of cytoplasm surrounding a large nucleus, this may
be expected. Oxidative phosphorylation has been measured
in a medium containing succinate or a-ketoglutarate as a
substrate. Adenylic acid was added to provide phosphate
acceptors, and hexokinase plus glucose were employed as a
trap for the terminal phosphate group of ATP. MgClg,
fluoride, ethylenediamine tetracetate and cytochrome c were
present in the system. DPN was added when a-ketoglutarate

80 D. W. VAN Bekkum

was used as a substrate, because washing causes a deficiency
of DPN in these mitochondria. Incubation was usually carried
out at 37°C for 24 minutes in oxygen. In recent experiments
the gas phase was air, which did not make any difference. For
a detailed description of these methods reference is made to
previous papers (van Bekkum et ah, 1954; van Bekkum and
Vos, 1955).

It should be pointed out that the properties of spleen and
thymus mitochondria are not identical with those of liver
mitochondria. Morphological differences have been observed:
spleen mitochondria are smaller and of less uniform size.
The number of mitochondria per cell is distinctly smaller in
spleen and thymus than in liver, and accordingly the yield of
mitochondria from the former tissues is much smaller. The
P/0 ratio of spleen mitochondria is about 1 when succinate is
oxidized. Under the same conditions the P/0 ratio of liver
mitochondria is usually about double that value. The P/0
ratio of thymus mitochondria was found to be well below 1.
Finally the phosphorylating activity of spleen mitochondria
may vary considerably between individual rats. This is
probably partly due to the marked variation of the cellular
composition of the spleen in rats. It has in fact been shown
that mitochondria isolated from spleens, in which the erythro-
poiesis has been greatly stimulated, display an increased
phosphorylation as well as elevated P/0 ratios (van Bekkum,
1955a). In order to minimize the effect of these variations,
the mitochondria were prepared from the pooled spleens or
thymus glands of 2-4 rats. Furthermore, in every experiment,
control and irradiated tissues were handled simultaneously.

Results

In our earlier experiments the relatively large dose of 1100 r
of total body irradiation was administered ; 4 hours thereafter
the rats were killed and the isolation of the spleen mitochon-
dria was started. A significant depression of the phosphate
uptake was present both when succinate and a-ketoglutarate

Oxidative Phosphorylation in Irradiated Cells 81

were used as substrate (Table I). In the experiments with
succinate the oxygen uptake was depressed to a less extent
than the phosphate uptake, which resulted in a decreased

Table I

Oxidative Phosphorylation of Rat Spleen IMitochondria 4 Hours

AFTER Irradiation (1100 r.)*

Phosphate O^ uptake PjO ratio

uptake [latomslmg.N

\imolelmg.N

Substrate: succinate 0-01 m

controls (18) 32-7±70 28-9±3-8 114+019

irradiated (19) 171+50 22-8±4-8 0-82±0-23

P of difference <0001 <0 001 <0 001

Substrate: cc-ketoglutarate 0 01 m

controls (7) 34-9±6-8 19-7±4-0 l-82±0-44

irradiated (6) 20-9±7-4 130±2-2 1-61 ±0-36

P of difference <001 <001 <0-2

* Figures represent means ± s.D. Figures between brackets indicate number of experiments.

P/0 ratio. With a-ketoglutarate the decrease in oxygen
uptake was more pronounced, and the decrease in the P/0
ratio was not statistically significant in this relatively small
series of observations (van Bekkum et al., 1954).

Effect of radiation dose

It was soon realized that the above experiments had been
performed on a tissue which contained large numbers of dead
and degenerating cells. Therefore the effect of the radiation
dose on the disturbance of the oxidative phosphorylation, as
well as on the amount of nuclear degeneration, was studied.
The interval between radiation and the kiUing of the rats was
kept at 4 hours. Doses down to 300 r were found to cause a
marked decrease in the phosphate uptake of both spleen and
thymus mitochondria. The minimal effective dose appeared
to be about 100 r in spleen and 50 r in the case of thymus mito-
chondria. The histological sections of these thymus glands,
after dosage with 50 r, showed definite changes at 4 hours.

82 D. W. VAN Bekkum

These consisted of pyknosis, fragmentation and vacuolization
of the nuclei of a small number of thymocytes. The percentage
of degenerated nuclei was 3-7 and 8-9 in two representative
sections against 1-0 and 1-7 in sections of control glands.
The number of mitotic figures was decreased by about 50 per
cent in the irradiated group. This amount of nuclear degenera-
tion seems rather small to explain the observed changes of the
mitochondrial functions. It was concluded that the oxidative
phosphorylation in these tissues represents a cytoplasmic
function the radiosensitivity of which is comparable to that
of the nuclei.

The interval between irradiation and the appearance
of the effect

Because of the possible relation between nuclear metabolism
and oxidative phosphorylation it seemed of interest to investi-
gate whether the disturbance of the latter becomes discern-
ible prior to or after the beginning of nuclear degeneration.
This was studied on spleen tissue after a total body dose
of 700 r (van Bekkum and Vos, 1955). Measurements were
made at 15 minutes, 30 minutes, 1, 2 and 4 hours after
irradiation. The earliest significant depression of phosphate
uptake was found at 2 hours (Fig. 1), when oxygen consump-
tion was only slightly depressed. The histological findings
may be summarized as follows: from 15 minutes up to 2 hours
after irradiation the mitotic frequency was greatly diminished,
at 4 hours some reappearance of mitotic figures was noted.
Signs of nuclear degeneration were absent or dubious at 15
and 30 minutes after irradiation. After 1 hour early stages of
nuclear degeneration became clearly discernible in a few
cells. At 2 hours nuclear degeneration was present in 10-20
per cent of the lymphocytes and at 4 hours the majority of
the cells showed pyknosis or nuclear fragmentation. Nuclear
damage could thus clearly be observed before a significant
decrease of phosphorylation was demonstrable.

This, however, cannot be taken as proof that nuclear degen-
eration precedes the mitochondrial lesion. Because individual

Oxidative Phosphorylation in Irradiated Cells 83

cells are inspected, the detection of cytological change is much
more sensitive than the estimation of oxidative phosphory-
lation which has to be carried out on the pooled mitochondria
from all the cells in a tissue. When, on the other hand, the total
number of damaged nuclei is compared with the overall
change of phosphorylation it may be stated that the develop-
ment of nuclear and mitochondrial damage runs roughly
parallel. In recent experiments results were obtained which

•/•

100--O-0

V4V2

T
2

r
4

IT
5 3

T
4

T
5

Fig. 1. Oxidative phosphorylation of rat spleen mito-
chondria at various periods after total body irradiation
(700 r). Substrate: succinate. Abscissae: hours after
irradiation. Ordinates: percentage of corresponding
control values. Wliite circles: oxygen uptake. Black
circles: phosphate uptake, n: number of experiments.

suggest that a biochemical defect in spleen mitochondria is
present as early as 30 minutes after total body irradiation,
when the only demonstrable cytological effect is the inhibition
of mitosis. These experiments will be described in one of the
following sections.

Tissue specificity of the effect

A rough survey has been made of the occurrence of the
disturbance of oxidative phosphorylation after total body

84 D. W. VAN Bekkum

irradiation in various tissues. The postirradiation interval
was 4 hours in all cases because after longer periods secondary
effects may be expected. So far, the disturbance has only been
found in spleen and thymus mitochondria. Results with
liver, regenerating liver and a number of transplantable
mouse tumours were negative even after doses of several
thousands of r. Intestinal mucosa and bone marrow did not
yield satisfactory mitochondrial preparations so that these
tissues could not be studied.

To investigate whether the effect in the mitochondria
occurs also after local irradiation of the spleen, the exterior-
ized spleen was irradiated with the rest of the body of the
rat shielded. A depression of oxidative phosphorylation was
found, which was comparable to that observed after total
body irradiation, and therefore the effect is at least chiefly
due to the action of X-rays on the spleen cells directly. As to
the nature of the cells containing these sensitive mitochondria,
evidence has been presented that the mitochondria from both
lymphoid and erythropoietic cells are susceptible to irradia-
tion (van Bekkum, 1955a).

It has not been possible to reproduce the effect of radia-
tion on the mitochondria in vitro, even with doses as large as
20,000 r. Similar negative findings have been reported by
Potter and Bethel (1952) and by Ord and Stocken (1955). In
:)ur opinion these negative in vitro findings carry little weight,
jince it is not possible to imitate even remotely the conditions
which exist inside the cell.

On the nature of the mitochondrial defect

The possibility has been considered that decreased phos-
phorylation after irradiation might be artificially induced
during the isolation process, as a result of some unknown and
completely unrelated change of the irradiated tissues, e.g. an
alteration of the viscosity of the homogenates. Therefore an
attempt was made to measure the rate of phosphorylation
in vivo by the use of radioactive phosphate. Rats were
injected intravenously with labelled inorganic phosphate

Oxidative Phosphorylation in Irradiated Cells 85

and after a suitable interval their spleens were excised under
Nembutal anaesthesia and immediately frozen in liquid air.
The frozen tissue was homogenized in perchloric acid and the
nucleotides were adsorbed from the neutralized perchloric acid
extract on charcoal. Elution was carried out with pyridine
solutions and AMP, ADP and ATP fractions were separated
by ion exchange on Amberlite IRA 400 according to a
modification of the method described by Cohn and Carter
(1950).

The specific activity of the ATP of the tissue can thus be
estimated, but the turnover rate of the phosphate groups of
ATP cannot be measured because no method is available to
determine the specific activity of the intracellular inorganic
phosphate which is supposedly the immediate precursor of
these groups. Therefore the specific activity of the total
inorganic phosphate was measured, and the specific activity
of the labile phosphate groups of ATP was expressed relative
to the former value (relative specific activity). Some of
the results obtained 4 hours after irradiation with a dose of
700 r are summarized in Table II.

Table II

The Effect of Total. Body Irradiation on the Incorporation of
Labelled Inorganic Phosphate in Rat Spleen ATP in vivo*

ATP specific activity: ATP relative specific
arbitrary units activity

Controls 10-9±l-5 0-66±006

4 hours after 700 r total

body dose 7-9±l-3 0-56±0-04

P of difference < 0 • 001 < 0 • 001

* The figures in the table represent means of 10 observations ± s.D.

The specific activity of ATP as well as the relative specific
activity of its labile phosphate groups were found to be
significantly depressed. Although this cannot be considered
to prove the existence of a disturbance of phosphorylating
reactions in vivo the data are at least in complete agree-
ment with the observations on isolated mitochondria. It is
noteworthy that no difference was observed in ATP content

86 D. W. VAN Bekkum

between the control and the irradiated groups. Similar results
have been obtained with this technique in thymus glands.

An increase of ATPase activity in spleen homogenates
after total body irradiation has been reported by several
authors (Ashwell and Hickman, 1952; Dubois and Petersen,
1954) and the question arose whether the depression of oxida-
tive phosphorylation might be related or even secondary to
this phenomenon. A more detailed investigation showed that
the increase of ATPase activity appears several hours after
the disturbance of oxidative phosphorylation has become
established. Furthermore, normal ATPase activities were
found in mitochondria that exhibited a markedly decreased
phosphorylating capacity (van Bekkum, 1955b). Finally
Petersen, Fish and Dubois (1955) reported that the ATPase
effect is absent after irradiation of the exteriorized spleen
exclusively and it should therefore be classified among the
secondary radiation effects.

The decrease in anaerobic glycolysis which was described
by Hickman and Ashwell (1953) in mouse spleen homogenates
one or more days after a total body irradiation, was found to
be absent at 2 and 4 hours after irradiation, when oxidative
phosphorylation was severely depressed (van Bekkum, 1955a).
It was concluded that different mechanisms are probably
involved in the development of these biochemical changes.

At present we are engaged in an investigation of the various
phosphorylative steps in spleen mitochondria from irradiated
rats. So far, some interesting data have been obtained on
the relation of cytochrome c to the depression of oxidative
phosphorylation. When cytochrome c is omitted from the
succinate reaction system both phosphate uptake and oxygen
consumption drop about 50 per cent in the control prepara-
tions. In other words the addition of cytochrome c causes an
appreciable stimulation (cytochrome c effect). This cyto-
chrome c effect is found to be consistently increased after
irradiation (Table III).

Even at 30 minutes after irradiation (700 r) a slightly
increased cytochrome c effect has been observed. If the

Oxidative Phosphorylation in Irradiated Cells 87

values obtained at 30 minutes and at 1 hour after irradiation
are pooled the differences between irradiated and control
preparations are found to be statistically significant both
in the case of phosphate and oxygen uptake. This demon-
strates an alteration of a mitochondrial function at a time
when nuclear degeneration is absent or limited to a few cells
only. Table III further shows that the cytochrome c effect
increases rapidly after the first hour, and that at 4 hours after
irradiation the very low phosphate uptake of the mito-
chondria is more markedly stimulated by added cytochrome c
than is the oxygen consumption. In this case cytochrome c

Table III

Influence of Total Body Irradiation (700 r) on the Cytochrome c

Effect in Spleen Mitochondria*

% stimulation by cytochrome cf
Hours after Phosphate uptake O^ uptake

control irradiated control irradiated

0-5 65(4) 77(4) 82(4) 95(4)

1 63 (4) 86 (4) 66 (3) 91 (3)

2 90 (4) 180 (3) 92 (4) 165 (4)
4 82 (2) 362 (2) 84 (2) 172 (2)

* Mean values are presented, the number of experiments is given between brackets.

t Stimulation is expressed in percentage of the values obtained in the absence of cytochrome
c. Substrate : succinate ; concentration of cytochrome c: 28 x 10 'M. The mitochondria were
washed twice with sucrose.

nearly doubles the P/0 ratio. These results suggest that in
spleen mitochondria a relative cytochrome c deficiency
develops very shortly after irradiation.

It was of course essential to know whether this reflects
an interference by radiation with some part of the cytochrome
system. It was not inconceivable that some radiation-
induced structural alteration of the mitochondria might cause
a leakage of cytochrome c during the isolation, in which case
washing of the mitochondria might be expected to increase
this leakage. In previous experiments with normal spleen
mitochondria it was found that repeated washing causes a
deficiency of DPN, which could be demonstrated in the
a-ketoglutarate system.

To test the possibility of the occurrence of a similar leakage

88 D. W. VAN Bekkum

of cytochrome c being aggravated as a result of irradiation,
the cytochrome c effect was estimated at 2 hours after
irradiation in unwashed mitochondria. A comparison of the
results (Table IV) wdth those obtained on twice washed

Table IV

Effect of the Addition of Cytochrome c and of DPN on Oxidative

Phosphorylation of Unwashed Spleen Mitochondria from Control

and Irradiated Rats (2 Hours after 700 r)*

Phosphate uptake Og uptake

control irradiated control irradiated

119 360 102 195

% stimulationt by cyto-
chrome c in succinate
system

% stimulationt by DPN
in a-ketoglutarate
system 8 5 7 13

• Values represent means of 3 experiments; 4 control and 4 irradiated rats were used per

t Stimulation is expressed in percentage of the values obtained in the absence of the stimulat-
ing substance. Concentrationof cytochrome c: 2-8 x 10-*m; DPN: 4 x 10-*M.

mitochondria (Table III) shows that the cytochrome c effect is
not increased by washing. Therefore this simple explanation
cannot be applied. It is also to be noted that irradiation does
not result in an increased leakage of DPN.

Conclusions

At present we are still far from a complete understanding
of the biochemical changes that take place in spleen mito-
chondria after irradiation. However, it seems fairly certain
that these changes occur shortly after irradiation and that the
cytochrome system is somehow involved. It is tempting to
connect the fact that irradiation affects this part of the respi-
ratory chain with the well-known observations on the oxygen
effect in irradiation experiments with living organisms.

In this connection it is of interest that Laser (1954) has
also suggested the involvement of the cytochromes in the
biochemical effect of radiation. However, much additional
information is required before the nature of this involvement
can be more accurately defined.

Oxidative Phosphorylation in Irradiated Cells 89

REFERENCES

AsHWELL, G., and Hickman, J. (1952). Proc. Soc. exp. Biol., N.Y., 80,
407.

Bekkum, D. W. van (1955a). Radiobiology Symposium, p. 201.
London: Butterworth.

Bekkum, D. W. van (19556). Biochim. hiophys. acta, 16, 437.

Bekkum, D. W. van, Jongepier, H. J., Nieuwerkerk, H. T. M., and
Cohen, J. A. (1953). Trans. Faraday Soc, 49, 329.

Bekkum, D. W. van, Jongepier, H. J., Nieuwerkerk, H. T. M., and
Cohen, J. A. (1954). Brit. J. Radiol., 27, 127.

Bekkum, D. W. van, and Vos, O. (1955). Brit. J. exp. Path., 36, 432.

CoHN, W. E., and Carter, C. E. (1950). J. Amer. chem. Soc, 72, 4273.

CoLWELL, H. A. (1935). The Method of Action of Radium and Alpha-
rays in Living Tissue. London: Oxford University Press.

Dubois, K. P., and Petersen, D. F. (1954). Amer. J. Physiol., 176, 282.

Hickman, J., and x\shwell, G. (1953). J. biol. Chem., 205, 651.

HiRSCH, G. C. (1931). Arch. EntwMech. Org., 123, 792.

Laser, H. (1954). Nature, Lond., 174, 753.

Maxwell, E., and Ashwell, G. (1953). Arch. Biochem., 43, 389.

Nadson, G. a., and Rochlin, E. J. (1934). Protoplasma, 20, 31.

Ord, M. G., and Stocken, L. A. (1955). Brit. J. Radiol., 28, 279.

Petersen, D. F., Fish, F. W., and Dubois, K. P. (1955). Proc. Soc exp.
Biol, N.Y., 88, 394.

Potter, R. L., and Bethel, F. N. (1952). Fed. Proc, 11, 270.

Trowell, O. a. (1952). J. path. Bad., 64, 687.

DISCUSSION

Loutit: It is very heartening to me to see this correlation between
biochemical and histological findings, for so rarely do we have the two
together. I would like to ask whether the cytologist or histologist was
always doing his work unseen as it were; did he know what he was
looking at, whether it was a half-hour, two-hour or four-hour section;
or could he have been biased by a previous knowledge of the time at
which the section was taken?

Van Bekkum: The histologist, Dr. O. Vos, only knew that some of the
sections were taken at say 30 minutes and others 1,2 or 4 hours after
irradiation, but he did not know which one. On the basis of nuclear
changes he could not differentiate between 15 minutes after irradiation,
30 minutes after irradiation and control sections, but he could differen-
tiate between them because of the decrease of mitotic figures in the
slices from irradiated rats.

Roller: I have seen lymphocytes, irradiated with quite small doses,
which are undergoing nuclear disintegration similar to that described
by Dr. Van Bekkum. The degeneration of the lymphocytes in extremely
high numbers cannot be explained by the old theory that the cells have
gone through mitosis after irradiation and die because of chromosome
fragmentation. That raises a very important point which is included

90 Discussion

in Dr. Van Bekkum's presentation: have we in the lymphocytes a
different system as far as sensitivity towards radiation is concerned ?

Louiit: In our laboratory we have seen, in other tissues besides lymph
tissues, the break-up of cells before mitotic division, with a dose of about
150 r. I do not think it is confined to this particular system.

Mitchell: This acute cytolysis is seen in some of the cells of the highly
radiosensitive and radio-curable basal-cell carcinoma of the skin ; even
after 50 r within about 45 minutes areas of liquefaction have been seen.

Holmes: It may be relevant to Dr. Roller's question to describe an
experiment we carried out for Dr. Trowell. He asked us to estimate the
DNA synthesis in lymph glands where he was quite sure there was
no mitosis. We found more uptake of ^^p into the DNA fraction than
one would expect in a resting tissue. It seemed just possible that even
if no division was occurring the DNA was undergoing more change than
it does in other tissues.

Bracket: Dr. Van Bekkum, is there any contamination of nuclear
material in your mitochondria, for instance is there any DNA? Could
it be that, when there is this destruction of nuclei, material originating
from degenerating nuclei might mix with your mitochondrial fraction?
According to Allfrey and Mirsky (1955, Nature, Lond., 176, 1042),
isolated thymus nuclei might be the site of some sort of oxidative
phosphorylation. It may be specially important, if that is true, to
know whether you have any nuclear contamination or not.

Van Bekkum: I cannot believe that it is possible, as Mirsky claims,
to completely separate the nuclei from the cytoplasm in a tissue such
as the thymus. As far as I can remember, Allfrey and Mirsky did not
demonstrate phosphorylation in isolated liver cell nuclei, which are
much easier to obtain, and in my opinion that is what should be done.

Bracket: I quite agree with you that that should also be done.

Van Bekkum: I am not convinced that the experiments of Allfrey
and Mirsky have proved beyond doubt that nuclei or nuclear fragments
are capable of oxidative phosphorylation. I am sure that even our
control preparations do contain a certain amount of nuclear fragments.
I have not been able to avoid that by varying the methods of homo-
genization and differential centrifugation. Therefore we have spent a
considerable amount of time on the in vivo studies with radioactive
phosphate and we have interpreted the results as supporting our
in vitro findings.

Howard: Coming back to the correlation of the histological effects in
the spleen, it is clear that lymphocytes can be killed by very small doses
of radiation. The dead cells take a little time to appear in the tissue,
but I do not think we have to assume that there is no change in the cell
population until the dead cells appear. There are changes taking place
as soon as there is a mitotic arrest. You only have to suppose that the
function of the mitochondria has something to do with the cell cycle,
or perhaps even that cytochrome c is bound up with the cell cycle in
some way, and you will expect that an hour or two hours after irradia-
tion, when there has been a blocking of cells entering mitosis, this might
be reflected in the activity of the mitochondria. So I think these histo-

Discussion 91

logical changes, even if we are not prepared to accept a lot of cell death
within the first two hours, are still very significant indeed to the inter-
pretation of the results.

Van Bekkum: What time do you suppose it takes for a lymphoid cell
to go through mitosis?

Howard: I think the cycle must be fairly rapid, of the order of a few
hours.

Van Bekkum: Well, in our spleen tissues, at least, the number of
mitotic figures is very low compared to the total number of cells, of
the order of 2 or 3 per thousand. I don't think that the explanation you
suggest can cover this if you consider what a small number of cells is
moved by it per hour.

Howard: The whole cycle probably takes a few hours, but there may
be a very short time spent in division. I would imagine that cell turn-
over is quite rapid.

de Hevesy: There is a large fraction of lymphocytes having a very
long life-time, as found by Ottesen, the existence of which may explain
Dr. Van Bekkum's finding.

Laser: Dr. Van Bekkum, I take it that you agree that irradiation of
isolated mitochondria has no effect on oxidative phosphorylation. In
this connection, I would like to mention one result which has rather
surprised us. Dr. Slater and I have measured the actual oxidative
phosphorylation during radiation. The system was so adapted that
within six minutes, during which we applied 30,000 r, we could measure
the disappearance of a-ketoglutarate and the phosphorylation. There
was no significant effect at all, which means that these processes went
on undisturbed during the actual application of 30,000 r to the isolated
material.

Van Bekkum: We have done the same sort of thing with similarly
negative results. Did you add cytochrome c to the system during the
measurement of phosphorylation ?

Laser: Yes.

Van Bekkum: So did we.

Popjak: Dr. Van Bekkum, have you an explanation for the rather
low P/O ratio in this particular type of mitochondria? It seems to me
that from spleen and thymus you are getting a P /O ratio of barely over 1 .
One normally gets higher P/O ratios with liver mitochondria.

Van Bekkum: I only know that Dr. Slater has also found, in the case
of heart mitochondria, a rather low P/O ratio of about 1 in the presence
of succinate. I don't know of any explanation for that.

Loutit: With this level of dose the period of two hours seems to me
a very critical one. There is also evidence, in addition to histological
and biochemical evidence, for cell death at this time. The ordinary
sodium pump mechanism has been shown to break doAvn at this time
and sodium leaks into the cell and potassium leaks out. Furthermore,
we have already discussed the loosening of the enzyme systems, that
enzymes are getting into places where they should not be, which might
be the preliminary stage for this final blow-up and deathblow to the
cell.

THE EFFECTS OF EXTRANEOUS AGENTS
ON CELL METABOLISM*

H. A. Krebs

Medical Research Council Unit for Research in Cell Metabolism,
Department of Biochemistry, University of Oxford

The question has been raised in the preceding contributions
whether or not a study of the effects of ionizing radiation on
isolated enzyme systems may be expected to solve some of the
problems of radiation biology. I would like to offer comments
on this question.

I think it is correct to say that all effects on living cells of
radiation, and of extraneous agents generally, are brought
about through interference with some chemical substance in
the cell. If this substance is an enzyme, the effect of the ex-
ternal agent (probably always a reduction of catalytic activity)
may cause a significant disturbance in cell metabolism, but
this is not necessarily the case. Whether a change of en-
zyme activity is of major consequence depends on whether
the enzyme concerned plays a role in the rate control of
metabolism.

Pacemaker reactions as vulnerable stages of cell

metabolism

The analysis of the circumstances which control the rates of
metabolic processes shows that the amount of enzyme present
is by no means always the controlling factor. It is true that

* Abbreviations used:

DPN diphosphopyridine nucleotide

DPNH2 reduced diphosphopyridine nucleotide

ATP, ADP adenosine triphosphate, adenosine diphosphate

GTP, GDP guanosine triphosphate, guanosine diphosphate

TPP thiamine pyrophosphate

P inorganic orthophosphate

CoA coenzyme A

Extraneous Agents and Cell Metabolism 93

the amounts of enzyme and substrate determine the maximum
rate of the process but maximum rates are exceptional under
physiological conditions. The limiting factor is usually
(though not always) the amount of substrate. The fact that
intermediate products generally do not accumulate shows
that the substrates of the intermediary enzymes are removed
as rapidly as they are formed. The average half-life of the
acids of the tricarboxylic acid cycle in a rapidly respiring
tissue is of the order of a few seconds (Krebs, 1954). Thus the
amount of enzyme in the tissue is sufficient to deal with the
intermediate as soon as it arises ; in other words, the amount
of available substrate is the factor limiting the rate at which
many intermediary steps proceed. Hence a partial destruc-
tion of an enzyme does not necessarily upset cell metabolism.

It is obvious, however, that this cannot apply to all steps
of metabolism. There are some reactions, small in number by
comparison, where the rates depend on factors other than the
amounts of enzyme or substrate. These are the " pacemakers "
of metabolism (Krebs, 1956).

The pacemaker reactions are the steps of metabolism which
are especially vulnerable to extraneous agents, because any
decrease in activity is liable to show itself in a diminished
overall rate of metabolism. Pacemakers are therefore the
enzyme systems towards which the study of the effect of
extraneous agents should be primarily directed. I would like
to elaborate these considerations on some examples, taken
mainly from the field of energy transformations.

Effects of extraneous agents on anaerobic glycolysis

The anaerobic glycolysis is known to involve some twelve
separate steps. The factors which control the overall rate of
glycolysis and adjust the rate to the physiological energy
requirements are by no means fully known but there are two
steps which have been identified as pacemakers. The first is
the hexokinase reaction (LePage, 1950) which probably
initiates all major metabolic reactions of glucose, such as the

94 II. A. Krebs

anaerobic fermentation, the complete oxidation and the
transformation into glycogen, pentoses, fat, amino acids, or
other cell constituents. The second is the triosephosphate de-
hydrogenase systems. This is a complex reaction in which, apart
from triosephosphate and the catalysts, at least three other
reactants — DPN, ADP and orthophosphate — are required:

triosephosphate + DPNl _^ f phosphoglycerate + DPNH2
+ ADP + P J ^ 1 + ^TP

As the coupling between the dehydrogenation of the sub-
strate and the synthesis of ATP is obligatory the reaction
cannot take place unless ADP and phosphate are available.
The concentrations of these two substances are in fact assumed
to be the rate-limiting factors (Lardy and Wellman, 1952).
The concentrations are bound to vary with the functional
state of the cell, especially with the rate of the expenditure
of ATP. Since oxidative phosphorylation removes ADP and
phosphate, respiration must inhibit glycolysis, an inter-
pretation of the "Pasteur effect" first suggested by Lynen
(1941) and Johnson (1941).

There must be additional pacemakers of anaerobic glycoly-
sis which decide the fate of glucose-6-phosphate. This inter-
mediate is placed at one of the branching points of metabolism.
Apart from giving lactate through glycolysis it can be trans-
formed into glycogen or oxidized to phosphogluconate and
pentose phosphate, but nothing definite is known about the
factors controlling the choice between these alternatives.

Many of the inhibitors of glycolysis are substances which
react with the two known pacemaker enzymes. Thus bromo-
and iodoacetate inhibit triosephosphate dehydrogenase, by
combining with the sulphydryl group of glutathione, the
prosthetic group of the enzyme. The hexokinase reaction is
inhibited by various hexosephosphates, in particular by the
product of the reaction, glucose-6-phosphate (Weil-Malherbe
and Bone, 1951), and by L-sorbose-1 -phosphate (Lardy,
Wiebelhaus and Mann, 1950). This inhibition is non-competi-
tive. The two pacemakers are also the points of attack of

Extraneous Agents and Cell Metabolism 95

glyceraldehyde, a powerful inhibitor of glycolysis (Mendel,
1929; Rudney, 1949). L-Glyceraldehyde is transformed in
glycolysing material into L-sorbose-1 -phosphate under the
influence of aldolase, d -Glyceraldehyde probably inhibits
triosephosphate dehydrogenase (Needham, Siminovitch and
Rapkine, 1951).

Whilst pacemakers are more vulnerable to extraneous
agents than non-pacemaker reactions, the latter are not
immune to inhibitors; but a substantial proportion of a
non-pacemaker must be inactivated before the overall rate is
affected. An inhibitor of glycolysis which interferes with a
non-pacemaker is fluoride. It inhibits enolase, the enzyme
which converts 2 -phosphogly cerate to enolphosphopyruvate
(Meyerhof and Kiessling, 1933; Lohmann and Meyerhof,
1934). However, fluoride also inhibits other enzymes, in
particular those dependent on magnesium ions as a cofactor,
such as adenosine triphosphatase and some phosphate-trans-
ferring enzymes. It is by no means established that the in-
hibition of glycolysis is solely due to the inactivation of enolase.

Effects of extraneous agents on cell respiration

When energy is released by the oxidation of carbohydrate,
fat and amino acids, there are over a hundred identifiable
intermediate steps, only a few of which are pacemakers. The
non-accumulation of intermediates indicates that those steps
which initiate the oxidation of a substrate, i.e. the reaction
between substrates and their dehydrogenase, must be among
the pacemakers of respiration. Once the oxidation has been
started, most of the subsequent reactions, leading to complete
combustion, follow at the pace set by the initiating step,
owing to the excess of enzymes dealing with the intermediary
products. The initiating reactions also decide which sub-
strate among a mixture is attacked preferentially — whether
carbohydrate, fatty acid or amino acids serve as a source of
energy. In addition, pacemakers are expected at two other
types of stages of the oxidative metabolism, at those where the

96 H. A. Krebs

rate of oxygen consumption is determined, and at those where,
after a partial degradation, more than one pathway is open;
in other words, where the pathways of metabohsm can branch.
Before I discuss the effect of inhibitors on the different
types of pacemaker reactions, I must make reference to the
mechanism by which the rate of oxygen consumption is con-
trolled. One of the decisive factors is the rate at which hydro-
gen atoms or electrons travel from reduced DPN, via flavo-
protein and cytochrome c, to molecular oxygen. Unless the
catalysts of the electron carrier chain are in the oxidized
form the substrates cannot be attacked. Further, the trans-
port of electrons under physiological conditions is coupled
with the synthesis of ATP from ADP and orthophosphate
( ' ' oxidative phosphorylation " ) : '

DPNH2 + 4O2 \ r DPN + H2O

ADP + P j "^ \ ATP

This coupling appears to be obligatory. Hence the rate of
oxygen consumption reaches a maximum value only if ADP
and orthophosphate are present above certain critical con-
centrations and it falls when the concentration of the phos-
phates falls below the critical level. In most tissues the physio-
logical concentrations of ADP and P are generally below the
critical level. The rate of oxygen consumption therefore
depends on the rate at which ATP is split in the tissue to ADP
and P, i.e. on the rate at which energy is spent. It is thus
evident that the component reactions of oxidative phos-
phorylation are pacemakers.

The following examples show, for the case of cell respiration,
that extraneous agents interfere with pacemakers rather than
with other intermediary steps of metabolism. Inhibitors of
respiration fall into three main classes, according to the type
of pacemaker which they inhibit. Substances of Class I
inhibit rate of oxygen consumption because they interfere
with electron transport. Class II interferes with the initiating
reaction of respiration and therefore affects the type of
substrate which is oxidized. Class III interferes with the

Extraneous Agents and Cell Metabolism 97

mechanism controlling the branching points of metabolism and
can therefore divert metabolism from one pathway to another.

Inhibitors belonging to Class I are hydrocyanic acid, azide,
carbon monoxide or sulphide which stop the electron transport
from iron porphyrin to molecular Og. Another example is
antimycin A which combines with an unidentified component
of the transport chain between dehydrogenase and cytochrome
c and therefore inhibits the oxidation of the substrate by
molecular oxygen whilst not preventing ferricyanide from
acting as an electron acceptor (Potter and Reif, 1952; Copen-
haver and Lardy, 1952).

Inhibitors of Class II interfere at the dehydrogenase level.
If the inhibitor is specific for one dehydrogenase, or one type
of dehydrogenase, it does not necessarily alter the overall rate
of oxidation because other substrates can take the place of
that which is prevented from reacting. Thus, cells exposed
to malonate which can no longer oxidize succinate at the
usual rate may still consume oxygen at the normal rate, if
another substrate, such as fumarate, is available.

The initial step of substrate degradation can be brought
about by several different types of reaction. In most cases
this is a more or less direct transfer to pyridine nucleotide
according to the general formula :

dehydrogenase .,. , i .. . ,
(1) substrate + DPN > oxidized substrate +

DPNH2

The a-ketonic acids require a more complex mechanism
which involves at least six additional cofactors : coenzyme A,
a-lipoic acid, cocarboxylase, ADP, GDP and inorganic phos-
phate. In the case of a-ketoglutarate the following reaction
mechanism has been formulated (Gunsalus, 1954). The initial
step is taken to be a reaction between a-ketoglutarate and
thiamine pyrophosphate (TPP) in which a succinic semialde-
hyde-TPP-complex is formed and COg liberated:

R . CO . COOH + TPP -> [R . COH . TPP] -f CO^
(R =C00H.(CH2),)

RAD.

98 H. A. Krebs

The succinic semialdehyde TPP complex then reacts with the
disulphide form of a-Hpoic acid in such a manner that (a) the
aldehyde group of the TPP complex is oxidized to the corres-
ponding carboxyl whilst the disulphide is reduced to the
dimercaptan; (b) the nascent carboxyl and one of the nascent
mercaptan groups condense to form succinyl lipoic acid. TPP
is regenerated in this reaction:

S-CH R.CO.S-CH

[R . COOH . TPP] + I ^CH2 -> /CH2 + TPP

S-C HS-C^

H2 Hj

(R' =COOH.(CH2)4)

The next stage is a transfer of the succinyl group from lipoic
acid to coenzyme A, yielding reduced a-lipoic acid and succinyl
coenzyme A:

/R yR

R.CO.S— CH HS-CH

')cH2 + HS.CoA-^R.CO.S.CoA+ ^CH2

HS-CH2 HS-C

The reduced lipoic acid interacts with DPN under the
influence of lipoic acid dehydrogenase to yield a reduced
pyridine nucleotide:

/R' yR'

HS-CH S-CH

:CH

yCU^ + DPN -> DPNH2 +
HS-C^ S-C"

H2 H2

Succinyl coenzyme A reacts with GDP and inorganic phos-
phate to regenerate reduced coenzyme A and to form GTP

Extraneous Agents and Cell Metabolism 99

and succinate, and GTP and ADP subsequently react to form
GDP and ATP :

succinyl CoA + GDP + P -> succinate + GTP + CoA
GTP + ADP -^ ATP + GDP

Other a-ketonic acids probably react analogously to a-keto-
glutarate, at least as far as the reactions with TPP, lipoic
acid and coenzyme A are concerned. In the case of pyruvate
and possibly other cases, the acyl coenzyme A arising in the

most substrates a-ketonic acids

a- lipoic acid

DPN

flavoprotein [succinate

(?or vitamin K}"* (acyl coenzyme A

iron porphyrin

Fig. 1. Pathway of hydrogen transport for
different types of substrates.

primary stages is assumed to react with AMP to form acetyl
AMP, and subsequently acetate and ATP (Berg, 1955).

Apart from a-ketonic acids there are a few other substrates
which do not react according to the common rule (reaction 1).
They are succinate, and fatty acids attached to coenzyme A,
possibly also some amino acids. In these cases DPN is not
involved in hydrogen transport. Instead there is a direct
transfer of hydrogen atoms to a flavoprotein, and thence to
iron porphyrins. These exceptions arise from the thermo-
dynamic properties of the substrates.

Fig. 1 summarizes the stages in hydrogen and electron

100 H. A. Krebs

transport from different types of substrates to Og. It differs
in two respects from earlier schemes. One concerns the role
of vitamin K. Martins (1956) has provided evidence suggest-
ing that vitamin K is an essential link which might replace
flavoprotein in some cases. The other concerns the role of
flavoprotein in hydrogen transport from succinate. That
flavoprotein is required in this case has recently been estab-
lished by Green, Mii and Kohout (1955) and Kearney and
Singer (1955).

The differences in the complexity of the various dehydro-
genase systems are reflected by differences in their behaviour
towards extraneous agents. It has long been known that the
oxidation of a-ketonic acids is sensitive to reagents, for
example, arsenite (Krebs, 1933), which do not affect other
dehydrogenases. Arsenite reacts with SH groups and the
specific action of arsenite on the oxidation of a-ketonic acids
can be understood on account of the special role played by
sulphydryl compounds in the dehydrogenation of a-ketonic
acids.

The relative simplicity of the succinic dehydrogenase
system explains the fact that the oxidation of this substrate
is more stable towards environmental changes than that of
other substrates. Depriving the tissue of soluble cof actors by
washing of minced material with water, inactivates all major
dehydrogenase systems except succinic dehydrogenase.

Examples of inhibitors of Class III acting at a branching
point of metabolism are agents inducing the formation of
ketone bodies in the liver. Among the pathways open to
acetyl coenzyme A in liver, there is the condensation with
oxaloacetate (i.e. entry into the tricarboxylic acid cycle) or
the condensation with another molecule of acetyl coenzyme
A [i.e. formation of acetoacetate ("ketogenesis")]. The
first requires oxaloacetate as a reactant, and much of the
evidence is in accordance with the view that the steady-state
level of oxaloacetate is a key factor in the control of keto-
genesis.

Agents which reduce the supply of oxaloacetate in the

Extraneous Agents and Cell Metabolism 101

liver are therefore expected to be ketogenic. This is in fact
the case. Malonate, which prevents the conversion of succinate
to oxaloacetate, or ammonium chloride which diverts the
metabolism of a-ketoglutarate to glutamate are both ketogenic
(Recknagel and Potter, 1951; Krebs and Kornberg, 1956).

Effects of extraneous agents on cell activities depending

on energy supply

What has been said so far all refers to the reactions supply-
ing energy. Another group of metabolic processes depends
on a supply of energy which must generally be available in the
form of ATP. To these belong all synthetic processes, (especi-
ally the formation of macromolecules from basic units),
active transport of solutes, active movement and chemical
processes associated with other specific functional activities
of the cells. Very little is so far known about the nature of
the enzymes concerned with these aspects of metabolism ; it is
not possible in this field to draw up schemes similar to those
representing the energy-supplying reactions.

However, one general feature appears to be shared by
many extraneous agents which interfere with processes
depending on energy. Although it may not be possible to
define the chemical reactions which are obstructed, many
effects can be explained by the assumption that the interfering
agent is chemically similar to a physiological agent, and that
owing to this similarity it occupies the physiological site, thus
displacing the physiological agent from its normal position.
To quote examples, this mechanism may account for :

(1) The growth inhibition by sulphonamides (which occupy
the position of ^-aminobenzoic acid) and by other anti-
metabolites like aminopterin (an antifolic acid agent), 6-
mercaptopurine (an antipurine agent) and halogen-substituted
phenylalanine derivatives (antiphenylalanine agents in pro-
tein synthesis).

(2) The anticoagulant effects of dicoumarol which interferes
with the conversion of vitamin K into prothrombin.

102 H. A. Krebs

(3) The action of many drugs, especially the blocking
agents. Cholinergic blocking agents (atropine, curare, tetra-
ethyl ammonium ions) are assumed to prevent the attachment
of the acetylcholine to the hypothetical receptor site, whilst
adrenergic blocking agents (ergotoxin, veratrin) analogously
block the adrenergic transmission.

(4) The toxic, or some of the toxic, effects of fiuoroacetate
which replaces acetate in the formation of citrate and there-
by yields fiuorocitrate which in turn is a powerful enzyme
inhibitor.

In view of the widespread occurrence of this type of inter-
ference the idea suggests itself that it might also be responsible
for some of the effects of radiation; that the decomposition
products of water arising from ionizing irradiation so modify
cell constituents that they become noxious and that the
noxiousness is due to the similarity to normal constituents.

Conclusions

To sum up, the main thesis put forward in this contribu-
tion is the concept that some stages of metabolism in living
cells are more vulnerable than others to attack by extraneous
agents. The vulnerable stages are those which control the
rates of metabolic processes — the "pacemaker" reactions.
The enzyme systems responsible for these reactions are
expected to work to full capacity under physiological condi-
tions, so that any change in the amount of active enzymes will
modify the rate of metabolic processes. In contrast, the
enzymes operating at other stages are present in excess of the
available substrate, and a partial destruction of the enzymes
therefore does not affect the rate of metabolism. Information
on the nature and mechanism of action of "pacemaker"
reactions is still limited, but the available information for the
case of energy transformations confirms that interference by
extraneous agents with cell metabolism is more often than not
due to interference with pacemaker reactions.

Extraneous Agents and Cell Metabolism 103

REFERENCES

Berg, P. (1955). J. Amer, Chem. Soc, 77, 3163.

CoPENHAVER, J. H., and Lardy, H. A. (1952). J. hiol. Chem., 195, 225.

Green, D. E., Mii, S., and Kohout, P. M. (1955). J. hiol. Chem., 217,
551.

GuNSALUS, I. C. (1954). Fed. Proc, 13, 715.

Johnson, M. J. (1941). Science, 94, 200.

Kearney, E. B., and Singer, T. P. (1955). Biochim. biophys. acta, 17,
596.

Krebs, H. a. (1933). Z. physiol. Chem., 217, 191.

Krebs, H. a. (1954). In Chemical Pathways of MetaboHsm, Greenberg
D. M., Ed. Vol. I., p. 109. New York: Academic Press.

Krebs, H. A. (1956). Dtsch. med. Wschr., 81, 4.

Krebs, H. A., and Kornberg, H. L. (1956). Ergehn. Physiol., in press.

Lardy, H. A., and Wellman, H. (1952), J. hiol. Chem., 195, 215.

Lardy, H. A., Wiebelhaus, V. D., and Mann, K. M. (1950). J. hiol.
Chem., 187, 325.

LePage, G. a. (1950). Cancer Research, 10, 77.

LoHMANN, K., and Meyerhof, O. (1934). Biochem. Z., 273, 60.

Lynen, F. (1941). Liehigs Annalen, 546, 120.

Martius, C. (1956). Conferences et Rapports, III Int. Congr. Bio-
chem., p. 1.

Mendel, B. (1929). Klin. Wschr., 8, 169.

Meyerhof, O., and Kiessling, W. (1933). Biochem. Z., 264, 40.

Needham, D. M., Siminovitch, L., and Rapkine, S. M. (1951). Biochem.
J., 49, 113.

Potter, R. van, and Reif, A. E. (1952). J. hiol. Chem., 194, 287.

Recknagel, R. O., and Potter, V. R. (1951). J. hiol. Chem., 191, 263.

Rudney, H. (1949). Arch. Biochem., 23, 67.

Weil-Malherbe, H., and Bone, A. D. (1951). Biochem. J., 49, 339.

DISCUSSION

Brachet: Prof. Krebs, would you comment on the possible role of cell
structure in this regulatory activity of the cell?

Krebs: A recent number of Nature contains a letter by Prof. Peters on
this point (1956, Nature, Lond., Ill, 426). He discusses the possible
role of what he calls the cytoskeleton of the cell, a hypothetical network
of structures which keeps enzymes and substrates in their places. He
suggests that hormones may change the nature of this cytoskeleton and
that hormones may not act directly on a specific enzyme ; that perhaps
one hormone can act on a number of enzymes at the same time, by
making substances accessible. This is perhaps a useful working hypo-
thesis, but it is difficult to visualize how one can test this idea by
experiments. I would certainly agree that the structure is a very
important point.

Alexander: I would like to ask a very similar question, but phrased
in a slightly different way. You have told us that in the whole chain of
enzyme reactions there are certain pacemakers. Now there must be a

104 Discussion

certain step which is rate-controUing for the pacemaking reaction. In a
heterogeneous S3^stem, as in the cell, there must be for each individual
enzymatic stage a number of steps which are physicochemically dis-
tinct, such as diffusion of substrate to the enzyme and diffusion of the
product away from the enzyme in addition to the actual chemical reac-
tion occurring on the enzyme. Any one of these steps could determine
the overall rate of the chemical reaction. Could you tell us whether the
rate-controlling step of the individual enzyme reactions in the cell is likely
to be the actual chemical reaction on the enzyme or a diffusion process ?

Krehs: I don't think that one can make a general statement about it.
What one can tackle experimentally is the identification of pacemaker
steps, by determining the steady-state level of the intermediate meta-
bolites, especially of the substrate of the pacemaker. If a substance
reacts at a variable rate, its steady-state concentration must vary,
because it will be produced at the same rate but removed at a different
rate. A number of people have started to determine the steady-state
level of intermediate metabolites, such as DPN, or reduced DPN,
organic phosphates. This may lead to the identification of a pacemaker.
But what changes the rate of such a reaction is a different matter.
Before this can be answered the mechanism of the pacemaker reaction
must be known. We just don't know enough about such mechanisms to
say what role diffusion might play.

Dale: Do you consider it useful to do model experiments which
may show effects on surfaces which bind enzymes and perhaps sub-
strates at the same time, e.g. to imitate perhaps internal cell boundaries
or cell surfaces with model experiments, such as burning of glucose on
charcoal ?

Krehs: In general I would think that the scope of model experiments
is nowadays limited. The earlier models, like the oxidation of sugars on
charcoal, would be largely irrelevant. W^e should study the real thing
for preference.

Dale: It must be extremely difficult to determine the steady state in
the cell for one of these recommended steps, because of their very small
amounts present at a given time.

Krehs: It is indeed a great problem but it is being tackled and is
being successfully solved in some cases. With paper chromatography
and isotope techniques it is possible to determine quantitatively
metabolites in very small amounts.

Dale: With regard to radiation it is difficult; you have to irradiate
practically at the same time, because the steady state may change as
soon as radiation stops.

Krehs: One can stop reactions very quickly under most conditions, by
liquid air and other means.

Iladdow: Did I understand j^ou to mention hexokinase in relation to
the cell surface?

Krehs: Yes, there is a good deal of evidence showing that the entry
of sugar into the cell is not by passive diffusion but an active process.
We have recently carried out experiments on the true sugar content of a
number of tissues, using chromatographic separation, and they confirm

Discussion 105

the older experiments that the sugar content of most tissues is indeed
extremely low, of the order of 10-15 mg. per cent in the case of muscle,
brain and testicle. Sugar was taken to diffuse very readily into cells, but
this low concentration indicates that there is some barrier at the cell
surface and that transport into the cell is something "active". Perhaps
phosphorylated sugar only is transported into the cell, in which case the
hexokinase would be required to be in the surface of the cell; but this is
merely an idea. The point I made is that the hexokinase reaction itself
or a step preceding it, dealing with the entry of sugar into the cell, is the
reaction which initiates the degradation of sugar in the cell.

Haddow: We have become very interested in the properties of the cell
surface in malignant cells and homologous normal cells. There are
charge differences.

Zamecnik: Prof. Krebs, would you comment on the reactions leading
toward synthesis of nucleic acid as possible rate-limiting steps ? We are
just beginning to get a more complete description of the series of
reactions leading to nucleic acid synthesis, and I wonder if they may not
be as likely a site as the steady-state conditions involved in glycolysis
and oxidative phosphorylation.

Krebs: There must, of course, be rate-limiting reactions for many
processes. What I have discussed in detail, because we have information
on them, are the energy-giving reactions. The synthesis of nucleic acids is
one of the processes dependent on energy supply. But its rate certainly
does not depend merely on how much ATP is available. There must be
some other mechanisms which control it. I have no idea of what these
might be. I certainly agree that every complex synthesis must have a
component which determines its rate. I should emphasize again the
principle that there may be some reactions which are of less interest, if
you study the effect of extraneous agents, than others which are more
relevant because they determine the overall rate.

Lajtha: In connection with that point we have found that uracil
deoxyriboside is readily methylated and gets into DNA thymine with
relatively low concentration of inorganic phosphate in the medium ; but
cytosine deoxyriboside needs a high concentration of inorganic phos-
phate in the medium to do the same. Could that inorganic phosphate
be already an energy-giving substance?

Krebs: I don't visualize any direct connection, but I must make it
clear that the ideas which I have put forward cannot explain everything,
they are merely meant to give some guidance in experimentation. They
do not throw light on why you need a high concentration of phosphate
in one case and not in another.

Cohn: In connection with the question on the biosynthesis of the
nucleic acids, it is quite clear now that they begin with rather small
molecules, e.g. with hexoses, trioses and dioses to build the ribose and
deoxyribose moieties, formate to fill in the place in the purine ring as
well as to add the methyl group, glycine, etc. So it seems to me that any
influence upon such steps as these must be reflected in the amounts of
substrates available for the build-up of nucleic acids, and thereby have
some effect, however remote.

THE INFLUENCE OF OXYGEN ON
RADIATION EFFECTS

H. Laser*

Molteno Institute, University of Cambridge

It is well established that the extent to which a variety of
cells may be damaged by ionizing radiation is greater if the
irradiation takes place in the presence of oxygen than if it
takes place under anaerobic conditions. This applies equally
to plant, insect and mammalian tissues (Barron, 1952;
Hollaender, 1952; Hollaender, Baker and Anderson, 1951;
Hollaender, Stapleton and Martin, 1951; Gray, 1953; Gray
et al.y 1953), and the mechanism of this so-called "oxygen
effect", which is not yet fully understood, may also hold the
key to the mode of action of ionizing radiation in general.

Broadly speaking, two disciplines, with their sometimes
widely varying train of thought and approach, have attempted
to explain the oxygen effect, those of the physicochemist and
the biochemist.

The former maintains that oxygen acts per se, e.g., that its
mere presence modifies the nature of the chemical inter-
mediates formed along the tracks of ionizing particles, thereby
apparently producing greater though not well defined damage
to the cells ; or it thinks in terms of strongly oxidizing radicals,
such as HO2, and of HgOg, which are only formed in presence
of dissolved oxygen, as causing increased radiosensitivity.

The difficulty in accepting a purely physicochemical ex-
planation lies, at least to my mind, in the fact that it creates
an unwarranted barrier between chemical and biochemical
causes. It fails to take into account the fact that the state of
the affected "entity " in the cell may determine its response to
irradiation. It furthermore assumes that oxidizing radicals or

* Member of the Scientific Staff of the Medical Research Council.

106

Influence of Oxygen on Radiation Effects 107

agents affect biological systems which are in any event in a
predominantly oxidized steady-state equilibrium ; or that they
oxidize reduced substances, e.g. SH groups (Barron, 1952)
which are normally reversibly oxidized and reduced. Although
such groups may be oxidized by radiation-produced radicals,
they will only be eliminated from participation in further
metabolic reactions if the oxidation thus produced is irrevers-
ible. That that is not generally the case follows, e.g., from the
fact that anaerobic fermentation of yeast is not affected by
fairly large X-ray doses which, however, strongly inhibit the
ability to reproduce. Similarly the work of Pirie, van Heyn-
ingen and Boag (1953) and van Heyningen, Pirie and Boag
(1954) on cataract induction by X-rays has shown that the
glutathione content, total protein-SH and the activity of
glutathione reductase in the lens were unaffected during the
first 20 hours after irradiation, while the activity of a number
of SH-enzymes begins to fall together with the onset of
clinical cataract only weeks after irradiation. The authors
believe that these changes may not constitute primary effects.
The biochemical approach which I propose to adopt with
regard to the oxygen effect visualizes that :

(1) only in the presence of oxygen is the enzymic equili-
brium within the cells such that they are most severely affected
by irradiation products of water, the primary step being
reduction by hydrogen atoms followed by secondary oxidation
through either molecular oxygen or oxidizing radicals, which,
however, leads to abnormal, irreversibly oxidized products;

(2) the oxygen effect, as expressed, e.g., by inhibition of
bacterial growth, occurs only if the cells maintain a certain
minimal metabolic activity and possess the entire enzymic
make-up (or at least its precursors) necessary for subsequent
growth and protein synthesis, during the actual irradiation;

(3) many substances and cell constituents which protect
the cell from irradiation do not do so effectively in the presence
of oxygen.

Experimental data in support of these three propositions
will be given.

108 H. Laser

It has previously been shown (Laser, 1954) with the bacter-
ium Sarcina lutea, that the oxygen effect on the rate of repro-
duction could be largely abolished by doses up to 26 kr if cell
respiration was inhibited by respiratory poisons during the
irradiation. Thus, after removal of the poison, the cells
behaved as if they had been irradiated in nitrogen, when
judged by the degree of growth inhibition. The effective
inhibitors were carbon monoxide, potassium cyanide, hydro-
xylamine and sodium azide. Urethane did not diminish the
oxygen effect (Fig. 1). The mode of action of the effective
poisons in their role as respiratory inhibitors is known (Keilin,
1933; Keilin and Hartree, 1939; Keilin and Slater, 1953). They
all block hydrogen transfer through the respiratory enzymic
system by combining with cytochrome % and stabilizing the
remaining respiratory enzymic chain in the reduced form.
Taking this mode of action as a guide in advancing a possible
explanation for the oxygen effect in irradiation, it is suggested,
at least for this bacterium, that the enhancement of irradia-
tion damage (1) involves the enzymic respiratory mechanism,
(2) requires at least part of the enzymic respiratory chain to
be in the oxidized form during irradiation. This supports the
view that the impedance has been caused by a reducing agent.
These results and their interpretation have recently been
corroborated by Tahmisian and Devine (1955) who have
shown that grasshopper eggs which show a certain regression,
*' negative growth", when irradiated in air are less affected if
the nitrogen of the air is replaced by carbon monoxide, i.e., in
20 per cent Og/CO. The protection by carbon monoxide is
light-sensitive, being effective only in the dark. That X-ray
induced inactivation of biological materia] is brought about
by reduction has also been shown for two different strains of
bacteriophage, by Ebert and Alper (1954) and by Bachofer
and Pottinger (1954). It should, however, be added that the
view that reducing agents are generally responsible for irradia-
tion damage has been contradicted. Thus, Forssberg's (1947)
claim, that catalase in aqueous solution is inactivated by
means of reducing hydrogen atoms produced by X-irradiation,

Influence of Oxygen on Radiation Effects 109

has not been supported by Sutton (1952), who found that
hydrogen atoms produced by means other than irradiation
did not inactivate catalase, and that irradiation in presence
of oxygen was more damaging than in an atmosphere of

Fig. 1. Percentage increase in oxygen uptake (= growth) of
Sarcina lutea during irradiation with 26 kr X-rays, in nitrogen
and in air ± potassium cyanide, hydroxy lamine and urethane.

110

H. Laser

hydrogen; nor has it been confirmed by Dale and Russell
(1956).

Furthermore, the involvement of haematin compounds fol-
lows from experiments with Escherichia coli (Fig. 2), in which
the influence of irradiation on respiration and on aerobic acid-
production of washed non-growing cells has been measured.

Fig. 2. Percentage inliibition of respiration and anaerobic glycolysis
of washed suspensions of Esch. coli irradiated in air with increasing dose

of X-rays.

The rate of oxygen uptake in the presence of glucose was
progressively inhibited with increasing doses of X-rays, up to
a maximum of approximately 65 per cent at 5,000 r. The
remaining 35-40 per cent respiration was found to be cyanide-
insensitive and could not be further reduced by increasing the
X-ray dose sixfold, although reproduction was progressively
more inhibited. Inhibition of anaerobic acid production
(after aerobic irradiation) follows a different course from that
of respiration but reaches the same plateau at about 30 kr.
Here, again, the remaining anaerobic acid production was

Influence of Oxygen on Radiation Effects 111

unaffected by sodium azide which, with Esch. coli, is an
inhibitor of anaerobic glycolysis.

It is conceivable that haematin compounds in the cell
behave in the same way as haemoglobin and cytochrome c,
which are first reduced by X-radiation and then partly
reoxidized in a secondary reaction by molecular oxygen and
by radicals to an unnatural, green pigment, to which extent
they lose their oxygen-carrying and catalytic properties (Laser,
1955). The fact that, with the exception of Esch. coli, the
aerobic and anaerobic metabolism of non-growing suspensions
of a fairly large and representative number of bacteria and of
yeast was not affected — even to the extent of uninhibited
adaptive enzyme formation (Baron, Spiegelman and Quastler,
1952-53) — by X-ray doses which inhibit growth by more than
90 per cent may be taken to indicate that the complement of
haematin compounds in the cell involved in these reactions is
not a limiting factor but may be so in relation to induction of
growth.

That the prerequisite for the oxygen effect is a certain state
and/or metabolic activity of the cell during irradiation is
demonstrated by the following two types of experiment :

(1) Spores of Bacterium suhtilis were irradiated under
varying conditions. They were then transferred to a growth-
promoting medium in manometer flasks, and the rate of
growth, i.e. the formation of vegetative forms and subsequent
reproduction, was determined by measuring the increase in
oxygen uptake with time, which under normal conditions
follows a logarithmic course and is a true measure of the
increase in the number of cells. Fresh spores, i.e., those har-
vested soon after sporulation, irradiated either dry (not dried)
or suspended in phosphate buffer^, showed about the same
radiosensitivity as vegetative forms and an oxygen effect of
the same order. However, if the spores, after thorough
washing, had been freeze-dried prior to being irradiated, they
were somewhat less radiosensitive and showed no oxygen
effect (with doses up to 36 kr) when resuspended in buffer
and irradiated in the liquid phase.

112

H. Laser

(2) The oxygen effect, which is very marked with fresh
yeast (Fig. 3), was similarly abolished with yeast {Candida
utilis) which had been starved of nitrogenous reserves ("low-
nitrogen" yeast) by depriving it, in presence of oxygen and a
carbon source (glucose), of added nitrogenous substrate for
1-2 days prior to irradiation (Fig. 4). Such a yeast has a very
low (resting) metabolism. When it is brought into a nutrient

lOO

Of 2 3 4 5 6 7

time(h)

Fig. 3. Percentage increase in Og uptake ( = growth) of yeast (Candida
utilis) after irradiation in air and in nitrogen with 24 kr X-rays.

(nitrogenous) medium the oxygen uptake per cell increases
before growth sets in. It is reasonable to assume that the
nitrogen-depleted cell has to replenish its relevant enzymic
make-up before starting to divide. As the depleted cell shows
no oxygen effect, it follows that the sensitive structure was
either not present or was in a state in which the presence of
oxygen did not affect its radiosensitivity.

Lastly, I would like to discuss experiments dealing with
protection from irradiation by means other than reducing
agents, such as cysteine. Dale (1940, 1942) has already shown

Influence of Oxygen on Radiation Effects 113

that carboxypeptidase and the prosthetic group (alloxazine-
adenine dinueleotide) of D-amino-acid oxidase could be largely
protected not only by their specific substrates but also by a
variety of substances which are in no way structurally
related to these substrates. However, the problem of protec-
tion does not seem to have been examined in relation to the

5000

iOO

4 5

time (h)

Fig. 4. Percentage increase in Og uptake (= growth) of untreated and "low-
nitrogen" yeast (see text).

(1) unirradiated control;

(2) unirradiated "low-nitrogen" yeast;

(3) "low-nitrogen" yeast irradiated in air and in nitrogen (24 kr.)

oxygen effect, except by allowing bacteria to deplete their
medium of dissolved oxygen by oxidation of added substrate,
e.g. succinic acid, without renewing the used-up oxygen
(Stapleton, Billen and Hollaender, 1952), a procedure which
in effect does not differ from the removal of oxygen by reduc-
ing agents or by physical means. The enzyme used for the

114

H. Laser

experiments to be described was a flavoprotein, namely notatin
(glucose oxidase) (Keilin and Hartree, 1948). By means of a
manometric micro-method for determining the initial rates of
glucose oxidation by small amounts of enzyme ( < 1 (jig.) in-
activation of the enzyme of the order of 10-20 per cent could
be reproducibly detected with accuracy. Table I, dealing with

Table I

Notatin, 20 [ig-jml. percentage
inactivation by 38-4 kr in

Addition

Air

A^2

95-100

Glucose

50-60

15- 20

Other sugars
Albumin, 01 %

15
15

15- 20
30

Glucose + albumin
Gelatin 01 %

the application of 38 kr X-rays reveals the, at first sight sur-
prising, result of a reversal of the oxygen effect, the enzymic
activity being practically completely inhibited by irradiation
in nitrogen but only slightly (15 per cent) depressed if oxygen
is present. However, the presence of glucose, the specific sub-
strate, during irradiation in air brought about a strong
inhibition (50 per cent) while the presence of glucose during ir-
radiation in nitrogen protected, lessening the inactivation from
100 per cent to 15 per cent. Catalase, in catalytic amounts, did
not alleviate the inhibition which glucose caused during irradi-
ation in air, i.e., the enzyme had not been partially inactivated
either by enzymically or radiochemically produced HgOg.
The inactivation of notatin on irradiation in air was observed
only in the presence of its specific substrate. A number of
other sugars, which are not oxidized by the enzyme, caused no
inactivation in air but exerted a high degree of protection
during irradiation in nitrogen. Similarly, added protein
(albumin), which greatly protected against irradiation in

Influence of Oxygen on Radiation Effects 115

nitrogen, decreased only slightly the inactivation which the
addition of glucose induces in air.

It may be legitimate to conclude that in this particular
case the enzyme seems most likely to be radiosensitive when
it is in the form of a semiquinone (Kuhn and Wagner- Jauregg,
1934; Michaehs, Schubert and Smythe, 1936; Haas, 1937)!
These data offer further support to the more general conclusion
that (1) the oxygen effect, as already pointed out above, is
related to enzymic activity during the actual irradiation;
(2) the points of attack within the cell or on an enzyme mole-
cule, as well as the damaging agents, differ widely depending on
the presence or absence of oxygen. The result will therefore
depend on the relative role which the affected group plays
in the economy of the cell.

REFERENCES

Bachofer, C. S., and Pottinger, M. A. (1954). Science, 119, 378.
Baron, L. S., Spiegelman, S., and Quastler, H. (1952-53). J. aen.
Physiol, 36, 631. v / s

Barron, E. S. G. (1952). Symposium on Radiobiology, p. 216. New

York: Wiley.
Dale, W. M. (1940). Biochem. J., 34, 1367.
Dale, W. M. (1942). Biochem. J., 36, 80.
Dale, W. M., and Russell, C. (1956). Biochem. J., 62, 50.
Ebert, M., and Alper, T. (1954). Nature, Lond., 173, 987.
Forssberg, a. (1947). Nature, Lond., 159, 308.
Gray, L. H. (1953). Brit. J. Radiol., 26, 609.
Gray, L. H., Conger, A. D., Ebert, M., Hornsey, S., and Scott,

O. C. A. (1953). Brit. J. Radiol, 26, 638.
Haas, E. (1937). Biochem. Z., 290, 291.
Heyningen, R. van, Pirie, a., and Boag, J. W. (1954). Biochem. J.,

Hollaender, a. (1952). Symposium on Radiobiology, p. 285. New

York : Wiley.
Hollaender, A., Baker, W. K., and Anderson, E. H. (1951). Cold

Spr. Harh. Sijmp. quanl Biol, 16, 315?
Hollaender, A., Stapleton, G. E., and Martin, F. L. (1951). Nature,

Lond., 167, 103.
Keilin, D. (1933). Ergehn. Enzymforsch., 2, 239.
Keilin, D., and Hartree, E. F. (1939). Proc. roy. Soc. B., 127, 167.
Keilin, D., and Hartree, E. F. (1948). Biochem. J., 42, 221.
Keilin, D., and Slater, E. C. (1953). Bril med. Bidl, 9, 89.
Kuhn, R., and Wagner-Jauregg, T. (1934). Ber. dtsch. chem. Ges., 67,

361.

116 H. Laser

Laser, H. (1954). Nature, Lond., 174, 753.
Laser, H. (1955). Nature, Lond., 176, 361.
MiCHAELis, L., Schubert, M. P., and Smythe, C. V. (1936). J. biol.

Chem., 116, 587.
Pirie, a., Heyningen, R. van, and Boag, H. W. (1953). Biochem. J.,

54, 682.
Stapleton, G. E., Billen, D., and Hollaender, A. (1952). J.

BacterioL, 63, 805.
Sutton, H. C. (1952). Radiation Chemistry, Disc. Faraday Soc, no. 12,

p. 281.
Tahmisian, T. N., and Devine, R. L. (1955). Radiation Res., 3, 182.

DISCUSSION

Alper: Mr. Paul Howard-Flanders and I have some results which
are relevant to what Dr. Laser has been discussing. We took Esch. coli B
in a starving condition and bubbled oxygen through them for a good
long time, so that they should have exhausted most or a good deal of
their endogenous substrate, in order to see what the radiosensitivity
under nitrogen would be. At first it seemed as if the nitrogen survival
curve was quite different if one had treated the bacteria in this way;
but a more rigorous examination of this phenomenon showed that it
was, in fact, due to the circumstance that the sensitivity of Esch. coli B
is affected by extremely low oxygen concentrations. This means, of
course, that in order to distinguish very clearly between the survival
curves you get under oxygen or under complete anoxia, you have to
have extremely rigorous conditions of oxygen exclusion. These survival
curves were all done with very dilute suspensions of bacteria, and with
suspensions in which the gas mixture was bubbling through con-
tinuously, so that one could feel fairly confident at least about the
oxygen tension in the fluid surrounding the cells. Now there are several
points relevant to Dr. Laser's argument about the necessity for the cells
to be metabolizing in order to show the oxygen effect. Longmuir (1954,
Biochem. J., 57, 81) gives a figure which is analogous to the Michaelis
constant, the oxygen concentration at which the bacteria are respiring
at half their normal rate. This concentration is 2-2 X 10"® m oxygen,
and on this basis they would be respiring at 95 per cent of their normal
rate at something like 4 x 10 ' m; the region within which they are
reaching full oxygen radiosensitivity, however, is something of the
order of 10-20 (jlm. We have also done survival curves with these
various gas mixtures in the absence and in the presence of substrate.
When we compared the curve which represents the points in the absence
of substrate, with the curve obtained when succinate was added, there
was no difference for the nitrogen point, for complete oxygenation, or
for 0 • 02 per cent oxygen. There was, in fact, only one point where the
succinate apparently made a slight difference, and since it was only at
the one point I think this will certainly need verification. Apart from
this experiment the succinate made no difference at all to the survival
curves.

Discussion 117

Gray: It is clear that the position of the bend in now very much in need
of being looked at again.

Previous studies with X-rays by Read, who observed root growth in
Vicia faba, by Giles, who observed chromosome structural damage in
Tradescantia microspores, and by Baker and others, who observed
chromosome structural damage in Drosophila sperm, all showed this
bend to occur when the partial pressure of oxygen in the gas phase was
about the same as in air, corresponding to a molarity of oxygen dissolved
in water at room temperature of around 250 (jiM/litre. This contrasts
with the situation indicated by Miss Alper for bacteria, where the bend
occurs at about 50 piM/litre dissolved oxygen. The contrast is the more
striking if expressed in terms of the molarities of dissolved oxygen which
confer half the difference between the anaerobic and the fully aerobic
sensitivities; these are around 7 [xM/litre for bacteria and 120 piM/litre
for the other materials. Each of the other materials referred to has been
irradiated as tissues, not as single cells, and the observed relation be-
tween sensitivity and oxygen tension must in each case have been in-
fluenced in some degree by the gradient of oxygen tension throughout
the tissue arising from cellular respiration. The point has been con-
sidered by the authors concerned and believed not to be one of major
importance, though definite experimental evidence is lacking. This
factor cannot have been operative, however, in the experiments of
Conger, who irradiated ascites tumour cells in a suspension in equili-
brium with known gas mixtures and obtained a relation essentially the
same as that which had been obtained for the roots, microspores, and
Drosophila sperm. The procedure used in Conger's experiments was
that the fluid in which the cells were suspended was stirred vigorously
by a jet of gas of known composition. The molarity of the dissolved
oxygen was not measured. In view of Miss Alper's results it becomes
a matter of importance to check Conger's observations. It will be re-
called that, using the same experimental procedure. Conger found only
a very slight influence of oxygen on the sensitivity of ascites tumour
cells to neutron radiation which, by analogy with the relatively slight
influence of oxygen tension on the chemical reactions induced in aqueous
solutions by high ion density radiations, lent support to a chemical — as
against a biochemical — type of explanation. In other cases the evidence
is less clearcut. Dr. Laser referred to the influence of carbon monoxide
on the X-ray sensitivity of his bacteria. It will be recalled that Sachs
found that carbon monoxide at four atmospheres pressure in the
presence of air increased damage to Tradescantia microspores. I found
similar effects in roots but only when the carbon monoxide pressure
was increased to about 20 atmospheres. Moreover, I found a much
smaller influence of carbon monoxide on growth inhibition induced by
exposure to neutrons. While these facts would be most readily accounted
for in terms of radiation chemistry, the fact that the enhanced radio-
sensitivity was a function of the period of carbon monoxide pretreat-
ment, which was only fully effective if applied for 30 minutes prior to
as well as during irradiation, suggests that metabolic factors were
involved.

118 Discussion

Alper: There is one point I would like to make. I don't know whether
our results at these extremely low oxygen concentrations are good
enough to be quite sure that we failed to demonstrate the effect I mean.
If you take the view that it is only after the respiratory chain is fully
oxidized that you get an oxygen effect, you should in fact have a little
tail to this curve. In the experiments on bacteria carried out by
Dr. HoUaender's group, quite a big tail is shown in the oxygen effect
curve. They worked with a closed system and a dense suspension of
bacteria so that they were able to use up the oxygen and did in fact
do so ; if the results are plotted out in this way you do get quite a tail
to that curve. We get the oxygen at these very low tensions by pro-
ducing it with an electrolysis cell and running the cell at currents from
10 mA upwards, so that we can get something of the order of 0-01 per
cent oxygen or less. You get a very slight but real increase at that
concentration, and increasing sensitivity as oxygen concentration
increases; there is some scatter in the points, but the results show this
clearly.

/ Stapleton: Dr. Billen in our laboratory followed the respiration of
several strains of irradiated Esch. coli. He measured the consumption of
oxygen by these irradiated cells, and compared this oxygen uptake with
that of non-irradiated cells, using glucose as substrate. He found that
the control cells, of course, consumed oxygen at a constant rate with
time of incubation. Irradiated cells, on the other hand, although the
final population as measured by ability to reproduce themselves was
something like one viable cell in 10^, consumed oxygen at approximately
the control rate for a period of something like 40-50 minutes. Then
there was a reasonably sharp break in the curve followed by a slow
steady decay of the respiratory ability of the cells. I think what this
curve means is that all the cells are viable during this period of time.
The break may represent a change or a beginning of death of the
population. Billen did some further experiments to see if some correla-
tion could be made with ATP synthesis in the cells and found that under
the same conditions the irradiated cells can synthesize ATP at approxi-
mately the same rate as control cells. At about the same time that this
break in the oxygen uptake occurs, although the cells were making
ATP at approximately the normal rate, something like 80 per cent of
ATP synthesized by the cell was found outside rather than inside the
cell. This again could mean that the population is changing with respect
vto viability.

Latarjet: We have material that could be of use to Dr. Laser.
Dr. Beljanski has treated several strains of Esch. coli with large amounts
of streptomycin on minimal medium, and finally isolated some stable
mutant strains which are unable to synthesize the porphyrin ring. They
are haemin minus and if you grow them on small amounts of peptone
you get bacteria which have none or at most 1 /500th the total porphyrins
of the normal strain. They have no catalase, no peroxidase, no cyto-
chrome, or at most very little. We were interested, not in the oxygen
effect, but in photorestoration. (Incidentally, these strains are as photo-
restorable as the wild ones.) But, hearing Dr. Laser, I wonder if such

Discussion 119

a material would not be of help in work with ionizing radiations, to see
what the oxygen effect would be in these strains.

Laser : It would certainly be of great help to me and I would be grateful
to obtain such a strain of Esch. coli. With regard to Dr. Stapleton's
statement that the respiration curve shows a sharp break after about
40 minutes, which amounts practically to a cessation of respiration, I have
never had that experience in extensive studies with a great variety of
bacteria.

THE INFLUENCE OF CHEMICAL PRE- AND

POSTTREATMENTS ON RADIOSENSITIVITY

OF BACTERIA, AND THEIR SIGNIFICANCE

FOR HIGHER ORGANISMS*

Alexander Hollaender and George E. Stapleton

Biology Division, Oak Ridge National Laboratory, Tennessee

Protection against ionizing radiation by chemicals can be
discussed on the basis of model experiments with polymers or
on experience with living materials. In some ways, experi-
ments conducted with chemicals and living cells fit into a
pattern related to model tests; however, for the purpose of
this discussion, model experiments are not sufficient since
they do not give information on metabolizing systems. The
most extensive work in the field of model experiments has
been done by Alexander, Charlesby, and Ross (1954). A
large number of compounds that protect readily against
radiation in the polymers are not necessarily effective on
biological materials, but they could serve as a guide to practi-
cal applications. Alexander and co-workers (1955) have
shown a close parallelism between the protective ability of
many compounds for polymers and the survival of irradiated
mice. The number of compounds that actually protect
mice to a highly significant degree, and with a minimum
of detrimental effects, is relatively small. Our interpre-
tation was that these compounds compete for the oxygen
present in the suspension and/or for radiation-produced
radicals and peroxides. Alexander and co-workers (1955)
beheve that these compounds act chiefly by competing for
HO2 radicals. On the basis of present knowledge, it is very
difficult to distinguish between these two mechanisms since

* Work performed under contract W-7405-eng-26 for the Atomic Energy
Commission in the Biology Division, Oak Ridge National Laboratory.

120

Influence of Chemicals on Radiosensitivity 121

oxygen removal is not always so efficient as chemical protec-
tion. As a matter of fact, the dose-reduction factor (DRF)
for Escherichia coli will go as high as 12 under the best condi-
tions for cysteamine protection (Hollaender and Doudney,
1955) in contrast to 3 for oxygen removal by replacement
(Hollaender, Stapleton, and Martin, 1951). The difference
between these dose-reduction factors probably is based on the
premise that replacement of oxygen by an inert gas (Doudney
and Hollaender, 1956) — for instance, nitrogen — might not
completely remove the oxygen, whereas sodium hydrosulphite
(Burnett et al., 1951) or cysteamine, since both are highly
water-soluble, can enter the cell and remove the oxygen,
especially when it is in loose association with certain com-
poimds or is adsorbed on the surface of particular cell struc-
tures. Several investigators have shown that cysteamine can
associate with nucleic acids and other compounds (Kluyskens,
1953).

Two compounds that appear to be of greater importance,
because of their striking protective ability, are p-mercapto-
ethylamine (MEA) and /S-p-aminoethyh'^othiouronium • Br •
HBr (AET). The former is apparently the most successful
compound for Esch. coli, and the latter for mice.

Cysteamine (MEA), first reported by Bacq and co-workers
(1951) to be protective for mice and since found to be an
effective chemical protective agent by workers in many other
laboratories, is an easily oxidized compound, has to be stored
under nitrogen, and has other characteristics that make it
somewhat difficult to handle. In freshly prepared water
suspension, there is increasing protection for Esch. coli B/r
(Fig.l) with increasing concentration until a plateau is reached
at 0-02 M. Phosphate buffer interferes somewhat with the
protection, and the protective ability is dependent on the
nutritional factors supplied after irradiation. The effect of
cysteamine on Esch. coli maybe summarized as follows (Fig. 2):

1. It protects in a concentration of 0-02 m and has a dose-
reduction factor of 12 if broth-grown cells are incubated in a
complete medium.

122 Alexander Hollaender and George E. Stapleton

2. Cells grown in inorganic salts and glucose do not require
a complete medium for highest protection.

3. Significant protection is still obtained if cysteamine is
washed off the cells immediately before irradiation, probably
a large part of the MEA is absorbed by the cell.

t.O-i

10--^

o
o
o

10^

o
<
cr

«o--5

.-4

— I ' 1 I I I

0.002 0006 001 0.02

MOLAR CONCN. OF CYSTEAMINE

-JJ-

0 04

Fig. 1. Effect of cysteamine on the survival

of Esch. coli IB/r during exposure to 60 kr of

250-kv X-rays.

4. Limiting concentrations of cysteamine do not change
the shape of the survival curves ; however, reduced survival is.
obtained at each dose level.

5. The protection is somewhat strain-dependent; a careful
technique must be used for the highest expression of protective
ability. Some points can easily be obscured by changing
some conditions of experimentation.

Influence of Chemicals on Radiosensitivity 123

The excellent protective ability of cysteamine for Esch, coli
and its limitations in animal experiments have stimulated
David G. Doherty (Doherty and Burnett, 1955; Doherty and
Shapira, 1956) of our laboratory to prepare more than 100
derivatives of this compound. Time is too short for a review

50 75 100

y-RAY DOSEIkr;

150

Fig. 2. Gamma-ray sensitivity of Esch. coli
B/r in the presence and absence of cysteamine.
0, Yeast extract; O, basal medium; A, cells
irradiated in H2O; B, cells irradiated in
HgO + 0 • 02 M cysteamine.

of the functions of all these compounds; therefore, only the
results are summarized below:

1. The protective ability disappears when the number of
carbons in the basic structure of AET is increased in excess of
3 (Fig. 3).

124 Alexander Hollaender and George E. Stapleton

2. Different groups may be added to the sulphur, provided
that their form is such that they do not unbalance the molecule.

3. The NHg group must be left free; otherwise, the toxicity
increases or the protective ability is drastically reduced.

4. The compound (AET) is stable at pH 3 and can be
hydrolysed only at pH's in excess of 8 with amino alkyl
mercaptans and dicyandiamide as the breakdown products.

5. Compounds found to be protective give a positive
-SH test at pH 7*5. Doherty's explanation for this is that
AET exists in multiple forms, and equilibria among these
forms can be changed by modifying the pH. Some of the
different stages that AET goes through are shown in Fig. 3.

CHg CHj CHj CH^ CHp CHp

I I 0H~ I I 0H~ I I

^"2 /S ^^ HN^ ^S ^=^ HN^ SH

O \ l> c

CHo — CHp
I I

Fig, 3. Multiple equilibrium structures of AET.

6. All compounds that are active in protecting mice appear
to be able to form a h type ring structure (Fig. 3) in solution
at a neutral pH similar to a thiazoline ring.

7. A solution of AET appears to be a mixture of different
types of isomers, whose forms are very similar to coenzyme A
(Bashford and Huennekins, 1955) and glutathione (Calvin,
1954), which are known to exist in several isomeric forms.

Unfortunately, AET is not one of the best compounds for
protecting 'Esch. coli but it is considerably more successful than
cysteamine in protecting mice. It almost doubles the LD50
and is now used routinely in our laboratory for protection
studies with mice.

From experiments- on bacterial cells, we are just beginning

Influence of Chemicals on Radiosensitivity 125

to understand some of the relationships of postirradiation
(recovery) systems on the cell level Some of the details of
experimentation and results of these experiments belong in
this discussion. Two points of view can be taken immediately :
(1) the recovery system may prevent the disruption of key

10--^

y RAYS, 80 kr

10"

z
o
t-
o
<
a:
u.

ID
2
>
>

.0--^

10-

TEXAS

— r—

INCUBATION TEMPERATURE {'O

— 1
42

Fig. 4. Survival of three strains of Esch. coli

at 80 kr at various temperatures. J^, Crooks ;

0, B/r; O, Texas. (Stapleton, Billen and

HoUaender, 1953.)

biological material or (2) it may allow the cell to replace
damaged material by synthesis. Both types of recovery may
be demonstrated.

1. Reduced postirradiation incubation temperature in-
creases the survival of several strains of Esch. coli. TheB/r strain
shows a true optimum for survival (Fig. 4) at 18° C after
X- or gamma-rays (Stapleton, Billen, and HoUaender, 1953).

126 Alexander Hollaender and George E. Stapleton

2. The effect is dose-independent, a true dose-reduction
phenomenon (Fig. 5).

3. The effect is related to inactivation by free radicals.
Dose-reduction factor is reduced for oxygen-free suspensions.

4. The survival of irradiated cells at any temperature may

-r

30
X-RAY DOSE (kr)

1
70

Fig. 5. Recovery at various X-ray doses

(aerobic B/r 20-hour culture cells irradiated).

O, Holding at 18° C; 0, holding at 37° C.

(Stapleton, Billen and Hollaender, 1953.)

be related to the relative rates of synthetic and radiation-
induced degradative processes.

An effect of the medium in which the cells are incubated is
displayed at all the temperatures studied (Stapleton, Sbarra,
and Hollaender, 1955) (Fig. 6). The pertinent results can be
summarized as follows:

1. Additivity can be shown between the temperature and

Influence of Chemicals on Radiosensitivity 127

medium effect. This finding illustrates that both points of
view may actually be correct.

2. Extracts of natural materials (e.g., beef, yeast, or other
tissue extracts) yield higher survival than that obtained on a

y RAYS. 85 kr

°\ BASAL MEDIUM ♦ YEAST EXTR

-1 1 r

18 30

INCUBATION TEMPERATURE (°C)

Fig. 6. Comparison of viability on basal
medium and basal medium plus yeast extract
as a function of postirradiation incubation
temperature. (Stapleton, Sbarra and Hol-
laender, 1955.)

simple inorganic salts-glucose (Fig. 7.) medium, although the
latter medium is not limiting for normal (non-irradiated)
cells.

3. Attempts to isolate the required materials indicated a
multiple requirement.

4. A complex, chemically defined plating medium (Table
I) will, to a high degree, substitute for the natural materials.

128 Alexander Hollaender and George E. Stapleton

Deletion experiments showed the minimal (Table II) organic
requirements to be guanine, uracil, and glutamic or aspartic
acids, precursors for ribonucleic acid and/or protein synthesis.

O
O

S
-t
<

100-

z
g

»-
o

>
>

10-

KIDNEY
TESTES

LIVER

INTESTINE

-1 1 1

10 100 1,000

LOG CONCENTRATION (^g/ml) "

10,000

Fig. 7. Relative activity of rabbit tissue homogenates ;
incubation at 37° C. (Stapleton, Sbarra and Hollaender,

1955.)

It seemed of the utmost importance to investigate the
effect of preirradiation growth conditions on the survival of
cells after irradiation, as well as their response to added

Table I

Complete Synthetic

Medium

Components

g.ll. of medium

Saltq A f K2HPO4
oaitSA IKH2PO4

0-5
0-5

rMgSO,

Salts B -

NaCl
FeSO 4

001
001

LMnSO^

001

Glucose

Plus or minus vitamins, amino acids, purines, and pyrimidines.

nutritional factors after exposure. Cells were grown in a
variety of media including: (1) basal medium — the inorganic

Influence of Chemicals on Radiosensitivity 129

salts-glucose medium, (2) basal medium fortified with amino
acids, (3) basal medium fortified with purines and pyrimidines,
(4) a combination of these, and (5) nutrient broth.

Table II

Chemically Defined Recovery Medium

Components

Amount

Glutamine

150 iig.

Uracil

30 [xg.

Guanine

30(i.g.

Salts A

1 ml.

Salts B

1 ml.

Agar

3-4 g.

Glucose

20 g.

Distilled Ufi

200 ml.

pH6-8

After irradiation, cells grown on the various media were
plated on the media described in Table III, and the following
results were obtained:

Table III
Effect of Culture Medium on Radiation Sensitivity

Surviving fraction of cells, after 85 kr of gamma-rays, plated

at 37° C on:

Basal Basal Basal

medium -{- medium -{- Complete medium +

Basal guanine -i- caseamino synthetic 20 mg. of

Culture medium medium uracil acids medium yeast extr.

Basal 2-2xl0-« 31xl0-« 40x10-6 2-2x10-5 1-3x10-5
Basal + guanine +

uracil l-OxlO"' l-OxlQ-^ 8-0x10-' 30xl0-« 2-6xl0-«
Basal + caseamino

acids 2-3 + 10-' 2-9 + 10-6 2-3x10-5 5-0x10-5 60x10-5

Nutrient broth 3 0x10-8 5 0xl0-« 8-^x10"' 2-7x10-6 3-5x10-6

1. Cells harvested from basal medium did not show a
striking response to added nutrilites. The survival as a func-
tion of dose indicated that they did not require the nutrilites
since essentially similar survival was obtained on basal

RAD.

130 Alexander Hollaender and George E. Stapleton

medium as was obtained on fortified media by cells harvested
from nutrient broth.

2. Cells harvested from basal media to which amino acids
or purines and pyrimidines were added showed requirements
for these materials after irradiation. Amino acids were most
effective.

3. Cells harvested from nutrient broth showed the most
clear-cut dependence of survival on the supplemented medium
following irradiation.

Data on the effect of growth conditions prior to irradiation
on the subsequent postirradiation requirements suggested
that a sizeable part of the process called "recovery" is related
to an adaptive process, and probably involves new enzyme
synthesis. Cells that have been grown in a simple medium
prior to irradiation might be expected to have a different
complement of enzymes from those grown in a nitrogen-rich
medium such as beef extract. The former cells have had to
synthesize not only all metabolic precursors and inter-
mediates from carbon fragments and ammonia but, probably,
also the enzymes necessary for these synthetic reactions.
The latter cells have had numerous intermediates supplied to
them continuously during growth and, therefore, have prob-
ably been able to by-pass many synthetic reactions. They are
essentially undeveloped or deficient cells. Non-irradiated
cells, if transferred from a rich medium to the basal medium
can adapt readily to the simple medium; but, interestingly,
irradiation appears to interfere with such an adaptation.
According to these studies it would seem that under optimal
conditions — (1) reduced incubation temperature and (2)
the presence in the postirradiation medium of precursors for
ribonucleic acid and protein synthesis — a reasonably large
fraction of the irradiated population appears to be able to
perform the required syntheses.

Short-term incubation of irradiated cells with extracts of
natural materials (15-30 minutes at 37° C) prior to plating,
results in loss of dependence of the cells on the plating
medium. Quantitatively similar survival is obtained on both

Influence of Chemicals on Radiosensitivity 131

media. This incubation period is not sufficient to allow cell
division to occur. Apparently, some nutritional factor is
present in these extracts which either partially reverses the
potential damage brought about by X- or gamma-rays or aids
in the adaptive process involved. These two suggested
mechanisms may actually be identical in the bacterial system.
The synthesis of ribonucleic and deoxyribonucleic acids,
and of proteins, was followed in non-irradiated and in irradi-
ated cells under the best and worst conditions for recovery,

6.0

4.0-

LJ
M

3 0-

± 2.0

o
o

1.0

30 60 90 120
ACID- SOLUBLE

30 60 90 120 30 60 90 120 30 60 90 120
RNA ONA PROTEIN-N

INCUBATION TIME(min)

Fig. 8. Relative rates of synthesis of nucleic acids and

protein by irradiated (0) and non-irradiated (□) Esch.

coli B/r on basal medium at 37° C.

i.e., incubation at 18° C in a complete medium and at 37° C in
basal medium, respectively (Fig. 8) (Stapleton and Woodbury,
1955). Similar aliquots of cell suspension were inoculated into
the two media at the two temperatures; and after various
incubation periods the cells were- harvested, washed, and
extracted with trichloracetic acid, and analysed by the
Schneider technique as used by Morse and Carter. Irradiated
cells held in the basal medium at 18° C show no net synthesis of
deoxyribonucleic acid or acid-soluble components and a
reduced rate of synthesis of protein and acid-insoluble ribo-
nucleic acid. On the other hand, irradiated cells incubated

132 Alexander Hollaender and George E. Stapleton

at 18° C in yeast extract show essentially normal synthesis of
all components (Fig. 9). To date, the results do not permit
one to decide whether these alterations in nucleic acid syn-
thesis are the cause or the result of the viability changes in the
irradiated population. Further control of growth conditions —
for example, the division cycle — might accentuate the re-
covery phenomenon. Synchronization of division might very
well produce an essentially homogeneous population for

4.0

o
d:

r-g

3.0

z2.0

1.0

All

60 120 180 240 360
ACID-SOLUBLE

60 120180 240 360
RNA

60 120 180 240 360
ONA

60 (20 ISO 240 360
PROTEIN-N

INCUBATION TIME(min)

Fig. 9. Relative rates of synthesis of nucleic acids and pro-
tein by irradiated (0) and non-irradiated (□) Esch. coli on
yeast extract at 18° C.

The acid-soluble fraction represents that fraction of the
cells soluble in cold 10 per cent trichloroacetic acid; only ribose
was estimated in this fraction by orcinol test.

recovery studies. Such studies are just beginning in the overall
programme of bacterial recovery.

Mention should be made of the results of our studies on
mammals, i.e., chemical protection during irradiation and
posttreatment with bone marrow or spleen. AET will raise
the LD50 30 days for C3H X 101 mice from 692 to 1148 r.
Bone marrow will raise the LD50 to 1292. A combination of
both treatments will bring the LD50 to 1863 (Congdon,
Upton, and Doherty, 1956, in preparation). Survival after
very high exposures (2400 r of gamma-rays) can be obtained
if, in addition to the combination treatment, daily injections

Influence of Chemicals on Radiosensitivity 133

of streptomycin are given and possibly some nutritional
support is supplied for the irradiated animals (Burnett and
Doherty, 1955).

A few generalizations in regard to the mechanism of pro-
tection by these different treatments are in order. Chemical
protection is apparently quite general, affecting many
different functions of the organism. This is obvious from the
ability of the chemicals to protect against graying and some-
what against cataract formation, to maintain body weight,
and to reduce the number of glandular disturbances. Post-
treatment with spleen or bone marrow, apparently, will
stimulate the function of the haematopoietic systems. Many
interesting findings could be discussed here in regard to the
protection by posttreatment, especially the immunological
aspects. These may come to light in the discussion. It
should be pointed out that most of these studies emphasize
only the immediate effects. Very little is known in mammals
about the effect of radiation protection on the long-delayed
effects; i.e., late-appearing malignancies and mutational
changes. Actually, these latter ones may prove to be the most
important ones. In bacteria, the situation is somewhat
clearer since genetic effects in micro-organisms may be readily
observed. This brings up one of the most important questions
in modern radiobiology ; namely, "Is the genetic damage
entirely dependent on the amount of ionizing radiation to
which the cell is exposed, or can the effect be modified by
chemical protection or by treatment after the exposure has
ended? " Bacterial cells are very well suited for checking this
since the chemical used will penetrate to most parts of the
cell and, after treatment, will help a large number of cells
to survive. In a number of strains of Esch. coli it has been ob-
served that, for certain mutations (nutritional reversions), the
mutation rate increases inversely to the survival ratio and is
not necessarily proportional to the amount of radiation to
which the cells are exposed. This means that the dose-
reduction factor for mutations approaches the DRF for
survival. In other words, it is possible in certain strains of

134 Alexander Hollaender and George E. Stapleton

Escli. coli to protect against mutation production by chemicals
or recovery factors. However, it is possible to recognize this
only if there is a minimum of population pressure effect.
Where the population pressure effect is very great, as in
reversion of a tyrosineless strain of Esch. coli, this population
pressure will obscure the entire phenomenon of protection
and recovery. In contrast to this, the arginineless strain
shows decreased mutation rate with increased survival
(Hollaender, Billen, and Doudney, 1955). It is important to
point out that the recognition of this phenomenon requires
careful analysis for each individual mutation. Very similar
results have been obtained in regard to chromosome breaks
in bean roots, where it has been possible to modify the number
of chromosome breaks by chemical treatment after exposure
(Wolff and Luippold, 1955). I hope this will be discussed at a
later meeting where cytological effects will be brought up.
In any case, it is fairly safe to say that the possibility exists
of the modification of genetic effects by radiation protection.

REFERENCES

Alexander, P., Bacq, Z. M., Cousens, S. F., Fox, M., Herve, A., and
Lazar, J. (1955). Radiation Res., 2, 392.

Alexander, P., Ciiarlesby, A., and Ross, J. (1954). Proc. roy. Soc,
223A, 392.

Bacq, Z. M., Herve, A., Lecomte, J., Fischer, P., and Blavier, J.
(1951). Arch. int. Physiol., 59, 442.

Bashford, R. E., and IIuennekens, F. M. (1955). J. Amer. chem. Soc,
77, 3878.

Burnett, W. T., Jr., and Doiierty, D. G. (1955). Radiation Res., 3,
217.

Burnett, W. T., Jr., Stapleton, G. E., Morse, M. L., and Hollaen-
der, A. (1951). Proc. Soc. exp. Biol., N.Y., 77, 036.

Calvin, M. (1954). Glutathione, A Symposium, p. 3. New York: Acad-
emic Press.

CoNGDON, C. C, Upton, A. C, and Doiiertv, D. G. (1956). In pre-
paration.

Doiierty, D. G., and Burnett, W. T., Jr. (1955). Proc. Soc. exp. Biol.,
N.Y.,S9, 312.

Doiierty, D. G., and Siiapira, R. (1956). In preparation.

Doudney, C. O., and Hollaender, A. (1956). In preparation.

Hollaender, A., Billen, D., and Doudney, C. O. (1955). Radiation
Res., 3, 235.

Influence of Chemicals on Radiosensitivity 135

HoLLAENDER, A., and DouDNEY, C. O. (1955). Radiobiol. Symposium,
p. 112, London: Biitterworth's Scientific Publications.

IIOLLAENDEii, A., Stapleton, G. E., and Maktin, F. L., (11)51). Nature,
Loud., 167, 103.

Kluyskens, p. (1953). C.R. Soc. Biol., Paris, 147, 733.

Morse, M. L., and Carter, C. E. (1949). J. Bad., 58, 317.

Stapleton, G. E., Billen, D., and IIollaender, A., (1953). J. cell,
comp. Physiol., 41, 345.

Stapleton, G. E., Sbarra, A. J., and IIollaender, A. (1955). J. Baet.,

70, 7.

Stapleton, G. E., and Woodbury, D. II. (1955). Presentation at
regional meeting of Kentucky-Tennessee Branch of the Society of
American Bacteriologists, Nashville, Tennessee, November, 1955.

Wolff, S., and Luippold, H. E. (1955). Science, 122, 231.

DISCUSSION

Stocken: Have you any long-term survival in the mixed treatment,
say comparing the thiouronium compound plus bone marrow alone or
against the thiouronium compound alone?

HoUaender: These experiments are going on now and as far as we have
gone (these have been going on for about six months), the mice are all
alive. This is if one uses isologous bone marrow; I have not discussed
the question of homologous or heterologous bone marrow. This is a
different problem and I hope that Dr. Lout it will bring some of these
questions up.

Laser: Dr. IIollaender, how do mice react to injection of thiouronium?
Do they seem unaffected or are they in a prostrate condition similar to
that frequently obtained in mice on application of protective doses of
cysteine, which produce pulmonary oedema and a high degree of
anaerobiosis ?

Secondly, I should like to refer to the notation used in your figures
where the ordinates are marked "survival." This is generally taken to
mean that the graphs indicate the percentage kill. This, however, is by
no means the case. Irradiated cells are not dead but appear to be quite
normal in many respects. They have, however, lost the ability to form
visible colonies within, say, 48-72 hours, although a certain percentage
of them may do so at a later period. In view of the importance of your
pioneer work, especially on the oxygen effect, for radiobiology in general
and lately also in connection with food technology, and in order to avoid
confusion in the literature, may I suggest that you substitute "viable
count" for "survival".

One further point: you describe recovery from radiation damage,
e.g. on addition of an amino acid. Recovery, to my mind, implies the
gradual disappearance of an induced and quantitatively determined
inhibition and necessitates the measurement of rates. Have you done so
or do you mean recovery to be synonymous with less inhibition ?

136 Discussion

Hollaender: In answer to your first question, I gave you the toxic
level for thiouronium. Routinely we use 6 mg. per standard mouse.
We get very good protection at 4 • 4 mg. If you go to 8-10 mg. the mouse
will be prostrate and a good percentage will die. The range in which it
is safe to use this compound is considerably greater than with cysteamine
or cysteine.

Now in regard to your second question, on survival of these bacteria :
we emphasize in all our reports that we have determined the survival on
the basis of colony-forming organisms. We are very cautious on this
point because, as Dr. Stapleton has shown earlier, during discussion
after your paper, these bacteria are not dead when they are unable to
form visible colonies. They still can respire for a considerable time. As
to the question of whether we get more colonies after 72 hours, we
check this very carefully. We usually make two counts; for instance
when we study the effects of lower temperatures the plates stay for 24
hours at 18° and then for at least 24 hours at 37°. The delayed effect on
B/r, i.e. the appearance of late colonies, is very small if X-rays are used.
Now if you go to the long u.v. you may have to wait as long as five
days before all colonies appear, but that is another point.

Alper: The same thing happens with Esch. coli B. I have checked this
in some, not all, experiments and found no more colonies after 48 than
after 24 hours.

Hollaender : If you use u.v. it is a different story, you have to be more
careful there.

Stapleton: With regard to Dr. Laser's question on why we call it
recovery, you can define recovery in many different ways and it is
apparently prevention of some lesion that would ordinarily lead to
death. It could be a lack of an inhibition.

Laser: You could call it prevention rather than recovery.

Stapleton: Yes.

Van Bekkum: Dr. Hollaender, you have commented in your paper on
the protective effects of some jS-alkyl and iV-alkyl derivatives of cyste-
amine. Dr. de Groot and I have recently studied a few similar com-
pounds with regard to their protective activity in vivo (mice) and in
vitro (isolated rat thymocytes). The compounds referred to are the
following :

(1) *S'-ethylcysteamine:— CgHs-S-CHa-CHa-NHa

(2) iV-ethylcysteamine :— HS • CHg • CHg • NH • Cg Hg

(3) iV-diethylcysteamine:— HS-CHa-CHa'N (C2H5)2

In vivo the *S'-ethyl derivative (1) has some protective activity, which is
less than that of cysteamine. The iV-ethyl derivative (2) is more toxic
and less protective than cysteamine, the iV-diethyl compound (3) is still
more toxic to mice and has practically no protective activity in vivo.
On the other hand, in the thyrhocyte system both compounds (2) and
(3) afford considerable protection, while the tS-ethyl derivative (1) is
completely ineffective. It seems, therefore, that the amine group is
most important for in vivo protective activity, while the sulphydryl
group seems to be required for in vitro protection.

Discussion 137

We have also investigated a number of amines, which are known to
protect mice excellently, like histamine, epinephrin and phenylethyl-
amine, and it was found that these compounds do not show any protective
activity with isolated cells. We think that the protection of mice by
these amines may be the result of their activity on some organ system
e.g. the cardiovascular or the respiratory system, and it seems possible
that part of the protective activity of cysteamine in vivo is due to a
similar mechanism. Have you any information on the pharmacological
effects of cysteamine ?

Hollaender : Yes, we have. This is work which has been done at the
University of Rochester, it is not completed and that is why I didn't
mention it. AET as well as cysteamine have a depressive effect on the
respiratory centres in the cat. AET is more toxic to the dog than to the
mouse. Preliminary tests in monkeys have shown that AET is less toxic
to the monkey than to the dog. The effect is different in different species
of animals. This, of course, requires very careful investigation before
we w ould say it is of practical significance.

Spiegelman: I should like to cite here an interesting experiment per-
formed by Norman (1953, J. Bad., 65, 151) which is relevant to some of
the points raised by Dr. HoUaender's paper. Norman grew cells in a
glucose minimal medium, irradiated them with u.v., and then plated
them on minimal media containing glucose in some cases and other
carbon sources in others. On plotting the log of the survivors measured
on these different plates, he found a much greater apparent kill if the
cells were plated on a carbon source different from the one on which they
had grown up. Thus, glucose-grown cells show a much greater kill when
plated on lactose as compared with glucose. That this apparent increase
in sensitivity is associated with a demand for the formation of new
enzyme is shown by experiments in which he grew the cells up in lactose
and plated them on lactose minimal medium. In such cases the apparent
kill was the same as when plated on glucose.

Latarjet: This is u.v. work — it destroys the adaptation.

Spiegelman: Yes, but the interesting thing to emerge from this
experiment is the following. First, it must be noted that all you are
asking the glucose cell to do, on being plated on lactose, is to make one
more enzyme, and this apparently makes an enormous difference. In
addition, when the killing curves obtained from the glucose and the
lactose plates are extrapolated back, they both give the same average
hit number required for kill and the value is about 1-2. This would
suggest that a nuclear phenomenon is involved. It would seem to me
that a more extensive investigation of this phenomenon is extremely
desirable. It is an experimental situation in which the comparative
conditions on the glucose and lactose minimal media are under experi-
mental control and where the enzymatic consequences are also pretty
well defined.

Haddow: Dr. Hollaender, have you done any tests on protection
against carcinogenesis with AET ?

Hollaender: These are in progress now. Many of these malignancies
appear fairly late, it takes a period of about two years to be sure that

138 Discussion

they don't have any. Dr. Upton in our laboratory is now maintaining a
large colony of mice which have survived this irradiation. We are
watching for leukaemias and also for other malignancies. But it may
take another year to be certain.

Laser: I have tried it with yeast and have obtained quite conclusive
results but with opposite effect. Yeast is grown in a synthetic medium
containing all the growth requirements, with glucose and ammonium
sulphate as the only sources of carbon and nitrogen. This yeast, when
grown in the same medium after irradiation, shows a strong irradiation
effect. However, if the irradiated yeast is transferred into a medium in
which the ammonium salt is replaced by another nitrogenous substrate
to which the cells may have to adapt, the radiation effect is diminished
and still more so if the glucose is also replaced by a different carbon
source, say galactose.

Spiegelman: I don't understand. What do you plate on?

Laser: I don't plate, but grow the yeast in a liquid medium and
measure growth rates either by optical or manometric methods. I find,
as already stated, that by changing after irradiation either the carbon
or the nitrogenous source or both — that is, by initiating adaptation —
the irradiation effect has become less apparent.

Spiegelman: Your killing is less?

Laser: Yes.

Popjak: I would like to raise some questions about the mechanism
of action of these chemical protectors. It has been said, in the case of
cysteamine for example, that it might be that it protects the "business-
end" of coenzyme A. Then it has been suggested that it acts like cysteine
and other readily oxidizable substances by virtue of taking up preferen-
tially oxidizing radicals, or by reducing tissue oxygen tension, this being
merely some kind of nitrogen effect. It seems to me that there might be
some other explanations for the protective action of cysteamine.
It appears that the free amine group is very important in protection,
because substitution on the nitrogen with alkyl groups eliminates the
protective action. Eldjarn and Pihl have reported that cysteamine
does combine very rapidly with the SH groups of proteins. If that is so,
then one might expect that cysteamine and analogous substances might
cause a reversible inhibition of enzymes. I have done some experiments
on this point and these support my assumption. It may be that cyste-
amine protects enzymes during the period of irradiation by combining
with some vital groups on the enzyme, rather than by catching oxidizing
radicals.

Hollaender : It just keeps the radicals from getting to a group by
protecting them?
Popjak: Yes.

Gale: There seem to be a number of analogies between the material
that we have just been given and the action of penicillin. I noticed
particularly one of the slides that Dr. Hollaender showed, where there
appeared to be an effect of radiation resulting in decreased synthesis
of RNA, and increased accumulation of the "acid-soluble RNA". I
wonder if you can tell us anything about the nature of substances which

Discussion 139

in fact form the so-called "acid-soluble RNA" in these irradiated
organisms.

Stapleton: No, we have not looked at this, but we certainly intend to
do so, especially on the basis of some of the work that has been done
with U.V., which indicates that irradiated cells pile up DNA precursors.
The point Dr. Gale refers to is the only indication in all these experiments
that there is any pile-up of anything.

Gray: In support of what Popjak has just been saying, Burns did an
experiment with yeast in which he obtained quite appreciable protection
with 2?-chloromercuribenzoate which did not, however, eliminate the
oxygen effect. The oxygen effect was still shov/n in the protected cells.
I think in the case of cysteine we have got to be extremely careful in
any given case to be sure that it is not operating simply by removal of
oxygen from the solution.

Whether or not cysteine protects mammals by inducing a state of
tissue anoxia I do not know, but an examination of cases reported in
the literature in which the addition of cysteine to solutions in which
bacteria were suspended or roots immersed reveals that the experimental
conditions were in each case such as to produce partial or complete
anoxia at the time of irradiation.

Popjak: The first oxidation product of cysteine would be cystine.
There are some enzymes which can reduce disulphides very effectively
and very rapidly, the typical example, of course, being the glutathione
reductase which has been described in wheat germ and pea seedlings,
but which Dr. Hele and I find to be present also in animal tissues and
which has a high specificity for TPN. So even though it may get oxi-
dized, in the cells it will get reduced again to cysteine.

Ch-ay: I am not speaking of possible chemical mechanisms of cysteine
protection, that is quite beyond me; I am just saying that before the
occasion for a chemical theory arises we must first be sure that we are
not dealing with simple anoxia in any given case. This has very often
not been established.

Hollaender: May I say that I don't want to leave the impression that
we have been able to reverse mutations. All we believe that we have
been able to do is to stop the effect before the mutation has been carried
through, before the damage has been completed. But what we have
done with these chemicals, either before or especially after irradiation,
is to interfere with the completion of the mutation process.

POSTIRRADIATION TREATMENT OF
MICE AND RATS

D. W. H. Barnes and J. F. Loutit

Medical Research Council Radiobiological Research Unit, Hanvell

From time to time there appear reports that specific treat-
ments given to mice after an acute lethal dose of radiation
have a significant effect in improving the proportion of
survivors at the conventional time of 30 days. Amongst the
chemical agents alleged to have this therapeutic activity
have been batyl alcohol (DL-a-octadecylglycerol-ether) (Ed-
lund, 1954); properdin (a natural euglobulin present in normal
serum) (Pillemer et al., 1954) polyvinylpyrrolidone (BUrger
et al., 1954) and carbon monoxide (Konecci et al., 1955). Up
to date none of these reports has been followed by confirma-
tory papers. In the case of polyvinylpyrrolidone the beneficial
effect has been denied (Becker and Kirchberg, 1955; Biirger,
1955; Rugh et al, 1953).

On the other hand, procedures which might be called
surgical are undoubtedly effective. For instance Brecher and
Cronkite (1951) showed that lethally irradiated rats could
recover if, after their irradiation, they were subjected to the
operation of parabiosis with an unirradiated rat. Following
this operation a cross-circulation between the two animals is
established. The survival of the damaged animal may be due
to the transfer of anything in the blood of the normal animal.
It could be due to the continuous transfusion of the formed
elements of blood — red corpuscles, leucocytes and platelets —
all of which are grossly deficient in the circulation of the rat
some days after a lethal dose of radiation. However, blood
transfusion as can be practised clinically has been without
effect except for the limited successes claimed on small series
by Salisbury and co-workers (1951) and Allen and co-workers
(1951). For such trials the rat is not suitable as the experi-

140

POSTIRRADIATION TREATMENT OF MiCE AND RaTS 141

mental animal and the dog is the animal of election. Moreover
the results of Brecher and Cronkite can equally well be attri-
buted to the transfer of some factor in solution or colloidal
suspension in the plasma exchanged. Such a substance could
"detoxify" the damaged animal — an hypothesis which did
not appeal to the authors — or act as a stimulant for that
early regeneration of the recipient's bone marrow which was
observed. This histological finding by itself suggests that the
effect was more than that expected from the symptomatic
treatment of continuous transfusion with whole blood. Still
another suggested alternative was the possibility of a transfer
of blood-forming cells from the donor.

Another surgical procedure has been much more widely
investigated. Jacobson and his colleagues (1949) first showed
that irradiation of mice with their spleens protected was much
less lethal than complete irradiation of the whole body plus
spleen. They proceeded to demonstrate (1951) that, in similar
fashion, a reduced mortality of the totally irradiated mouse
could be attained by implanting intraperitoneally spleens
from normal mice, accelerated regeneration of the haemo-
poietic tissues being identified as the fundamental effect of
this procedure. Similar, dramatic changes in mortality were
reported by Lorenz and his colleagues (1951, 1952) following
the injection of suspensions of bone marrow into irradiated
mice. Jacobson (1952), in a comprehensive review of the work
of his group and of Lorenz and colleagues, marshalled the
evidence in favour of his hypothesis that a humoral factor in
normal spleen and bone marrow (both haemopoietic tissues in
the mouse) was responsible for stimulating the recovery of
the irradiated animal's damaged haemopoiesis. Only one argu-
ment seemed to the present authors to be incontestably in
favour of this hypothesis, namely that heterologous material
from guinea pigs had, in the hands of Lorenz and colleagues
(1952), been effective. The principle was confirmed later by
Congdon and Lorenz (1954) who obtained positive results
using bone marrow from rats.

Further evidence in favour of the humoral hypothesis

142 D. W. H. Barnes and J. F. Loutit

came later from San Francisco. Cole and co-workers (1952)
showed that homogenization, with its attendant severe
damage to cells, did not destroy the therapeutic effect.
Furthermore, fractionation of these homogenates in sucrose
by ultracentrifugation (Cole et a/., 1953) localized the effective
principle in the layer of greatest density composed of cell
nuclei (and a few whole cells). The activity of this nuclear
fraction could be destroyed by preparations made from
crystalline desoxyribonuclease and trypsin (Cole and Ellis,
1954) which were said to be without action on intact cells
(Cole and Ellis, 1955).

Against the humoral theory we have argued on the follow-
ing grounds.

(1) On repeating the original observations of Jacobson
and co-workers, we were impressed that, in those mice
which survived the critical month but died subse-
quently, some of the spleens implanted had "taken"
and had become accessory spleens.

(2) We confirmed that an intact organ was not an essential,
that suspensions were effective on injection, and that
the intravenous route gave results superior to the
intraperitoneal (Barnes and Loutit, 1953). (We were
not at that time able to obtain improved survival from
heterologous material from guinea pigs or rabbits.)
This superiority of the intravenous route would not be
expected if a soluble hormone were involved but would
be in the case of cells.

(3) When we adopted intravenous injections as a routine
we noted that there was an approximate threshold of
material above which no further improvement was
obtained. Jacobson and co-workers (1955) have made
similar observations. Depending on the material used
this threshold may be 10^-10^ cells.

(4) The active principle is extremely thermolabile : it is
inactivated in a few minutes at 50°C (Cole, Fishier and
Ellis, 1955) and in a few hours at room temperature, at
4° C and at - 15° C (Barnes and Loutit, 1954). This is

POSTIRRADIATION TREATMENT OF MiCE AND RaTS 143

compatible with its being cellular. To test this further
we have adopted one of the schedules recommended for
the preservation of living cells (Smith, 1954), i.e. storage
of the material in glycerol at — 79° C, and found that
the activity is preserved for 80 odd days at least (Barnes
and Loutit, 1955).

(5) The activity is destroyed by a dose of a few hundred
rontgens of X-rays in vitro (Cole et al., 1953) and in
vivo (Barnes and Loutit, 1954) which again is more in
favour of its being of a cellular rather than a chemical
nature.

(6) In most laboratories it is not possible to keep the sur-
vivors of animal experiments for the rest of their lives.
We have had sufficient accommodation to allow us
to do so. We have thus accumulated information on
the overall survival of the normal unirradiated CBA
mice of our colony (these are not strictly controls in
the temporal sense), and mice irradiated with 950 r
and treated with isologous (CBA) spleen, spleen from
homologous strain A mice and heterologous bone
marrow from Wistar rats. The median survival time
of the unirradiated mice is 900 days. For the irradiated
animals only those which survive the conventional
30 days are included for scoring. Routinely they
come to experiment at about the age of 100 days. The
median survival time for those given isologous spleen
is a further 400 days and for those given homologous
spleen approximately 40 days. From the Hmited data
for heterologous transfer, the survival seems much the
same as for homologous.

(7) The previous result suggests .that antigenic differences,
such as occur between cells of various origin, are im-
portant. We have another similar observation in that
CBA animals, previously immunized by intravenous
injections of tissues from mice of strain A, will no
longer recover following irradiation with 950 r if treated
with spleen from strain A (Barnes and Loutit, 1954).

144 D. W. H. Barnes and J. F. Loutit

However, until an accredited cell-free material is shown to be
potent, or until cells from the donated material are shown
unequivocally to repopulate the host, there can be no absolute
confirmation of either hypothesis.

Two sets of recent observations from the Radiobiological
Research Unit allow us now to be dogmatic that, unlikely as it
originally seemed, repopulation from the donor does in fact occur.
1. Mitchison (1956) used the inbred strains of mice, A and
CBA, with which our previous work had been done. CBA male
mice of about three months of age were irradiated as in
previous experiments with 950 r X-rays (240 kv. ; 15 mA.;
HVL =1-2 mm. Cu; dose rate 43 r/min.). This dose is almost
invariably lethal to untreated mice. On the same day, after
irradiation, the CBA mice were injected intravenously with a
suspension of cells from the spleen of infant mice of strain A.
The fresh spleens were cut into small pieces, suspended in
fresh rabbit serum and macerated with an electrically driven
mincer. Each mouse received 0-4 ml., the equivalent of
two-fifths of a spleen — that is about 15 X 10^ cells estimated
from counts made in a haemocytometer. These mice were
sacrificed at intervals and tissues were taken for test of their
content of antigens specific for strain A.

The method of test involved administration of the respective
tissues to normal CBA mice. Each tissue for test was macer-
ated and injected intraperitoneally. If it contained, in
adequate numbers, cells derived from strain A it would thereby
induce in the normal CBA mouse, within a period of 8-12
days, the state of transplantation immunity. The injected
CBA mouse was, after the interval of 8-12 days, inoculated
subcutaneously with a suspension of tumour cells specific for
mice of strain A — sarcoma 1 (Dunham and Stewart, 1953).
This tumour had been maintained in the ascites form by
repeated passage in strain A mice. Ascites fluid was diluted
with isotonic sodium citrate until it contained approximately
10® cells in the 0-1 ml. used for injection. The reaction was
scored after a further 8 days when the tumour, or its remnant,
was carefully excised and weighed.

POSTIRRADIATION TREATMENT OF MiCE AND RaTS 145

In control tests carried out in parallel with the experiments
it was shown that CBA mice, previously inoculated with
tumour to give full transplantation immunity, on re-injection
with the test-dose of tumour returned weights for the im-
plants which varied from individual to individual and from
batch to batch within the range of 2 to 42 mg. On the other
hand normal, non-immunized CBA mice, while also varying
between individuals and between batches, had tumours
weighing 48-877 mg. Within batches there was in each case
a clear-cut difference, usually one to two orders of magni-
tude, between individuals of the positive control group and
individuals of the negative control group. The weights of the
tumours derived from the test-animals were compared with
these controls. The tumour-weight in an experimental animal
was taken to be significantly different from weights of the
non-immune controls if it was less than the mean minus twice
the standard deviation.

In addition to the tissues, spleen, lymph nodes and liver,
the tissue-fluids, blood and peritoneal exudate, were also
assayed for the antigen of strain A. Peritoneal exudates were
induced by prior injection of sterile paraffin and consisted of
lymphocytes and mononuclear cells.

The results of the tests for A antigen in the tissues
and tissue-fluids of the irradiated CBA mice treated with
intravenous injections of strain- A spleen are shown in
Table I, overleaf. These figures show that the spleen and
lymph glands of these animals usually give significantly
positive results from the earliest time tested — 4 days. The
positive results persist until the latest time of test — 51
days. Peritoneal exudates also gave positive results through-
out the time of test, 16-51 days. On the other hand
liver and blood, tested only at 14 days, gave negative
results.

Similar tests were conducted with tissues from unirradiated
CBA mice injected intravenously with suspensions of spleen
from strain-A mice. The results are given in Table II, over-
leaf. These results indicate that the injected A cells may

146

D. W. H. Barnes and J. F. Loutit

Table I

Results of Tests for Strain-A Antigen in Tissues and Tissue-fluids

From CBA Mice Irradiated with 950 r X-rays and Promptly Injected

Intravenously with a Suspension of Strain-A Spleen.

Fractions indicate the number of positive results out of the total.

Interval after

irradiation

and injection

Tissue or tissue-fluid tested

Spleen

Lymph
nodes

Peritoneal
exudate

Blood

Liver

4 days
7 days

13 days

14 days
16 days
19 days
36 days
51 days

2/2
2/5

4/5

5/6

4/5
3/5
4/5
3/5

5/6

2/2
1/3

2/2
2/2

0/5

persist and perhaps multiply in the unirradiated CBA
mouse for a few days only.

In ancillary experiments it was demonstrated that the
critical dose of materials from mice of strain A to give positive
results was: peritoneal exudate, 10^ cells; suspension of spleen,
10^ cells. It is possible that the trauma to cells attendant on

Table II

Results of Tests for Strain-A Antigen in Tissues and Tissue-fluids
FROM Unirradiated CBA Mice Injected Intravenously with a Suspen-
sion OF Strain-A Spleen

Fractions indicate the number of positive results out of the total.

Intei'val

after
injection

Tissue or tissue-fluid tested

Spleen

Lymph nodes

Blood

4 days

7 days

14 days

53 days

3/5

1/5
0/5
0/5

0/5

0/5
0/5

POSTIRRADIATION TREATMENT OF MiCE AND RaTS 147

making the suspension of spleen cells accounts for the increased
number necessary compared with the exudate which could be
obtained without damage to the cells.

Whereas 10^ strain- A spleen cells are necessary to produce
a state of transplantation immunity, in the experiments above
15 X 10^ spleen cells were injected intravenously into the
CBA mice. It is possible that these cells were favourably
distributed in the normal or irradiated recipient and accounted
for the positive results recorded for tissues in the first days
after injection. However, in the unirradiated animals these
positive results were soon reversed to negative as was expected
on the basis of immunity following the implant of foreign and
incompatible tissue. In the irradiated animals the positive
results not only persisted in tissues that were manifestly and
measurably enlarged and hyperplastic, but even showed
evidence of increase for which the original paper should be
consulted. Moreover, it is noteworthy that a peritoneal
exudate induced at a late stage was also positive.

One must conclude that the persistence of the A antigen
against all the laws of tissue grafting is a result of the massive
dose of radiation ; and that the apparent increase in A antigen
is the result of the growth of the grafted strain-A cells or the
incorporation of the A antigen by the CBA host. Recent work
from other laboratories leads to the same conclusions. Main
and Prehn (1955) demonstrated that mice injected with
homologous cells after lethal doses of X-rays survived and
would then take skin grafts which normally would be incom-
patible. Lindsley, Odell and Tausche (1955), using rats, could
identify red blood cells characteristic of the donor in rats
irradiated with near lethal doses and treated with homologous
bone marrow. Finally, No well and co-workers (1955) have
reported that mice injected with rats' bone marrow develop
myeloid cells and circulating leucocytes which give positive
histochemical reactions for alkaline phosphatase — a property
of the rat but not of the mouse.

2. The rather improbable explanation involving incorpora-
tion of the donor's antigens into the host need no longer be

148 D. W. H. Barnes and J. F. Loutit

invoked in view of the second line of approach to be reported.
This is the use of marked chromosomes in the donated
material. Two different markers have been employed (Ford
et al, 1956).

(I) The rat chromosomes. It has already been noted that
administration of heterologous material can allow the lethally
irradiated mouse to survive (Lorenz et al., 1952; Congdon and
Lorenz, 1954). We have lately been able to repeat this result
using as donors inbred rats from our colony which stems from
the Wistar strain. Our CBA mice are given 950 r as before
and then injected intravenously with a suspension of bone
marrow obtained from the two femurs of a young rat. The
recipient CBA mouse has cells with a complement of 40
chromosomes with terminal centromeres (Fig. 1). The rat has
cells which contain 42 chromosomes, a number of which have
a characteristic cruciform appearance in metaphase (Fig. 2),
since their centromeres occupy a central position. In a squash
preparation it is therefore easy to differentiate those cells in
metaphase which are derived from the host, i.e. the mouse,
from those which stem from the donor-rat.

(II) Mouse-translocation T6. Carter, Lyon and Phillips
(1955), by irradiating the testes of male mice and promptly
breeding from them, were able to select offspring that were
semi-sterile as a result of inheriting a pair of translocated
chromosomes from the irradiated parent. Ford and Hamerton
in this laboratory examined cytologically the available stocks
carrying these translocations and showed that one (denoted
by Carter and co-workers as T6) had a chromosome which was
readily differentiated at metaphase from any of the normal
chromosomes of the mouse. It was about half the length of
the smallest normal chromosome (Fig. 3). Dr. Lyon has bred
for us young mice carrying this translocation in the hetero-
zygous state and we have used the spleens of these mice aged
7-10 days as donor material for the irradiated CBA mice.

The irradiated CBA mice, treated with either the rat
bone marrow or the spleen of the T6 mouse, have been sacri-
ficed at intervals after irradiation and treatment and their

ash preparations of cells in metaphase of mitosis after colcliicine treatment :

J " » J •/

' fi

#H^

■^j^niTjBJ^mjtHpnUlijym^ "•»

1. 1. From normal CBA mouse — 40 chromosomes, all centromeres effec-
tively terminal.

J. 2. From normal Wistar rat — 42 chromosomes, some centromeres occupy

central position.

r ffinirtn nnnp, 1 4.S

Fig. 3. From T6 mouse with one characteristic ultra-short chromosome due

to a translocation.

Fig. 4. From CBA mouse irradiated with 950 r and treated ^^t^^ ^^^f .^^".^"^

iniection of spleen from T6 mouse; showing typical damage from radiation^

dicentric chromosomes and acentric fragments.

POSTIRRADIATION TREATMENT OF MiCE AND RaTS 149

tissues have been examined cytologically by Ford and
Hamerton. The technique employed was an adaptation of
the Feulgen squash method. The animals were injected with
colchicine one hour before sacrifice and the tissues were
handled as suspensions.

The results to date may be summarized as follows. Of the
bone marrow cells in metaphase which could be scored, the
great majority have been identified by means of the marker
as originating from the donor (Table III). This holds also for

Table III

Preliminary Data of Ford and Hamerton on Identification of Markers
IN Cells at Metaphase (+ Present, — Absent, ? Doubtful)

Time of
sacrifice

No. of
ani-
mals

scored

Cells in metaphase — Identification of marker T6

Bone Marrow

+ - ?

Spleen

+ - ?

Lymph node

+ - ?

Thymus

+ - ?

5 days
14 days
28 days
49 days
70 days

3
1
2
2
2

201 1* 104
185 1* 13
114 0 12
197 0 84
299 0 52

113 1* 11

35 2* 4

140 0 41

105 0 8

21 0 13

62 2* 14
10 0 0

28 1* 7
45 0 6

51 0 8

5 days
11 days
19 days
28 days
49 days
49 days
55 days
65 days

2
2
2
2

2 r

Cells in metaphase — Identification of marker — rat

127 4* 0
549 0 1
695 0 11
219 0 23
739 0 20

0 599 5
163 43 9

0 9 0

184 0 0

8 1* 0

929 13 0

205 12 38

436 0 48

4 492 0

16 51 0

7 49 0

-^ — —

* Mostly cells showing classical signs of damage from radiation.

the much more limited examination of spleen, lymph gland
and thymus, the only tissues as yet of which satisfactory
preparations have been made. For each time of examination

150 D. W. H. Barnes and J. F. Loutit

where more than one animal has been sacrificed, the cell-
counts reported in Table III are the sum of the individual
counts. On the one occasion when one differed from its mate
or mates the count on that animal is recorded separately.
Preparations were made of liver but no mitoses were seen.
Testes were examined but were completely atrophic as judged
by the naked eye and without mitotic figures in the cytological
preparations.

The exceptions to the broad generalization made above are
as follows:

(i) In the process of making the squash some cells are
violently disrupted and the full complement of 40 chromo-
somes of the mouse's cell or 42 of the rat's cell may not be
traced. While the rat's cell may be identified by the charac-
teristic cruciform members of the set, even in the absence
of a full count of chromosomes, to diagnose with certainty
in the homologous transfer the presence or absence of the
marker, necessitates the visualization of all 40 chromosomes.
In some cells with counts of less than 40 chromosomes
the marker was not seen. These cells have been scored as
"doubtful".

(ii) Cells may not be sufficiently well spread for all 40
chromosomes to be separated and seen. If the marker is not
identified, it is not possible to say whether such a cell is from
donor or host and the cell is also recorded as "doubtful".

(iii) Occasional cells are seen in the early days after irradia-
tion with multiple lesions of the chromosomes of the kind
well known to be induced by radiation e.g. dicentric chromo-
somes and acentric fragments. These cells can be attributed
to the host, but they do not persist after the first few days
(Fig. 4). .

(iv) While in the early days and weeks after such irradia-
tion it appears as if the great majority at least of the cells in
division are attributable to the donor, it may well be that in
the course of time the host's tissues under investigation will
show some recovery. Thus, after seven weeks in the case of
the heterologous transfer of rat bone marrow to mouse, we

POSTIRRADIATION TREATMENT OF MiCE AND RaTS 151

have seen the reappearance of mitoses of murine cells. It is,
however, premature to do more than record the findings so far.

In preliminary experiments where less than the 99 per cent
lethal dose of 950 r has been given to CBA mice, we see
this reversal of cellular types at a comparatively early time
after irradiation. When CBA mice are given 545-575 r and
treated with T6 spleen, the regenerating tissues are mainly
of the T6 type in the first week, but later belong mainly to
the normal CBA type. In further preliminary experiments
CBA mice were irradiated over only part of the body. The
hind third was given 1200 r and T6 spleen was then injected
intravenously. The regenerating bone marrow of the femora
and inguinal lymph glands at 5 days corresponded in cell-
type to the normal CBA host.

The interpretation of these experiments is clear. In the
mouse given the LD99 of X-rays the regenerating cells, seen
in mitosis, of the haemopoietic tissues are almost without
exception characteristic of the material from the donor. The
living cells in the preparation injected must, therefore, be
dividing and colonizing the empty spaces of bone marrow and
lymphatic tissue. The alternative explanation of the former
experiment of Mitchison — that the host had incorporated
antigens — can no longer be maintained. In the homologous
transfer it is inconceivable that the host's cells had accepted
whole chromosomes (translocations at that), rejected some of
its own to maintain a normal complement and still had a
balanced set for division. Equally it is unnecessary in the
heterologous transfer to postulate complete exchange of
chromosomes by the host.

The preliminary results of experiments involving sub-
lethal doses of irradiation suggest that the length of the
symbiosis of donor's and host's cells may be dependent on the
dose of radiation given. It seems that the immune mechan-
isms which normally determine compatibihty of tissue grafts
are in abeyance following massive doses in the lethal and
supralethal range. This refractoriness is long lasting. From
the direct evidence of Table III, we show it has lasted for

152 D. W. H. Barnes and J. F. Loutit

ten weeks in the case of acquired tolerance to homologous
tissue. From indirect evidence of experiments as yet incom-
plete we infer that it may be more or less permanent. Chim-
aeras, formed bv irradiation of CBA mice with 950 r and
treatment with spleen of strain A, have been kept, as noted,
routinely until death. Such animals as were available have
been challenged with subcutaneous inoculations of the
tumour sarcoma-1 ; ten, having survived 18 to 246 days after
irradiation, have all "taken" the tumour and succumbed.

REFERENCES

Allen, J. C, Moulder, P. V., and Emerson, D. M. (1951). J. Amer.

med. Ass., 145, 704.
Barnes, D. W. H., and Loutit, J. F. (1953). Proc. R. Soc. Med., 46, 251.
Barnes, D. W. H., and Loutit, J. F. (1954). Nucleonics, 12, No. 5, 68.
Barnes, D. W. H., and Loutit, J. F. (1955). J. nat. Cancer Inst., 15,

901.
Becker, J., and Kirschberg, H. (1955). Strahlentherapie, 98, 343.
Brecher, G., and Cronkite, E. P. (1951). Proc. Soc. exp. Biol., N.Y.,

77, 292.
Burger, H. (1955). Strahlentherapie, 98, 348.
Burger, H., Grabinger, H., and Lehmann, J. (1954). Strahlentherapie,

95, 399.
Carter, T. C, Lyon, M. E., and Phillirs, R. T. C. (1955). J. Genet.,

53, 154.
Cole, L. J., and Ellis, M. E. (1954). Radiation Res., 1, 347.
Cole, L. J., and Ellis, M. E. (1955). In Radiobiology Symposium,

p. 141. Ed. Bacq and Alexander. London: Butterworth.
Cole, L. J., Fishler, M. C, and Bond, V. P. (1953). Proc. nat. Acad.

Sci., Wash., 39, 759.
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Proc. Soc. exp. Biol., N.Y., 80, 112.
Congdon, C. C, and Lorenz, E. (1954). Amer. J. Physiol., 176, 297,
Dunham, L. J., and Stewart, H. L. (1953). J. nat. Cancer Inst., 13,

1299.
Edlund, T. (1954). Nature, Lond., 174, 1102.
Ford, C. E., Hamerton, J. L., Barnes, D, W. H., and Loutit, J. F.

(1956). Nature, Lond., 177, 452.
Jacobson, L. O. (1952). Cancer Res., 12, 315.

Jacobson, L. O., Marks, E. K., and Gaston, E. O. (1955). In Radio-
biology Symposium, p. 122. Ed. Bacq and Alexander. London:

Butterworth.
Jacobson, L. O., Marks, E. K., Robson, M. J., Gaston, E. O., and

ZiRKLE, R. E. (1949). J. Lab. din. Med., 34, 1538.

POSTIRRADIATION TREATMENT OF MiCE AND RaTS 153

Jacobson, L. O., Simmons, E. L., Marks, E. K., and Eldredge, J. H.

(1951). Science, 113, 510.
KoNECCi, E. B., Taylor, W. F., and Wilks, S. S. (1955). Radiation

Res., 3, 157.
LiNDSLEY, D. L., Odell, T. T., and Tausche, F. G. (1955). Proc, Soc.

exp. Biol., N.Y., 90, 512.
LoRENz, E., CoNGDON, C. C, and Uphoff, D. (1952). Radiology, 58,

863.
LoRENZ, E., Uphoff, D., Reed, T. R., and Shelton, E. (1951). J. nat»

Cancer Inst., 12, 197.
Main, J. M., and Prehn, R. T. (1955). J. nat. Cancer Inst., 15, 1023.
MiTCHisoN, N. A. (1956). Brit. J. exp. Path., 38, 239.
NowELL, p. C, Cole, L. J., Habermeyer, J. G., and Roan, P. L.

(1955). USNRDL-TR-59.
PiLLEMER, L., Blun, L., Lepow, I. H., Ross, O. A., Todd, E. W., and

Wardlaw, a. C. (1954). Science, 120, 279.
RuGH, R., SuESS, J., and Scudder, J. (1953). Nucleonics, 11, No. 8, 52.
Salisbury, P. P., Rekers, P. E., Miller, J. H., and Marti, N. F.

(1951). Science, 113, 6.
Smith, A. U. (1954). Proc. R. Soc. Med., 47, 57.

DISCUSSION

Latarjet: I would like to add a comment to Dr. Loutit's paper. It
seems to me that at the present state of our knowledge there is a critical
question which concerns this 50 days: does the proliferation of homo-
logous or even heterologous material still occur at 50 days because the
immunity is still down, or does it occur because a state of tolerance has
been established by adding this homologous material when the im-
munity has been broken down? If we were to wait longer, could we
reach a state where full immunity has been recovered and the homo-
logous cells still occur, or not ? Do total body irradiations transform an
adult into a newborn animal?

Loutit: I think it is probably a bit too early to be dogmatic; but at any
rate we have had some of these homologous animals from our previous
work as long as eight months after receiving homologous tissue, and at
eight months after receiving homologous tissue they will still take
tumours specific to the donor and not the host. Main and Prehn's skin
graft work was of about the same duration. I think the foreign skin
graft survived up to 190 days.

Hollaender : Our mice have received rat bone marrow; we have quite
a number now which have been kept for 160 days, the erythrocytes are
still full red. What usually happens is that about 50 per cent die
between 30 and 60 days, another 25 per cent apparently get some kind
of a disease. The 25 per cent which then survive seem to be as per-
manent as we can have (this work has been done by Dr. Makinodan in
our laboratory).

Haddow : Do you know the cause of death ?

Hollaender: The first 50 per cent probably died because they finally

154

Discussion

produced antibodies against the rat bone marrow. We have no explana-
tion for the next 25 per cent. They lose weight, some go down to 9 g.
and then die.

Van Bekkum: I'm very glad that Dr. Loutit has been able to demon-
strate so admirably that his original point of view with regard to the
mechanism of bone marrow therapy is the correct one. I should like to
mention some of our results in this field which are in essential agree-
ment with Dr. Loutit's findings. The point is that we have employed
quite different techniques to identify the origin of the haematopoietic
cells in mice which survived a lethal dose of total body irradiation as a
result of homologous or heterologous bone marrow injections. These
experiments were carried out in collaboration with Dr. O. Vos, Mr.
J. A. G. Davids and Mr. W. W. H. Weyzen. The methods are summar-
ized in Table I. The first technique is based on the observation that

Table I (Van Bekkum)
Evidence for the Cellular Hypothesis

Combination

Homologous

Heterologous
Heterologous

Surviving mice,
identification of

Bone marrow

Erythrocytes
Granulocytes

Method

Therapeutic effect
in irradiated donor
and receptor strain

Specific antisera

Histochemical

isologous bone marrow cells are about 20 times more effective than
homologous bone marrow cells when injected into lethally irradiated
CBA mice (Fig. 1). In other words, you need about 20 times more

^t SUHVIVALS
AT JO DAYS

too'

90-
80-
70-
60"

50'

40-

30-

20-

10-

10'

• o o-

•o c-

• t

I '"I

10»

"T 1 1 I I I I 11

I I I I ■ I mniBSR OP CELLS

■n

10'

10"

Fig. 1. (Van Bekkum). Therapeutic effect of i.v. bone marrow cells in

irradiated mice.
O Isologous. 0 Homologous. A Heterologous (rat).

Discussion 155

(nucleated viable) homologous cells than isologous cells to obtain the
same therapeutic result. We wanted to know whether the regenerated
bone marrow of irradiated C57 Bl. mice, that had been treated with
CBA bone marrow cells, consisted of CBA cells or of C57 Bl. cells. The
question could be answered by injecting this bone marrow into both

CBA cells

irradiated C57 Bl.
U d.

cells

/ \

'^ irradiated '^
CBA C57BI

effect: -f-

Fig. 2. (Van Bekkum).

irradiated CBA and C57 Bl. mice and by comparing the therapeutic
effects in the two strains (Fig. 2). The results of this experiment
(Table II) strongly suggest that the bone marrow tested consisted
predominantly of CBA cells.

Table II (Van Bekkum)
Therapeutic Effect of Regenerated Bone Marrow

Number of % survivals at 32 days

cells X 105 CBA C57BI

10 0

2 30 . 0

4 100 0

8 90 0

The second method employed specific antisera, which permitted the
identification of rat and mouse erythrocytes. In this case lethally
irradiated CBA mice were injected with rat (WAG) bone marrow cells
and agglutination reactions were carried out with both antisera in
samples of the peripheral blood of the survivals. Fig. 3 shows that from
the tenth day after irradiation onward a gradual replacement of the

156

Discussion

mouse erythrocytes by rat erythrocytes occurs. These results also
demonstrate that the injected rat bone marrow has survived and pro-
liferates in the irradiated mice. Results of a similar nature have been
reported by Lindsley, Odell and Tausche (1955, Proc. Soc. exp. Biol.,
N.Y., 90, 512) in the case of homologous bone marrow injection into
irradiated rats, although in these experiments no significant effect of the
bone marrow on survival was observed.

ANTI

RAT

SERUM

43+-

2-f- -

SB
l-l

o
a

ANTI

MOUSE

SERUM

o o

o o
o

OOP

y/-

2 + -

3-1-

r
o

-T—
10

-I— //-

20 30

— I—
70

— I—
90

—1 —
110

DAYS A?TEH
IHRADIATIOl

Fig. 3. (Van Bekkum). Identification of erythrocytes in irradiated and rat

bone marrow treated mice.

In our third approach a histochemical method was used which
permits differentiation between rat and mouse granulocytes. Wachstein
(1946, J. Lab. din. Med., 31, 1) observed that rat granulocytes show a
strongly positive alkaline phosphatase reaction while mouse granulo-
cytes are consistently negative in this respect. Irradiated mice were
injected with rat bone marrow and on various days after the irradiation
smears of the peripheral blood were prepared and studied with the
alkaline phosphatase reaction. Table III shows the percentage of posi-
tive granulocytes in irradiated mice after treatment with rat bone
marrow. In most cases the granulocytes were predominantly identified
as rat cells. It should be noted that No well. Cole and Habermeyer
(1955, USNRDL report T. R. 59) have independently obtained iden-
tical results using Wachstein' s method.

Finally, I want to comment on the survival of irradiated mice after
treatment with homologous and heterologous bone marrow. As shown

Discussion 157

Table III (Van Bekkum)
Alkaline Phosphatase Reaction in Granulocytes

% positive cells

Control mice

Control rats

Irradiated and rat bone marrow treated

mice

46th day after irradiation

> 94 (13)
17(1)

76th day

> 93 (8)
0(1)

111th day

> 97 (4)

68(1)

0(1)

170th day

71(1)
86(1)

The figures in brackets represent the number of observations.

in Table IV, there is practically no delayed mortality in the case of
isologous bone-inarrow therapy, but after homologous and heterologous
therapy a large percentage of the mice die between the thirtieth and the
sixtieth day after irradiation. These data are also in agreement with
those of Dr. Loutit, although I think that our 1 00-day s survival rate is
slightly better.

Table IV (Van Bekkum)
Delayed Mortality of Irradiated and Bone Marrow Treated Mice

Bone marrow Number of survivals

30 rf. 60 d. 80 d. 100 d.

Isologous
Homologous
Heterologous (rat)

Spiegelman: I might note first that I consider the transformation
hypothesis quite unlikely. However, it seems to me that your test for
its existence is insensitive, because you are demanding that the trans-
formation result in a complete conversion of the chromosomal apparatus,
and this is highly improbable. VV^e know that in bacterial transforma-
tions only a minute portion of the chromosomal material is actually
incorporated, and the number of characters involved is invariably
small, one or two. It would be better then to look at somewhat more
restricted changes in phenotype, for example, perhaps with respect to
the antigenic properties of the cell. A simple experiment perhaps is to

158 Discussion

take rat cells which have established themselves in a mouse and intro-
duce them into a normal, non-irradiated mouse and see if they disappear.
Has this been done?

Van Bekkum: Not yet.

Spiegelman: I think this would test it very cleanly and directly.

Lajtha: Can these irradiation and bone marrow transfusion experi-
ments be repeated before the mice die, in those mice which die, say after
50 days? Those mice which die only after 30 days will show some of
their original cells returning, which probably means that their immune
reaction starts killing off the donor cells. Now, could a repeat irradia-
tion and transfusion then be performed and thus elongate the life of the
animal? Furthermore, do you think that such a treatment could
"cure" an experimental leukaemia in the animals?

Loutit: I suppose it could, but I have not done it, and I don't know
what the answer would be.

Stocken: I would like to show a slide which is relevant to Cole's
proposition that nuclear material is concerned in the curative factor.
This slide (Table I) show s decreasing numbers of whole cells and varying

Table I (Stocken)

30 Days Survival of CBA Mice after 950 r X-rays
AND I.V. Injection of Spleen Fractions

clei X 10«

Whole cells x 10^

Survival

4>f4>

6-5

4/4

3/4

1-3

3/4

4/4

1/4

2-2

0/20

1-4

0/20

< 0-6

0/10

<0-2

0/10

amounts of nuclei. I think it is reasonably clear that when the number of
whole cells is reduced to less than 30,000, the recovery does not take
place. You will notice that where 36,000,000 nuclei were put into the
mice no benefit was obtained.

Hollaender: I would like to keep an open mind with regard to cell-free
extracts still possibly producing recovery. Even if you establish the
bone marrow and have tissue growing in the animal, the recovery
process as such may be a product of something which is given up by the
cells. I think we should keep searching, and I think some of the work
which Dr. Stapleton has done may be pertinent at this point.

Stapleton: While we were studying prevention of death or recovery
in bacteria, we had some indication that precursors for RNA synthesis
might be involved. We wondered if an RNA polynucleotide fraction
from Esch. coli would stimulate recovery in bacteria. We prepared a

Discussion 159

rather crude polynucleotide RNA fraction from Esch. coli and found it
was stimulatory in recovery of Esch. coli. We tried this same preparation
injected into irradiated mice, and it seemed to work in mice too. These
experiments are extremely preliminary, but it looks as if the same sort
of preparation injected repeatedly into animals might bring about
something of the order of 20 per cent survival in mice irradiated with a
supralethal dose. We have had a lot of variability among samples pre-
pared similarly. Some fractions have been toxic. We hope that when
we iron out some of the difficulties concerning toxicity of some of this
material prepared from bacteria, we can then give a better answer than
we can at the present time. There is no possibility here, of course, of
repopulation.

Stocken : I think that Dr. Loutit used very careful but accurate words
when he said that the humoral factor was not the initiating mechanism.
I think that this completely covers it.

Stapleton: One reason why I hesitated to say anything about this
bacterial fraction was the fact that people have found increased survival
somewhat like we have found by injecting materials such as ground
glass, charcoal, and so on, following irradiation.

Butler: Then what is the status of Cole's nucleoprotein fraction? Is
the effect due to cells?

Loutit: With Stocken we have made preparations according to the
Cole recipe, using one of these marker chromosomes and the recovering
marrow in those animals contains the marker. That suggests to me
either that the residual number of whole cells are the effective thing, or
if it is the nuclei, that they can reform their own cytoplasm and get
cracking again from that. I find that a little far-fetched.

Stocken: In self-defence, I think that this preparation here has been
reasonably well done. All the cells have been counted. We have used
various techniques for looking at nuclei, and I don't think that Cole had
these advantages. I do think that his material was probably contamin-
ated with whole cells.

Alexander: An impressive argument which Cole used was that he said
that his "nuclear" preparations could be inactivated by treatment
with DNAse, whereas the cellular preparations could not. This would
show that the activity of the "nuclear" preparations is not due to con-
tamination with whole cells, since these should not be inactivated by
DNAse. But Dr. Jacobson told me last year in Chicago that although
DNAse does not inactivate preparations containing large numbers of
cells (of the order of several million), it does destroy the activity of
suspension containing 10* or so cells per mi. Since this number of cells
is sufficient to produce appreciable effects and is also the amount of
contamination to be expected in preparations of nuclei, the DNAse test
can no longer be considered as decisive proof for the nuclear hypothesis.

Stocken: We have done these experiments too, on the same prepara-
tion and there is no recovery with the DNAse-treated preparation.

Spiegelman: Have these cells been stained to see whether they still
have DNA material?

Stocken: No. They have been examined by phase contrast microscopy

160 Discussion

in 28 per cent protein solution as described by Baner, Joseph and
Esnouf (1955, Science, 123, 24).

Stapleton: Someone in Jacobson's group did a very nice experiment
using leukaemia cells, making a nuclear fraction, and from the nuclear
fraction they were able to transplant leukaemia to another animal. I
think they found also that the leukaemia cells were acted upon by the
same enzymes that Cole proposed were not effective on whole cells.
This seems to me to be good proof that there were cells in the nuclear
fraction, plus the fact that the enzymes did work on some whole cells.

Stocken: Yes, Jacobson came to the conclusion that he needed 100,000
cells.

STUDIES ON THE MECHANISM OF
PROTEIN SYNTHESIS*

P. C. Zamecnik, Elizabeth B. Keller, M. B. Hoagland,

J. W. LiTTLEFIELD AND R. B. LOFTFIELD

Medical Laboratories oj the Collis P. Huntington Memorial Hospital of Harvard
University, Massachusetts General Hospital, Boston

The investigation of protein synthesis received a great
stimulus ten years ago, when the ready availabihty of radio-
active isotopes made it possible to pursue in greater detail the
line of research initiated by the Schoenheimer school (Schoen-
heimer, 1942). After a few fruitful years, however, this
approach reached a plateau. During the past two or three
years there has again occurred great progress in this field,
due to the application of other techniques — centrifugal,
microbiological, and cytochemical — to the problem. Two good
reviews (Tarver, 1954; Borsook, 1956) have appeared recently,
covering early as well as current work in the field.

Brachet (1941) and Caspersson (1941) first called attention
to the high concentration of ribonucleic acid in the cytoplasm
of cells of rapidly growing tissues, and in cells of tissues
engaged in the secretion of proteins. The work of Palade
(1955) and of Sjostrand and Hanzon (1954) has utihzed the
high resolving power of the electron microscope to call
attention to details of structure of the fine reticular network
found in the cytoplasm of many cells. Two principal con-
stituents of this cytoplasmic network are (1) the double-
walled membranes, and (2) the small ^ense granules visualized
in high concentration both on the exterior walls of the tubules,
and also as unattached particles. These structures have been
particularly evident in secretory cells such as those of the
pancreas.

* Supported by grants in aid from the U.S. Atomic Energy Commission and
the American Cancer Society. This is a pubHcation of the Harvard Cancer
Commission.

RAD. 161 7

162 Zamecnik et al.

Work in this laboratory has become confluent with this
cytochemical stream as a consequence of our interest in par-
ticulate cell constituents concerned in protein synthesis. In
agreement with the initial observation of Borsook and co-
workers (1950), we have found the microsome fraction of the
rat liver cell to be that centrifugally separable component of
the cytoplasm most rapidly labelled with ^^C-tagged amino
acids (Keller, Zamecnik and Loftfield, 1954).

By means of sodium deoxycholate it has been possible to
separate the two above-mentioned principal components of
the "microsome" fraction, and to determine that the small,
dense granule (ribonucleoprotein particle) has a higher
specific radioactivity in its protein than does the vesicular or
fragmented membranous component (Littlefield et al., 1955).
The careful work of Palade and Siekewitz (1956) in tracing the
lineage of these microsomal constituents back to the small,
dense granules and membranes of the cell serves as a bridge
between cellular topography and cell fractionation studies.
Our conclusion about the high rate of labelling of the ribo-
nucleoprotein particle is based on experiments which were
carried out on whole rats. Their distinctive features were

(1) the intravenous injection of ^*C-leucine or ^*C- valine and

(2) the employment of labelling times of 2-10 minutes. These
conditions brought out maximal differences between the
specific activities of proteins located in the various cell
fractions, differences which became less distinguishable with
increased time periods.

These studies also suggested that the ribonucleoprotein
particle in vivo was engaged in a rapid turnover process, in
which protein or large peptide fragments synthesized therein
were passed on to other parts of the cell. It has been calcu-
lated (Littlefield and Keller, unpublished) that the rate of
labelling of the cytoplasmic ribonucleoprotein particles of the
liver is sufficient to account for most of the protein synthesis
of the rat liver. This is not to imply that protein synthesis
may not occur also in the nucleus, but it does strengthen the
thought that the main pathway of synthesis of a protein

Studies on the Mechanism of Protein Synthesis 163

molecule may pass through the cytoplasmic ribonucleoprotein
particle.

In parallel with these in vivo studies, we have investigated
the incorporation of labelled amino acids into proteins in cell-
free fractions of rat liver. One has little hesitancy in calling
the in vivo work a study on the mechanism of protein synthesis.
In the cell-free preparations, however, there is no net protein
synthesis as measured by the usual procedures, and the term
"incorporation" encompasses this degree of uncertainty.
In our context, however, this term "incorporation" does have
a rather precise meaning, which begins with the choice of
labelled amino acid. On the basis of past experiments in our
own laboratory (Zamecnik and Frantz, 1949) and of others
(cf. Tarver, 1954), we consider it safest to employ labelled
amino acids which are least likely to engage in other known
metabolic processes of the cell. For example, glutamic acid,
aspartic acid and alanine are located at the gateway to the
citric acid cycle ; glycine is a precursor of the purine molecule,
and is readily converted to serine and phosphatidyl serine;
lysine may become bonded to protein in side-chain linkage
by its £-amino group (Schweet, 1955); and the sulphur-
containing amino acids can attach to protein by disulphide
bonding (Tarver, 1954). The possibility that glutathione is
being synthesized in a cell-free system is further reason for
avoiding when possible glutamic acid, cysteine, and glycine as
amino acid labels for experiments on incorporation into pro-
teins. No doubt in time a particular pitfall will be found for
the use of every amino acid as a labelled precursor for protein.

These considerations, however, have led us to the general
use of L-leucine and L-valine in our recent incorporation experi-
ments (Loftfield and Harris, 1956). Partial hydrolysis of
labelled protein at the end of typical in vitro experiments has
indicated the presence of a variety of labelled peptides
(Zamecnik and Keller, 1954; Keller and Zamecnik, 1956;
Zamecnik et al., 1956), and provides evidence of alpha-
peptide bonding.

We have several types of evidence bearing on the question

164 Zamecnik et al.

of whether this peptide bonding represents de novo synthesis
of a peptide chain or exchange of a single amino acid for its
non-radioactive isotope within the interior of an existing
peptide chain. To begin with, Littlefield and Keller (1956) have
shown that biologically active microsomes, labelled by incuba-
tion of ascites tumour cells with ^^C-leucine, do not lose their
protein label when incubated in a complete cell-free system
containing 0 • 01 m ^^C-L-leucine. In another type of experi-
ment, liver microsomes, labelled by 3-minute cell-free incu-
bation with ^*C-leucine or ^^C- valine of high specific activity,
did not lose this radioactivity when ten times as much inert
leucine or valine was added (Littlefield et at., 1955) for a
further incubation period. These specific pieces of data
argue against a simple exchange reaction as the mechanism of
labelling. Reasoning along more general lines, the hydrogen
bonding and specific three-dimensional patternization of a
completed protein molecule would appear to prohibit exchange
of a single amino acid for another located in the interior of the
peptide chain. The data are more compatible, therefore, with
the conception that the labelled amino acids measure a small
amount of de novo synthesis of long chain peptides in the
ribonucleoprotein particulate fraction of this cell-free system.
During the 10-minute cell-free incubation at 37° in which the
labile ribonucleoprotein particle retains its biological activity,
up to 0-2 per cent labelling of the ribonucleoprotein leucine
occurs.

There are five essential components of the cell-free incor-
poration system: (1) the microsome fraction, (2) the pH 5
precipitable enzyme fraction, (3) ATP (and usually an ATP-
regenerating system), (4) GTP or GDP, and (5) the labelled
amino acid. The general method of preparation of the
protein fractions is indicated in Fig. 1. If any one of these
constituents is omitted, the incorporation suffers (Keller and
Zamecnik, 1956). It has recently been possible (Littlefield
and Keller, 1956) to simplify this system a little by using
cellular fractions prepared by 0-5 M-NaCl extraction and
centrifugal fractionation of distilled water lysates of Ehrlich

Studies on the Mechanism of Protein Synthesis 165

mouse ascites tumour cells. Here it is possible to obtain good
incorporation into the ribonucleoprotein particulate fraction
of the microsomes, in the almost complete absence of the mem-
branous lipoprotein fraction. While these two fractions of the
microsome pellet can also be separated by means of sodium

HOMOGENIZATION in 2.5 volumes of medium A

I5,000x^ lOmin.

ppt. (discard)
SUPERNATANT

+ 3 volumes of medium B

105,000 x^ 60 min,

microsome pellet
SUPERNATANT

+ I volume of medium B

pH to 5.2 and then centrifuge

Supernatant
(discard)

PRECIPITATE

Supernatant
(discard)

Resuspend in same volume
of medium B and
centrifuge.

PRECIPITATE

Dissolve in

'dium A to 8 mg./ml.

= pH

enzyme fraction.

Fig. 1. Fractionation scheme for rat liver.
Medium A: 0-35 m sucrose, 0 035 M-KHCO3, 0 004 M-MgCla, 0 025 m-KCI.
Medium B: 0-9 m sucrose, 0 004 M-MgClg, 0 025 m-KCI.

deoxycholate, after such treatment the ribonucleoprotein
particles are biologically inactive, presumably due to denatur-
ation. This newer separation method is a gentler procedure,
which preserves the fragile synthetic activity of this particle.
Another advantage in using cellular fractions from the
mouse ascites tumour is that 10 (jlm of ATP per ml. in itself is
adequate for energy generation, and the ATP regenerating

166 Zamecnik et al.

system (phosphopyruvate plus pyruvate kinase) may be
omitted; however, GTP (or GDP) is still required.

An amino acid activating mechanism has recently been
found which is catalysed by the "pH 5 enzyme" fraction of
the rat liver protein in the presence of ATP (Hoagland, 1955;
Hoagland, Keller and Zamecnik, 1956). The reaction appears
to proceed as follows :

ATP + amino acid + enzyme :^ (aminoacyl-AMP) enzyme

4" pyrophosphate

The evidence points to several separate activating enzymes,
rather than to a single enzyme or activation site capable of
activating all the amino acids.

The role of GTP (or GDP) in this incorporation process is
puzzling. It has thus far been unable to substitute for ATP in
Hoagland's amino acid activation reaction. GTP has been
found to be an essential cofactor for the incorporation of all
six labelled amino acids tested (Keller and Zamecnik, 1956).
It is a very specific cofactor, the only active one among many
nucleoside tri-, di- and monophosphates tested. Through the
kindness of Dr. Waldo Cohn we were able to test the following
dinucleotides of guanine, obtained from partial hydrolysis of
RNA and possessing a free 3'-phosphate: GC, CG, GU, UG,
GA and AG. All of these compounds, in roughly 0 • 1 [xm per
ml. concentration, were unable to substitute for GTP.
Addition of 0-25 mg. of AGUC ribonucleotide polymer,
kindly furnished by Dr. Severo Ochoa, to the in vitro test
system caused a slight inhibition of the incorporation. In
order to test for the GTP requirement, it is necessary to use a
pH 5 enzyme fraction washed quite free of endogenous GTP,
and to employ a microsome fraction prepared by centrifuga-
tion of a diluted 15,000 X g supernatant fraction of rat liver
homogenate (Keller and Zamecnik, 1956).

Our present conception of the sequence of events in the
process of protein synthesis as observed in rat liver cytoplasm
is summarized in Fig. 2. The role of the ribonucleoprotein
particle is considered to be the sequentialization of activated

Studies on the Mechanism of Protein Synthesis 167

amino acids. In the schemes suggested by Bounce (1952) and
by Koningsberger and Overbeek (1953), the amino acids are
attacked by covalent bonding of the amino or carboxyl group
respectively of the amino acid with the phosphate of the
ribonucleic acid. Since, however, the amino acids appear
to be activated in the soluble enzyme fraction of the cell, it
would be reasonable to consider that activated aminoacyl
nucleotide compounds line up along a ribonucleoprotein

amino acid + ATP-^

aminoacyl to AMPp+ pp

-rcraw

../"■■■ 'o

Activation Sequentialization Cross-linking

and
(soluble enzymes) (ribonucleoprotein particle) potternization

( elsewhere —

Pendoplasmic
reticulum )

Fig. 2. Postulated steps in protein synthesis in rat liver cytoplasm.

template, with their side-chain R groups determining the
sequence by their ability to fit into particular spaces occurring
on the ribonucleoprotein surface, rather than that there
occurs a formal triester linkage of amino acid to nucleic acid.
Van der Waal's forces and electrostatic charges would serve
as the binding forces of the side chains of the activated amino
acids to the ribonucleoprotein template. This scheme has
been drawn up in detail by our colleague Loftfield, and will
be published elsewhere.

In the in vivo experiments of Littlefield and co-workers
(1955), the ribonucleoprotein fraction of the liver cell appears
to pass on its radioactive protein or large peptide chain to

168 !^AMECNiK et al.

other fractions of the cell. This is particularly so for the mem-
branous, lipid-rich, deoxycholate-soluble portion of the
microsome fraction. In electron micrographs of intact liver
cells, these membranes are in close juxtaposition to the
ribonucleoprotein particles, where they may serve as acceptor
for a formed peptide chain, and as a site for its transformation
into a completed protein molecule or lipoprotein complex.

Our evidence suggests therefore that protein synthesis in
the rat liver cytoplasm may be divided into three steps, as
indicated in Fig. 2. Further subdivisions remain tasks for the
future.

REFERENCES

BoRSOOK, H. (1956). J. cell. comp. Physiol., 47, Suppl. 1, p.35.
BoRSOOK, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and

LowY, P. H. (1950). J. hiol. Chem., 187, 839.
Bracket, J. (1941). Arch. hiol. {Liege), 53, 207.
Caspersson, T. O. (1941). Naturwissenschaften, 28, 33.
DouNCE, A. (1952). Enzymologia, 15, 251.
HoAGLAND, M. B. (1955). Biochim. biophys. acta, 16, 288.
HoAGLAND, M. B., Keller, E. B,, and Zamecnik, P. C. (1956). J. biol.

Chem., 218, 345.
Keller, E. B., and Zamecnik, P. C. (1956). J. biol. Chem., 221, 45.
Keller, E. B., Zamecnik, P. C, and Loftfield, R. B. (1954). J.

Histochem. Cytochem., 2, 378.
KoNiNGSBERGER, V. V., and OvERBEEK, J. Th. G. (1953). KoninJd.

Nederl. Academic Van Wetenschappen Proc, Series B, 56, 248.
LiTTLEFiELD, J. W., and Keller, E. B. (1956). Fed. Proc, 15, 302.
LiTTLEFiELD, J. W., Keller, E. B., Gross, J., and Zamecnik, P. C.

(1955). J. biol. Chem., 217, 111.
Loftfield, R. B., and Harris, A. (1956). J. biol. Chem., 219, 151.
Palade, G. E. (1955). J. biophys. biochem. Cytol., 1, 59.
Palade, G. E., and Siekevitz, P. (1956). J. biophys. biochem. Cytol., 2,

171.
Schoenheimer, R. (1942). The Dynamic State of Body Constituents.

Harvard University Press.
ScHWEET, R. (1955). Fed. Proc, 14, 277.

Sjostrand, F. S., and Hanzon, V. (1954). Exp. Cell Res., 7, 393.
Tarver, H. (1954). In The Proteins, Vol. II, Part B, p. 1199. New

York: Academic Press.
Zamecnik, P. C, and Frantz, I. D., Jr. (1949). Cold Spr. Harb. Symp.

quant. Biol., 14, 199.
Zamecnik, P. C, and Keller, E. B. (1954). J. biol. Chem., 209, 337.
Zamecnik, P. C, Keller, E. B., Littlefield, J. W., Hoagland, M. B.,

andLoFTFiELD,R.B.(1956). J. cdZ. comp. P%sioZ., 47, Suppl. 1, p. 81.

Discussion 169

DISCUSSION

Work: We too have been following uptake of radioactive amino acids
into liver preparations in the intact animal, and have adopted a similar
centrifugal fractionation scheme followed by a scheme of salt fraction-
ation of the guinea pig liver microsomes. We found also that we had
peak activity in the microsomes. A nucleoprotein fraction obtained by
salt fractionation of the microsome material showed a quite clearly
defined peak thirty minutes after injection; we could separate out two
nucleoprotein fractions, one of which showed this peak of activity and
one which did not. Why there should be a peak at thirty minutes in our
guinea pigs and a peak at a very much shorter period in Dr. Zamecnik's
rats, I don't know. It looks as though we are both handling very
similar types of material.

On the additional steps in the reaction, the activation of the carboxyl
group, we have been able to confirm Hoagland's work. Hoagland very
kindly wrote and gave us details before he published this.

I was interested in your remarks about the use of leucine AMP
anhydride ; we have made alanine AMP anhydride, and have found that
it is unstable ; in aqueous solution it polymerized rapidly to its peptide.
I would be interested in hearing if anybody had managed to get a suf-
ficiently stable preparation to work with. It seems to me that the amino
group must be protected in some way or other in the enzymic carboxyl
activation process. As you say, it may be attached to the enzyme
surface in some way which protects the amino group. Another distinct
possibility, I think, which is worth consideration, is that the amino
group may also be protected by a phosphoramide linkage. We made
some phosphoramides and found that metabolically they were indis-
tinguishable from the free amino acids; in other words the phosphor-
amide group obviously comes off very quickly in a biological system, and
it seems to me that it is a possible intermediate which we ought to
consider.

Another point that I think we ought to keep in mind is that these
mixed anhydrides are so unstable that if you add any acceptor which is
potentially capable of forming an anhydride group, even in the absence
of any enzyme, you get mixed anhydride. Thus if you take AMP and,
say, leucine phosphate anhydride you get ATP from it without any
enzyme.

Zamecnik: De Moss, Genuth and Novelli have just published (1956,
Fed. Proc, 15, 241) their results, and thfeir yield of this leucyl AMP
anhydride was very poor, about 7 per cent. The half-life of this com-
pound was several minutes at pH 7, making possible a study of its con-
version to ATP by the enzyme in the presence of pyrophosphate. With
regard to your work on guinea pigs, the technical details of our experi-
ments with the rat and yours with the guinea pig may be a little dif-
ferent. I suppose we both injected intravenously?
Work: Yes.

Zamecnik: In one type of experiment we gave a small dose of
^*C-leucine of a very high specific activity, about 9 mc per m-mole (1 -4

170 Discussion

mc to a 270-g. rat), in the hope that we would get a high level of radio-
activity incorporated into protein in a few minutes (cf. Fig. 3, Little-
field et ah, 1955, loc. cit.).

Work: We may have missed a peak which you picked up in using a
higher dose. We used about 35 mc/kg. animal. Ours was a comparable
specific activity, but a much smaller dose.

Zamecnik: As regards the two different ribonucleoprotein fractions, in
the case of the rat liver we obtained particles such as this using sodium
deoxycholate fractionation. Ultracentrifugally, the ribonucleoprotein
fraction behaves as one peak, although I don't think that necessarily
implies very much. But when my colleagues Littlefield and Keller pre-
pared ribonucleoprotein particles from the Ehrlich mouse ascites tumour
they found three closely parallel ribonucleoprotein peaks. I don't
know whether the specific activity in those were all the same.

Work: We also found that if we fractionated the supernatant with
ammonium sulphate we got very considerable variation in activity.

Zamecnik: We have carried out some fractionation of this 100,000 g
supernatant protein too. Dr. Hoagland has rather good evidence that
there is not just one enzyme or one enzymatic site involved in activation
of all amino acids. I don't know whether there is one enzyme for each
amino acid, but there is evidence for three or four separate ones so far.
De Moss and Novelli (1955, Biochim. biophys. acta, 18, 592) have con-
firmed this finding in bacteria, and they found evidence for activation of
8 separate amino acids, and think there is more than one enzyme
involved.

Work: That would fit in with our experience. Whenever we fractionate
we divide it into a lot of fractions with less activity than the original.

Zamecnik: We have not had good luck in fractionating this pH 5
enzyme and then using these fi-actions separately for the whole incorpor-
ation process. We seem to lose the activity on fractionation, and my
impression is that we have several enzymes involved, which are going
into different fractions.

Popjak: Is it not possible that the aminoacyl nucleotide or amino-
acyl AMP is only a first intermediary so to speak, and that the hydrox-
amate that you get in these preparations perhaps really comes from
another type of activated amino acid ; in the acetate activation reaction
it is also postulated that it is the acetyl AMP which is the first inter-
mediate and then acetyl coenzyme A is formed. I think that this is very
likely, particularly in view of what Dr. Work has said about the instabil-
ity of mixed anhydrides of amino acids with AMP.

Zamecnik: That fits in with the idea that maybe one has an enzyme-
bound activated intermediate.

Work : One point I should mention : we found that the rate of appear-
ance of ATP is very much faster than the rate of appearance of hydrox-
amate. Does that agree with your findings?

Zamecnik: Yes, our preparations contain our "ATPase", unrelated
to amino acid activation, in addition to the ATP splitting involved in
this process.

Popjak: It is very likely that you have more than one activating

Discussion 171

enzyme, because even in the case of simple homologues like fatty acids
several activating enzymes are known, all of which have different chain-
length specificity; so that in a group of more diverse substances like
amino acids it is even more likely that you should have more than one
activating enzj^me.

Bracket: Do you know (a) whether the protein present in these very
small particles has any of the enzymatic activity which is usually
associated with microsomes; (b) whether it is something like a histone
or any of the basic proteins ; and (c) whether removal of the ribonucleic
acid from these very small granules has an inhibitory effect on incorpora-
tion?

Zamecnik: We have added ribonuclease in very small concentrations,
about 1 yLg./mh, and have found it inhibits the incorporation reaction
completely. There is some breakdown of RNA, but the inhibition
appears to exceed it enormously. We have done some preliminary frac-
tionation experiments on these particles. If we add ribonuclease at a
concentration of 5 or 10 [ig. /ml. and incubate it with these particles for
10-15 minutes, everything comes down in a coagulum, all stuck together.
We were disappointed with that experiment. But we have also incubated
ribonucleoprotein particles with between two-tenths and 1 y.g. of
ribonuclease in a cold room at 4°C for 3 days, in the presence of a fairly
high salt concentration; under those circumstances the ribonucleo-
protein broke down into several fragments. A fraction which did not
centrifuge down at 100,000 g for 2 hours had about twice the specific
radioactivity of the fraction which did spin down. This soluble protein
fraction was placed in the electrophoresis machine (we have only done
this once). There were three components. We cut between the slowest
moving peak and the other two peaks and found that the slowest
moving peak had half the specific activity of the others. That is a crude
fractionation, but it suggests that we may be able to break this ribo-
nucleoprotein down by some procedure. I have no doubt that it consists
of more than one protein. We suspect that only a small portion of the
protein components ds involved in the rapid synthetic mechanism.

Holmes: I should like to know if Dr. Zamecnik has any evidence of the
RNA itself being broken down and rebuilt during amino acid incorpora-
tion.

Some years ago we prepared a crude cytoplasmic ribonucleoprotein
fraction from the tumours of rats injected during life with ^ss-methio-
nine and ^sp. This fraction was prepared by precipitation at pH 5-0
after removal of the nuclei and contained, all the remaining RNA and a
considerable amount of protein. The uptake of ^^p into the RNA
seemed to parallel the incorporation of methionine into the protein.
X-ray irradiation of the tumour in vivo had no effect upon the uptake
of either of the labels, whereas the injection of shock-producing chemicals
caused a proportionate reduction in the uptake of both.

Zamecnik: In Potter's laboratory experiments were performed on in
vitro incorporation of labelled orotic acid into RNA in rat liver homogen-
ates; they used almost the same components that we have used here,
and found that the orotic acid does make its way into the RNA molecule.

172 Discussion

We have not done definitive experiments in our laboratory, but I suspect,
from the few we have carried out, that RNA is being synthesized, since
i*C-labelled ATP makes its way into RNA during that same time.

Spiegelman: Along these lines it seems to me very worth while, in
order to define the nature of this system, to enquire more closely into the
relation between nucleic acid synthesis and protein. As you know, in
the study of enzyme formation there seems to be no doubt that there is a
mandatory coupling between RNA synthesis and synthesis of the new
protein molecule. It would be interesting to see whether for example an
agent like hydroxyuridine would affect this incorporation phenomenon
as it does the synthesis of the protein molecule. Dr. Zamecnik, in your
in vivo incorporation experiments have you ever simultaneously
injected a complete mixture of amino acids, in addition to the labelled
one, to see whether there was any effect ?

Zamecnik: We have injected a "quenching" dose of the inert amino
acid and then one has a drop off.

Spiegelman: The point of that experiment is to see whether, if you
make protein synthesis much easier, your incorporation will also
increase.

Zamecnik: There is an adequate intercellular supply of free amino
acids in the whole animal, isn't there?

Spiegelman: There is a supply, but is it adequate?

Zamecnik: But you can calculate that it is adequate for about 5-10
minutes of protein synthesis in the rat liver.

Spiegelman: The yeast cell has a very high internal supply too but you
can stimulate protein synthesis in yeast cells immediately and consider-
ably by providing an external supply of amino acids. I should also like
to know whether you have tried a reconstitution experiment of your
ribonuclease-treated material.

Zamecnik: Yes, we have tried this type of experiment ; it doesn't work.

Pirie: Have you got any evidence of intermediate peptides of any
size, large or small?

Zamecnik: No. My colleague Dr. Loftfield has been specially interested
in that question and has been studying ferritin synthesis in the rat liver.
If you give colloidal iron oxide to a weanling rat, the weanling rat
synthesizes 10 mg. of ferritin within the next two days, whereas pre-
viously he had none. That is a case of de novo synthesis of protein.
The specific activity of leucine or valine in the ferritin is approximately
the same as that of the intracellular free leucine or valine concentrations,
and Loftfield concludes that the ferritin molecule is not supplied with
any appreciable amount of peptide fragments from the large amount of
liver protein already existing, but that the ferritin molecule appears to
be formed from free amino acids exclusively. Furthermore, in the amino
acid activating system there is no evidence that amino acids can react
with an activated amino acid to form a peptide.

I might mention that it is still possible to preserve a modicum of
doubt as to whether there is a real distinction between "exchange" and
"synthesis" in amino acid incorporation experiments. It is hard for us
to make a distinction in the animal system we use. We seem to be

Discussion 173

dealing with what we interpret to be de novo synthesis. I think one
could even say that in dealing with bacterial systems, where one does
not add a complete complement of amino acids, there may be enough
residual amino acids to provide a complete supply of amino acids for a
short time, whereas over a longer period of time they would eventually
be exhausted. There is a certain amount of proteolysis going on also.
One may define "exchange" as a simultaneous opening of two peptide
bonds in the interior of a long peptide chain with a substitution of another
amino acid. I wonder whether that does in fact happen.

Work: I feel fairly convinced on purely chemical grounds that it does
not. Once the peptide bond is formed it is a completely different order
of stability and it is extremely unlikely that it would open again.

NUCLEIC ACIDS AND AMINO ACID
INCORPORATION

E. F. Gale

Medical Research Council Unit for Chemical Microbiology, Department of

Biochemistry, University of Cambridge

In order to investigate the biological functions of nucleic
acids, it is necessary to devise preparations of cells in which
it is possible to modif}^ the nucleic acid components without
affecting other factors involved in relevant biochemical
activities. Although the actions of transforming principles
are demonstrated in growing cultures, intact cells in general
do not appear to be permeable to nucleic acids and, conse-
quently, the actions of the latter must be studied in subcellular
preparations. Since the demonstration that suitable prepara-
tions could be obtained from broken staphylococcal cells
(Gale and Folkes, 1953a), a number of other subcellular
materials have been obtained in which direct investigation of
nucleic acid function is possible (Allfrey, 1954; Beljanski,
1954; Lester, 1953; Littlefield et al., 1955; Nisman, Hirsch and
Marmur, 1955; Nisman, Hirsch, Marmur and Cousin, 1955;
Webster and Johnson, 1955; Zamecnik and Keller, 1954).
As a result of investigations with these preparations it has
been found that the incorporation of labelled amino acids is
dependent upon the presence of nucleic acids and that, in
appropriate structures, the process can be mediated by ribo-
and by deoxyribonucleic acid (Gale, 1956a). The purpose
of this contribution is to discuss what we ourselves have
learned of the function of nucleic acids in amino acid incor-
poration studied in disrupted staphylococcal cells, and
consists largely of a summary of material recently pub-
lished elsewhere (Gale and Folkes, 1955a and b; Gale, 1956a
and b).

174

Nucleic Acids and Amino Acid Incorporation 175

Disrupted Staphylococcal Cell Preparation

If a suspension of Staphylococcus aureus is incubated in the
presence of glucose and a single amino acid, such as glutamic
acid, labelled with ^^C, the protein of the preparation acquires
radioactivity. This radioactivity is not removed by pro-
longed washing of the protein fraction with trichloracetic
acid, acetic acid, alkali, alcohol, ether or acetone and can be
released from the protein fraction only by such chemical
measures as result in hydrolysis of the peptide bonds. If the
amino acid in the incubation medium is glutamic acid, hydro-
lysis and separation of the protein residues after incubation
show that the radioactivity is confined to the glutamic acid
residues. Treatment of the protein with ninhydrin or by
application of Sanger's method for N-terminal groups (Sanger,
1945) shows that less than 3 per cent of the incorporated
residues occur as end-groups. It seems that the situation is
essentially similar to that studied by Borsook and his col-
leagues (reviewed by Borsook, 1954) in other tissues, and that
the incorporated radioactive amino acid has become part of
the structure of certain proteins of the preparation.

If cells rendered radioactive, by incubation with glucose
and a labelled amino acid, are disintegrated by exposure to
supersonic vibration and the disintegrated material then
separated on the centrifuge into four fractions: (a) material
sedimented in 10 minutes at 800 g, (b) material sedimented in
20 minutes at 4000 g; (c) material sedimented in 60 minutes
at 16,000 g, and (d) "soluble" material, it is found that
Fraction (b) has the highest specific activity. Furthermore,
if freshly harvested cells are suspended in a buffered salt
solution containing 10 per cent sucrose, disintegrated, frac-
tionated in the same fashion, and the various fractions in-
cubated with ^^C-glutamic acid or glycine, together with
ATP as energy source, it is found that Fraction (b) is able
to incorporate the labelled amino acid rapidly, having
markedly greater activity than (a) while the activity of (c)
and (d) is negligible. Electron microscope examination of

176 E. F. Gale

Fraction (b) shows that it consists of disrupted cells with a torn
outer wall and a greatly decreased content of electron-dense
material. This fraction constitutes the "disrupted cell
preparation" used for the work to be described.

Properties of the disrupted cells

The disrupted cell is non-viable and possesses no measurable
respiration but retains many of the enzymic and synthetic
activities of the intact cell. It can metabolize glucose with the
production of acid and can utilize hexosediphosphate as a
source of energy for protein synthesis. If provided with an
energy source and a complete mixture of the naturally
occurring amino acids, it can synthesize protein as shown by
increase in protein-nitrogen, in catalase and in glucozymase
activity; if galactose is also supplied, as inducer, it can
synthesize p-galactosidase although this enzyme is completely
absent in the initial preparation. If provided with a mixture
of purines and pyrimidines, including i^C-uracil, and a source
of energy, synthesis of ribonucleic acid (RNA) can be demon-
strated by incorporation of radioactivity into the RNA
fraction of the preparation, and this synthesis is increased by
the further addition of a complete mixture of amino acids.
As shown in earlier work with intact cells (Gale and Folkes,
19536), the synthesis of RNA is markedly increased by the"
presence of chloramphenicol at a concentration which limits
protein synthesis.

When disrupted cells are incubated with a i*C-labelled
amino acid and a source of energy (normally ATP + hexose-
diphosphate), the labelled amino acid becomes incorporated
into the protein of the preparation. If the labelled amino acid
is one component of a complete mixture of amino acids
(condition 2), incorporation proceeds linearly for some hours
and is accompanied by measurable increase in protein. If the
labelled amino acid is the only amino acid present in the
incubation mixture (condition 1), then the rate of incorpora-
tion, which may initially be greater than that occurring in the

Nucleic Acids and Amino Acid Incorporation 177

presence of the complete mixture of amino acids, decreases
with time and incorporation eventually ceases ; the amount of
amino acid incorporated when the process ceases corresponds
to only a small proportion of the corresponding residues
initially present in the protein fraction. In condition 1, no
change in the protein content of the preparation can be
demonstrated and it has been shown elsewhere (Gale and
Folkes, 1955a; Gale, 1956a and b) that such incorporation
cannot be taken as a measure of protein synthesis or as an
indication that such synthesis is occurring unless supporting
evidence is forthcoming. As a working hypothesis, it has
been suggested that incorporation under condition 1 takes
place as a result of an exchange reaction between amino acid
added to the medium and corresponding residues in certain
of the proteins present in the preparation. It may be that
such exchange incorporation is an activity of a part or parts
of the protein-synthesizing mechanism and that this activity
can occur when total protein synthesis is not possible.

Effect of Nucleic Acid Removal

Nucleic acid can be removed from the disrupted cells by
extraction with M-NaCl or incubation with ribo- or deoxyribo-
riuclease. After such treatment the preparation is no longer
able to synthesize protein unless the incubation mixture is
supplemented by appropriate mixtures of nucleic acids or
their precursors (Gale and Folkes, 19556). The treatment
also results in a decrease in incorporation of amino acids under
condition 1 ; the degree of decrease varies with the amount of
nucleic acid removed and also with the particular amino acid
whose incorporation is being studied. The incorporation of
glycine is particularly sensitive; removal of nucleic acid to
the point where the content is less than 10 per cent of that in
the initial cell results in reduction of glycine incorporation to
10-15 per cent of that which takes place in the intact cell.

Incorporation in nucleic acid-depleted preparations can be
restored by addition of staphylococcal nucleic acid to the

178 E. F. Gale

incubation mixture. The action of added nucleic acid is to
increase both the rate of incorporation and the amount of
amino acid incorporated by the time the process ceases.
Restoration can be brought about by the presence of either
DNA or RNA but the latter is always less effective than the
former; optimal concentrations of RNA normally restore
incorporation to 50-70 per cent of the value obtained in the
presence of optimal concentrations of DNA. Of a variety of
nucleic acids tested in the staphylococcal system, only those
obtained from Staph, aureus proved to be effective.

Modification of Nucleic Acid Component

Whatever may be the mechanism of incorporation under
condition 1, it provides an experimental system in which a
relationship between proteins, amino acids and nucleic acids
can be investigated.

Effect of digestion of nucleic acid. If staphylococcal DNA is
digested with deoxyribonuclease, the activity of the digest in
restoring glycine incorporation is less than that of the intact
nucleic acid and is further reduced on dialysis. If staphylo-
coccal RNA is digested with ribonuclease, the effect of the
digest in restoring glycine incorporation is greater than that
of the undigested RNA, the digest promoting incorporation
to the same level as that obtained in the presence of optimal
concentrations of DNA. Whereas the restoration by intact
RNA appears to be species specific, ribonuclease digests of
RNA from a variety of sources prove to be effective and the
active material in all cases is dialysable.

Attempts have been made to fractionate RNA digests,
and activity in promoting incorporation has been found
associated with two types of fraction. When fractionation is
carried out according to the methods described by Markham
and Smith (1952) for the separation of small polynucleotides,
activity towards specific amino acids is found associated with
specific polynucleotide fractions, whereas activating sub-
stances of a relatively non-specific nature ("incorporation

Nucleic Acids and Amino Acid Incorporation 179

factors") are found in fractions which, from their physico-
chemical properties, are not nucleotides. It may be that
specific polynucleotides are activated by substances related to
the "incorporation factors" but definite information on this
point awaits the characterization of the latter factors.
Effect of X -irradiation on the nucleic acid response. Table I
shows that exposure to 150,000 r has no significant effect

Table I

Restoration of Glycine Incorporation by Staphylococcal Nucleic
Acids Before and after X-irradiation

Increase in glycine incorporation {c.p.m.jmg.)

Nucleic add added:

mg.lml.

0-2

X-radiation dose

(r)

Deoxyribonucleic acid

None

50,000
150,000

154
179
180

260
265

287

Ribonucleic acid

430
372
347

None

50,000
150,000

75
74
70

158
142

197
141
158

Incorporation in absence of added nucleic acid =218

on the ability of staphylococcal RNA or DNA to promote
glycine incorporation. These results provide a further indica-
tion that the ability to promote incorporation under con-
dition 1 resides in portions of the nucleic acid structure rather
than in the complete polynucleotide.

Inhibitors

Antibiotics. Chloramphenicol inhibits the incorporation of all
amino acids so far tested ; in no case does the inhibition reach
completion. In all cases investigated, inhibition increases with
increasing concentration of antibiotic but reaches a plateau
value which varies with the amino acid whose incorporation
is studied (see Table II). The synthesis of protein, whether
measured by increase in protein-N, catalase or ^-galactosidase,

180 E. F. Gale

is markedly more sensitive to chloramphenicol than any
incorporation reaction and, moreover, inhibition of protein
synthesis proceeds to completion.

Table II shows that penicillin and bacitracin inhibit the
incorporation of certain amino acids and that the inhibition
resembles that by chloramphenicol in reaching a different

Table II

Inhibition of Amino Acid Incorporation (Condition 1) by

Antibiotics

% Inhibition by

Chloramphenicol

Penicillin

Bacitra
cin

300

003

0-3

100

Amino acid

[Ig.lml.

[Ig.l.ml

. [Ig.lml.

[Ig.lml.

Glutamic acid

Glycine

Alanine

Aspartic acid

Leucine

Threonine

Phenylalanine

Lysine

Arginine

Tyrosine

Proline

Valine

plateau level in each case but differs in that not all incorpora-
tion reactions are affected. The inhibitions by the three anti-
biotics are significantly the same whether incorporation is
promoted by RNA or DNA, consequently the plateau effects
are not due to differentiation between the nucleic acids.
Chelating agents. Attention has been paid to the possibility
that metal activation is involved in the promotion of incorpor-
ation by nucleic acids. A preliminary survey of the action of
chelating agents indicated that glycine incorporation was
highly sensitive to inhibition by 8-hydroxyquinoline. No
significant inhibition was obtained with 0-01 M-versene or
a:a-dipyridyl.

8-Hydroxyquinoline. The inhibition by 8-hydroxyquinoline
(oxine) is unusual in that 10 ~^ M-oxine produces 80-90 per

Nucleic Acids and Amino Acid Incorporation 181

cent inhibition whereas 10"^ m is markedly less inhibitory.
Fig. 1 shows the effect of oxine on glycine incorporation in
nucleic acid-depleted cells incubated in the presence and
absence of staphylococcal DNA; 10 ~^ M-oxine prevents the

ISOOt

1600-

1400

2 1200

o
o

z
o

1000

800-

<
a:

2 600 ^

O
o

400

^ 200
o

+ DNA

O CONTROL

r
6

5 4

LOG MOLAR C0NC.8-KYDR0XYQUIN0LINE

-I
3

Fig. 1. Effect of 8-hydroxyquinoline on the incorporation of glycine
in disrupted staphylococcal cells depleted of nucleic acid and in-
cubated in the presence and absence of added staphylococcal

deoxyribonucleic acid.

stimulation of incorporation by DNA. The effect is not specific
for DNA since stimulation by RNA or by purified preparations
of the "glycine incorporation factor" is abolished in the same
way and at the same concentration. Albert, Gibson and
Rubbo (1953) found that the bactericidal action of oxine
depends upon the presence of heavy metal ions in the medium
and that toxicity was maximal when equimolar quantities of

182 E. F. Gale

oxine and heavy metal were present. A similar situation
appears to hold in the present instance since oxine is found to
have no inhibitory action on glycine incorporation if the
reagent solutions are "stripped" of heavy metals by the
procedure of Waring and Werkman (1942) prior to test.
Metal effects. Glycine incorporation is highly sensitive to
inhibition by copper ions and the toxicity of copper is greater
in the absence of other metals such as iron or cobalt. In
"stripped" incubation mixtures, glycine incorporation is
80 per cent inhibited by 10 ~^ m Cu"^^ while the promotion of
incorporation by added DNA is abolished by concentrations
of 10"'^ to 10"^ M Cu"^"*". Although there is as yet no direct evid-
ence for the participation of metals in amino acid incorpora-
tion, it may be that copper ions or oxine complexes of heavy
metals block sites of reaction between nucleic acids and
proteins and so prevent incorporation.

REFERENCES

Albert, A., Gibson, M. I., and Rubbo, S. D. (1953). Brit. J. exp.Path.,

34, 119.
Allfrey, V. G. (1954). Proc. nat. Acad. Sci., Wash., 40, 881.
Beljanski, M. (1954). Biochim. biophys. acta, 15, 425.
BoRSOOK, H. (1954). Chemical Pathways of MetaboHsm. New York:

Academic Press.
Gale, E. F. (1956a). Symp. Biochem. Soc, in press.
Gale, E. F. (19566). Harvey Lectures, in press. Springfield, 111.: Charles

C. Thomas.
Gale, E. F., and Folkes, J. P. (1953a). Biochem. J., 55, xi.
Gale, E. F., and Folkes, J. P. (19536). Biochem. J., 55, 721, 730.
Gale, E. F., and Folkes, J. P. (1955a). Biochem. J., 59, 661.
Gale, E. F., and Folkes, J. P. (19556). Biochem. J., 59, 675.
Lester, R. L. (1953). J. Amer. chem. Soc, 75, 5448.
LiTTLEFiELD, J. W., Keller, E. B., Gross, J., and Zamecnik, p. C.

(1955). J. biol. Chem., 217, 111.
Markham, R., and Smith, J. D. (1952). Biochem. J., 52, 558.
NiSMAN, B., HiRSCH, M. L., and Marmur, J. (1955). C.R. Acad. Sci.,

Paris, 240, 1939.
NiSMAN, B., HiRSCH, M. L., Marmur, J., and Cousin, D. (1955). C.R.

Acad. Sci., Paris, 241, 1349.
Sanger, F. (1945). Biochem. J., 39, 507.

Waring, W. S., and Werkman, C. H. (1942). Arch. Biochem., 1, 303.
Webster, G. C, and Johnson, M. P. (1955). J. biol. Chem., 217, 641.
Zamecnik, P. C, and Keller, E. B. (1954). J. biol. Chem., 209, 337.

Discussion 183

DISCUSSION

Spiegelman : I wonder whether staphylococcal RNAse can break down
non-homologous RNA to active fragments. This could explain the
specificity of the intact RNA, i.e. the RNA is actually broken down in
those cases where it exhibits activity. Were this the case the specificity
would react with the enzyme, not the RNA.

Gale: I cannot contradict that suggestion. I can only say that there
is no detectable RNAse activity in the disrupted cell preparation.

Butler: I should like to mention results of some experiments by my
colleague Dr. Hunter, which were actually begun in Dr. Gale's labora-
tory. He found an inhibiting effect of some nitrogen mustards on the
incorporation phenomenon. This was with the intact bacterium under
starved conditions. He has got very good correlation between the
inhibition of incorporation and the inhibitory effect of these com-
pounds on the Walker tumour. But if the examination is done under
protein synthesis conditions there is no great inhibition, it is only found
under the cell exchange conditions.

Pirie: What enzyme was it whose formation was inhibited by radi-
ation of the DNA preparation, and did you get inhibition with any lower
doses ? Did you find any physical changes in your preparation of DNA
after radiation with these doses?

Gale: The enzyme was ^-galactosidase. I have not tested any other
enzyme system. I have concerned myself principally with amino acid
incorporation, where irradiation of the staphylococcal nucleic acids has
no effect on their ability to promote glycine incorporation.

Cohn: This might be an appropriate time to make a few comments
on nucleic acids, and in making them I imply no criticism of any
particular work here or otherwise, but rather in the. light of more exact
interpretation of evidence which has been accumulated. There is no
doubt that nucleic acids and the deoxyribonucleic acids, polydiesters of
sugars and phosphates, do exist in tissue, and that they also exist in the
preparations that are made from tissues. But it is a long step from there
to assuming that the preparations that have been made (and this
applies to all the preparations of which I have any knowledge) are any-
where near as clean as the letters or the formulae used to represent them.
There is a great deal of doubt as to whether even crystalline proteins are
100 per cent what they are reputed to be, and certainly with respect
to such a characterization as crystallinity the nucleic acid field is far
removed. I think it was Gulland who said that "Nucleic acids are not
compounds, they are methods of preparation ". Now we know that there
are impurities in nucleic acid preparations, and no one has reported much
better than 90 per cent purity by any reliable means on any nucleic acid
preparation. If we overlook the possible biological significance of 5 or
10 per cent, we overlook all that we know about trace elements and trace
compounds such as enzymes. Furthermore, it is exceedingly difficult to
remove ribonuclease or ribonuclease-like enzymes from nucleic acid
preparations. Many nucleic acid preparations will autolyse themselves
if given a chance, showing symptoms of contamination with their own

184 Discussion

specific nucleases, whatever they may be. The heterogeneity, in many
different ways, of nucleic acids is rapidly becoming apparent; hetero-
geneity according to site, as Prof. Davidson could attest; to metabolic
activity in a specific site; to size. I am reminded of an observation
coming from Dr. Markham's laboratory with respect to 5' ends in nucleic
acids and particularly in tobacco mosaic virus. The evidence of these 5'
ends depends on how you precipitate the virus. If you do it one way
you get evidence of 5' ends, if you do it another way you do not. Here
is an apparent heterogeneity in terms of size or structure or admixed
material which appears to depend on the method of precipitating the
virus from the plant extract. Dr. Gale's evidence indicates that some-
thing (probably non-nucleotide as he himself says) which is carried by
nucleic acids has a pronounced effect, an effect in which we are all
interested. But in interpreting these effects we must remember that in a
preparation of RNA and DNA the major component may indeed be
what the letters stand for, but there is no guarantee that that is all that
is present in the preparation.

Bracket: Dr. Gale said that, as a rule, intact cells are not permeable to
nucleic acids. I think that the nucleic acids, at any rate when they are
not very highly polymerized, can get into certain cells. They certainly
can get into the amoebae. RNA can also get into the cells of the onion
root : cytological evidence shows that it first produces extensive mitotic
activity, followed by an inhibition. Most people still believe, and I
believed up to two years ago, that a large molecule like a nucleic acid
would not penetrate into a cell. I now think that we can no longer
accept that this is always true.

With regard to the question of the possible role of copper, has copper
any effect on RNA and protein metabolism, producing for instance a
dissociation of the two? It has been found that there may be an
accumulation of RNA in bacteria treated with cobalt under conditions
where growth stops; I wonder whether copper can produce such an
effect.

Gale: A dissociation of protein synthesis and nucleic acid synthesis
has been shown with cobalt, but not, as far as I know, with copper. In
the glycine incorporation system cobalt will antagonize the toxic action
of copper, but whether or not this is due to differences in the affinity of
their chelating systems, I don't know.

PROTEIN SYNTHESIS IN PROTOPLASTS*

S. Spiegelman

Department of Bacteriology, University of Illinois

Introduction

Weibull (1953) observed that exposure of Bacillus
megaterium cells to lysozyme under hypertonic conditions
leads to the formation of structures he labelled as protoplasts.
Usually each rod-like cell yields two or three of the spherical
protoplasts. Osmotically stabilized suspensions of the proto-
plasts were metabolically active, possessing a high endogenous
respiration (Weibull, 1953) and capable of glucose oxidation
at constant rates for extended periods of time (Wiame,
Storck and Vanderwinckel, 1955). It was quite generally re-
cognized that a subcellular system had been uncovered which
could be potentially useful in the analysis of cell function. A
number of laboratories immediately undertook a study of
the synthetic capacity of protoplasts. It is the purpose of the
present paper to summarize the results obtained to date.

The Synthetic Potentiality of Protoplasts

While we shall be mainly concerned with the synthesis of
specific proteins, it is of interest to begin with studies demon-
strating that protoplasts can support rather involved and
extensive synthetic processes. It Was shown independently
in two laboratories (Brenner and Stent, 1955; Salton and
McQuillen, 1955) that bacteriophage multiplication occurs in
protoplasts of B. megaterium if the bacteria are infected or
induced prior to the removal of the cell wall. Virus yields

* The original investigations described stemming from the author's labora-
tory were aided by grants from the National Cancer Institute of the U.S.
Public Health Service and from the Office of Naval Research.

185

186 S. Spiegelman

were in the neighbourhood of 30 per cent of those obtainable
with intact cells. Single burst experiments demonstrated that
virus synthesis was occurring in a major proportion of the
infected protoplasts.

That protoplasts do indeed retain a major proportion of the
synthetic potentiality of the cells from which they are derived
is dramatically exhibited by Salton's (1955) experiments on
spores. A modification of Hardwick and Foster's (1952) pro-
cedure for "committing" cells to sporogenesis was employed.
The cells were then converted to protoplasts with lysozyme
and incubated further. Approximately 1 per cent of the
protoplasts were thereby converted to viable spores detectable
by suitable plating procedures. Thus far no one has achieved
direct conversion of protoplasts to viable cells by resynthesis
of the cell wall. McQuillen (1955c) has, however, provided
evidence indicating that protoplasts are capable of limited
division. When properly supplemented, and incubated with
aeration for periods extending between four and six hours,
protoplasts take on dumb-bell shapes which are highly sugges-
tive of the occurrence of incipient division.

Incorporation Studies

Attempts to study protein synthesis by tracer methods were
actually made prior to the appearance of WeibuU's publication
and when it was not realized that under certain conditions
treatment of sensitive cells with lysozyme results in the
appearance of microscopically visible structural elements.
Lester (1953) exposed Micrococcus lysodeikticus to lysozyme
in the presence of sucrose and found that such "lysates"
could still incorporate ^*C-labelled leucine into the protein
fraction. The addition of deoxyribonuclease enhanced the
incorporation, whereas ribonuclease abolished it. Similar
findings were reported by Beljanski (1954), who used labelled
glycine. Here again stimulation with DNAse and inhibition
with RNAse were observed. It seems probable that both
of these investigators were dealing wholly or in part with

Protein Synthesis in Protoplasts 187

protoplasts. We shall return subsequently to the significance
of the results with the two nucleolytic enzymes.

The first extensive investigation of incorporation in defined
protoplast preparations was performed by McQuillen (1955a).
A variety of ^^C-labelled compounds was used, and a com-
parison of intact cells and protoplasts was made. The results
obtained in the two were qualitatively similar. Thus, ^^C-
carboxyl-labelled glycine made its way into the protein
glycine and also into the adenine and guanine of the nucleic
acids. The rate of incorporation in protoplasts was between
50 and 100 per cent of that observed in intact cells.

About the only striking difference between cells and proto-
plasts which emerged in these studies was a curious and un-
explained dissimilarity in response to uranyl chloride. It was
found that UO2CI2 suppressed the incorporation of glycine
into the nucleic acids of protoplasts but had no effect on the
metabolism of intact cells.

Induced Synthesis of Protein

The induced synthesis of enzymes in suspensions of proto-
plasts was simultaneously achieved in three laboratories.
Wiame and his collaborators (1955) showed that arabinokinase
was formed in protoplasts prepared from Bacillus subtilis when
they were incubated aerobically in the presence of arabinose,
(NH4)2S04, yeast extract, and NaCl at 0* 5m as a stabilizing
agent. McQuillen (19556, 1956) and Landman and Spiegelman
(1955) demonstrated that protoplasts of B. 7negaterium strain
KM can be induced to synthesize a (B-galactosidase. I should
now like to summarize the principal properties of this latter
system. Unfortunately, McQuillen' s findings have not as yet
appeared in extenso and so comparison of the data obtained in
the two laboratories is impossible.

Since they have already been detailed elsewhere (Landman
and Spiegelman, 1955), we need not entertain here an exten-
sive description of the conditions and stabilizing medium
which were found to permit p-galactosidase formation in

188 S. Spiegelman

protoplasts of B. megaterium. We may note, however, that,
in addition to inducer, a supply of amino acids, hexose-
diphosphate, and aerobiosis were found to be essential. As
with other synthetic functions, the properties of enzyme
formation in protoplasts and intact cells were remarkably
similar, providing the comparisons were carried out under
hypertonic conditions.

The interest in the protoplast as a possible tool in the further
analysis of enzyme synthesis stems essentially from the
possibility that it would be more amenable to specific enzyma-
tic resolution than the intact cell from which it is derived.
Fortunately, this possibility is potentially attainable, for it is
when one examines responses to various enzymes that striking
differences between protoplasts and cells begin to emerge.
This, for example, is clearly exhibited in Table I in the case

Table I

Effect of Trypsin and Lipase on Enzyme Formation in

Cells and Protoplasts

Cells or protoplasts were suspended in inducer-free induction medium
(O-SM-KgHPOi at pH 7-8; 2 % amino acids, 0-6% hexosediphosphate) and
incubated with the indicated enzyme for 1 hour, subsequent to which inducer
(0 • 06 M lactose, final concentration) was added. The enzyme formed in the next
two hours is recorded in terms of the m[j,M of o-nitrophenyl-^-D-galactoside
hydrolysed per ml. per minute.

Enzyme Present

Cells

Protoplasts

None

Trypsin (100 (Jig./ml.)

Lipase (100 jjig./ml.)

1,090
1,140
1,020

620
0
0

of the response to lipase and trypsin. Intact cells are com-
pletely insensitive to the enzymes whereas the synthetic
ability of protoplasts is completely abolished.

These results illustrate a point worthy of the attention of
those concerned with performing and interpreting experiments
with subcellular fractions. One might perhaps be led to
conjecture that a lipid is a key component of the enzyme-
forming mechanism, based simply on the observation re-
corded with lipase. However, the fact is that the loss of
enzyme-synthesizing ability is a simple consequence of the

Protein Synthesis in Protoplasts 189

physical dissolution of the protoplasts. After incubation with
either lipase or trypsin at the levels indicated, few protoplasts
can be recovered. It is thus important in any given case to
demonstrate that an inhibition of enzyme synthesis which is
observed to follow a particular treatment is not the result
of a generalized destruction. This caution is also relevant to
experiments involving ribonuclease (RNAse) and deoxy-
ribonuclease (DNAse). Lysis of protoplasts by RNAse has
been observed by Brenner (1955) and in our own laboratory.
To be interpretable, experiments of this nature must be
accompanied by evidence that the enzyme treatment has
resulted in the selective removal of the homologous compound.

An extensive examination has been made (Spiegelman and
Li, unpublished) of the effects of both RNAse and DNAse on
the synthesis of p-galactosidase in both intact cells and
protoplasts. No inhibitions were observed with intact cells
under any conditions of test. Striking effects were, however,
obtained with protoplasts. A few words may be interposed
here on the conditions necessary for consistent results. Cells
for an experiment were customarily prepared by inoculation
with 2 per cent peptone and incubation with shaking overnight
at 30°C. By morning, the cultures were in stationary phase
and were put through a "rejuvenation" prior to use. This
consisted in diluting the cultures fivefold with fresh medium
and reincubating until they had entered logarithmic growth
as determined by periodic examination of the optical density.
It was noted that extraordinary care had to be exercised in
controlling the extent of this rejuvenation if protoplast pre-
parations were to be obtained exhibiting uniform behaviour
with respect to enzyme-forming ability and response to
enzymatic resolution.

Investigation of the cells during the course of the rejuvena-
tion revealed that our procedure had inadvertently led to
extensive phasing of the culture. This finding made under-
standable the extreme precision with which the timing of the
rejuvenation had to be carried out. It also made possible the
preparation of protoplasts which responded homogeneously

190 S. Spiegelman

to the action of enzymes. These observations may be related
to the recently published experiments of Thomas (1955) who
noted a periodic variation in permeability of Pneumococci to
such large molecules as DNA and DNAse.

It was also found that consistent removal of RNA and DNA
by the corresponding enzymes can be achieved only if treat-
ment is instituted during the formation of the protoplasts.
Once protoplasts have been formed and have been incubated
for a while in the stabilizing medium, they become relatively
impervious to enzymatic resolution. It may be noted that in
addition they become more and more resistant to the dis-
ruptive effects of lipase, although never completely so. The
procedure employed for resolution may be outlined as follows.
The cells are suspended in the hypertonic medium containing
all supplements necessary for synthesis including amino acids
and HDP but lacking the inducer. Lysozyme is added at a
level of 200 {ig. per ml. to convert the cells into protoplasts.
In addition, at the same time, the enzyme to be tested for
ability to resolve the protoplasts is included. The incubation
is carried out for a period of 45 minutes at 30°C with constant
shaking, by which time the cells will have been converted
completely into protoplasts. The protoplasts are then re-
covered by centrifugation. An aliquot is removed for test of
enzyme-forming ability; the remainder is retained for chemical
analysis. Residual capacity to synthesize enzyme is examined
by suspending the treated protoplasts in hypertonic medium
containing amino acids, HDP, and inducer. The resulting
suspension is incubated on a roller-type device for 2-3 hours
with periodic sampling for enzyme assay. Controls are always
run in parallel.

Table II summarizes a typical series of experiments in
which the effect of DNAse on protoplasts is examined in
terms of the percentage removal of DNA, RNA, and the
residual enzymxC-forming capacity. It will be noted that in
some cases the treatment with DNA has led to the removal
of some RNA. The reasons for this are still under investiga-
tion. The results, in so far as enzyme-forming abilities are

PnoTEiN Synthesis in Protoplasts

191

concerned, are clear-cut. It is quite evident from the data
summarized in Table II that considerable amounts of DNA
can be removed, up to 99 per cent, without loss of enzyme-
forming capacity. However, it will be noted that in those
cases where 30 per cent or more of the RNA is lost, serious
inhibitions of enzyme-forming ability resulted.

Table II

The Efect of DNAse on Enzyme Synthesis and DNA and RNA Content

DNAse (400 [j.g./ml.) was present in the experimental flasks during proto-
plast formation (45 minutes). Protoplasts were then recovered by centrifuga-
tion and washed. An aliquot was used for determination of DNA, RNA and
protein. The extent of removal of each nucleic acid is determined in terms of
ratio to protein in the protoplast pellet and comparison with untreated control.
This corrects for loss due to lysis during treatment. Enzyme-forming ability
is examined with another ahquot of the protoplasts which is resuspended in an
induction mixture (0-5M-K2HPO4, pH 7-8; 2% amino acids, 0-6% hexose-
diphosphate, and, 0-06m lactose). Samples are removed periodically for
enzyme assay. Enzyme activity is determined in terms of the muM of o-
nitrophenyl-^-D-galactoside hydrolysed per mg. of protein per minute. The
rate of enzyme formation is obtained as the number of enzyme activity units
synthesized per mg. of protein per hour.

•

Percentage

removal

Enzyme formed

(in percentage of

Experiment

DNA

RNA

untreated controls)

IO2OC1

400

1029C,

420

IO2OC3

540

IOI9D2

104

IOI9C2

120

IOI9C3

100

718

715

719

712

Table III gives a comparable series of experiments in which
the protoplasts were treated with RNAse, and here the picture
is also clear. In most cases, there is relatively little con-
comitant loss in DNA. Again, one observes that wherever the
removal of RNA exceeds 35 per cent, drastic inhibitions of
enzyme-forming capacity results.

The data obtained in the experiments just described support

192

S. Spiegelman

Table III

The Effect of RNAse on Enzyme Synthesis and DNA and RNA Content

RNAse (500 (jig./ml.) was present in the experimental flasks during proto-
plast formation (45 minutes). Protoplasts were then recovered by centrifuga-
tion and washed. An aliquot was used for determination of DNA, RNA and
protein. The extent of removal of each nucleic acid is determined in terms of
ratio to protein in the protoplast pellet and comparison with untreated
control. This corrects for loss due to lysis during treatment. Enzyme-forming
ability is examined with another aliquot of the protoplasts which is resus-
pended in an induction mixture (0-5M-K2HPO4, pH 7-8; 2% amino acids,
0-6% hexose diphosphate, and 0 06m lactose). Samples are removed periodi-
cally for enzyme assay. Enzyme activity is determined in terms of the m^xM of
0- nitrophenyl-p-D-galactoside hydrolysed per mg. protein per minute. The
rate of enzyme formation is obtained as the number of enzyme activity units
synthesized per mg. protein per hour.

Enzyme synthe-

sized ( in per-

Percentage

removal

centage of
untreated

Experiment

DNA

RNA

controls)

1013

930B

926B

1004D

1004C

1029B

1004F

1014B

1020B2

1020B3

the conclusions derived from the study of other subcellular
systems, such as those developed by Gale and Folkes (1955a
and h) and by Zamecnik and his collaborators (Zamecnik and
Keller, 1954). They suggest that the molecular integrity of
RNA is essential for the synthesis of new protein molecules.

REFERENCES

Beljanski, M. (1954). Biochim. biophys. acta, 15, 425.

Brenner, S. (1955). Biochim. biophys. acta, 18, 531.

Brenner, S., and Stent, G. S. (1955). Biochim. biophys. acta, 17, 473.

Gale, E. F., and Folkes, J. P. (1955a). Biochem. J., 59, 661.

Gale, E. F., and Folkes, J. P. (19556). Biochem. J., 59, 675.

Hardwick, W. a., and Foster, J. W. (1952). J. gen. Physiol., 35, 907.

Protein Synthesis in Protoplasts 193

Landman, O. E., and Spiegelman, S. (1955). Proc. nat. Acad. Sci.,

Wash., 41, 698.
Lester, R. L. (1953). J. Amer. chem. Soc, 75, 5448.
McQuiLLEN, K. (1955a). Biochim. biophys. acta, 17, 382.
McQuiLLEN, K. (19556). J. gen. Microbiol., 13, iv.
McQuiLLEN, K. (1955c). Biochim. biophys. acta, 18, 458.
McQuiLLEN, K. (1956). Symp. Soc. gen. Microbiol., in press.
Salton, M. R. J. (1955). J. gen. Microbiol, 13, iv.
Salton, M. R. J., and McQuillen, K. (1955). Biochim. biophys. acta,

17, 465.
Thomas, R. (1955). Biochim. biophys. acta, 18, 467.
Weibull, C. (1953). J. Bact., 66, 688.
Wl\me, J. M., Storck, R., and Vanderwinckel, E. (1955). Biochim.

biophys. acta, 18, 353.
Zamecnik, p. C, and Keller, E. B. (1954). J. biol. Chem., 209, 337.

DISCUSSION

Davidson: There is one point I should Uke to ask in connection with
technique. How, if you are working to a matter of minutes, can you
know when to take your sample ? By the time you have got your DNA
estimation it is too late.

Spiegelman: We take our 10-minute samples blindly. The plateaus are
30 minutes long and sampling at 10-minute intervals is adequate to
exhibit them.

Gale: Is there no change in the turbidity associated with the stepwise
increase in the DNA?

Spiegelman: You do not see cycling of overall protein synthesis but
there is apparent cycling of induced enzyme formation. There is a very
interesting possibility here which I think fits in with the hypothesis
proposed by Gale, namely that the RNA templates of the induced
enzymes are unstable. They would therefore require recharging from
the nucleus, and in this event formation of such proteins would be more
closely tied to nuclear events than would constitutive protein formation.
However, it is not possible at the present time to make any statement
that such a difference is actually real. It should be noted that the
absence of cycling in the constitutively formed proteins does not mean
that none exist, because one can imagine that they are all cycling but
are out of phase with each other; and so the effect is cancelled out.
What one has to do is follow the synthesis of particular constitutive
proteins. We have been looking for such, but we haven't as yet found
any that possess all the properties that would make them suitable
material for study.

Alexander: Does the actual numerical increase in bacteria also follow a
plateau if you take them out ?

Spiegelman: No, not in this system. That is difficult to do because this
is multicellular ; each cone is not a single cell, we have had to shake them
apart to get good correspondence.

RAD. 8

194 Discussion

Alexander: Therefore it is possible that the actual number of cells may
also go stepwise.

Lajtha: Does this cycling imply that the lag-phase which we usually
see is only apparent, and that in the first few cycles the number of cells
are so few that one does not see them ? We were worried about this some
time ago and started cultures with high numbers initially, and then we
did not see any lag-phase at all. Furthermore, functionally these
bacteria in the so-called lag-phase were sensitive to very small concen-
trations of nucleic acid analogues, and to very high concentrations, about
50 times that much, in the log-phase. If we started the culture with the
same high numbers of bacteria that were present in the log-phase, we
had to use again high concentrations of analogues to achieve comparable
inhibition of growth.

Spiegelman: I don't think that all lag-phases are going to be explained
on this basis. I do, however, think that this illustrates a rather interest-
ing point. Many biologists have used an enormous amount of ingenuity
with microbial populations to get phased cultures. In actual fact it
may be difficult to avoid them. We just let the thing go to a stationary
phase and start it off again and it is phased ; but it does not stay phased
very long. We obtain four good cycles and that is about all.

Gale: How long are your inoculum cells in the stationary phase before
rejuvenation?

Spiegelman: About five hours.

Krebs: How do the protoplasts obtain energy? Do they respire?

Spiegelman: Yes, they do respire, but we give them HDP, which seems
to be the thing they like the best.

Krebs: Am I wrong in assuming that this is one of the organisms that
does not ferment anaerobically ?

Spiegelman: It is essentially an aerobic organism.

Bracket: What do you know about the relationship between this
cycling and the time of DNA synthesis ? Is there any correlation between
the stage where DNA is being synthesized and the stage where the
enzyme is being synthesized?

Spiegelman: It is very difficult to get really accurate information on
that, although we have discovered one thing which may help a lot.
You can freeze this thing in whatever stage it is by simply raising the
osmotic pressure. I don't know why this should work, but it does. It
will freeze it for a matter of hours. Our data suggest that you don't
get complete coincidence of the enzyme and DNA-synthesizing plateaus.

Bracket: Have you studied further the stimulating effect of the
removal of DNA ? It is of course rather reminiscent of what happens in
Acetabularia after removal of the nucleus.

Spiegelman: It is tempting to imagine that the removal of the DNA
actually decreases the ability of the preparation to synthesize certain
proteins. This may give added advantage to the one which is being
induced, since the inducer is present, and consequently leads to an
increase in its formation. I should like to emphasize that the experiments
I have described are not as decisive as yours, because when I say I
remove the DNA all I can mean is that I remove the DNA as measured

Discussion

195

by a chemical operation in which all the soluble components are ex-
tracted with cold perchloric acid. I should like to ask how big a piece
of DNA would be precipitated by, say, cold 0 • 2 N-perchloric acid.

Cohn: I should say from four nucleotides onwards you are running
into danger of such precipitation ; and since DNAse does leave pieces of
four, five and six nucleotides, there could be DNA polynucleotide in
such a precipitate.

Spiegelman: In that case, my conclusion is strengthened. The data
reported are based on the chemical analysis of the precipitate. It
would suggest that I don't have anything larger than four nucleotides.
It would appear then that I must have broken the DNA down into very
small pieces.

Cohn: Yes, if there is nothing in the precipitate.

Spiegelman: We do look in the precipitate. We give an enormous dose
of DNAse, in order to get the treatment through in time. It should
degrade very fast if it is going to go at all.

INFLUENCE OF RADIATION ON
DNA METABOLISM

Alma Howard

British Empire Cancer Campaign Research Unit in Radiobiology , Mount Vernon

Hospital, Northzvood

Since the discovery by Hevesy and his colleagues (for

references, see Hevesy, 1948) that the incorporation of ^^p

into DNA of Jensen sarcoma and other tissues of the rat was

markedly reduced by X-irradiation, it has been recognized

that interference with DNA synthesis is one of the most

general and important biological effects of radiation. It has

been generally supposed that ionizing radiation interrupts the

DNA biosynthetic chain by altering some reaction along its

course, and it was natural therefore to examine the possibility

that some step or steps in the biosynthesis would prove to be

especially radiosensitive, and that the effects of irradiation

might be altered by manipulation of the metabolites involved

in such steps. Hevesy (1949) showed that the uptake of

i*C-acetate into DNA of rat tissues was depressed by X-rays,

as was that of ^^P. A number of papers since have indicated

that a large part, perhaps the whole, of the biosynthetic

process is affected by radiation. It has been claimed, however,

that the incorporation of labelled adenine is not reduced by

doses that have a marked effect on the entry of formate,

glycine or orotic acid into DNA. This claim has been made

from two sources :

(1) Harrington and Lavik (1955) found that the incorpora-
tion of [8-i4C]adenine into DNA purines of rat thymus, during
the period 30 minutes to 24 hours after 100 r whole body
X-rays, was significantly greater than in controls. In the
same experiment the incorporation of [2-i*C]orotic acid
into DNA pyrimidines and of ^^C-formate into purines was

196

Influence of Radiation on DNA Metabolism 197

depressed. Bennett and Krueckel (1955) have repeated this
experiment and observed a marked depressing effect of
irradiation on the incorporation of [8-i*C]adenine, the DNA
specific activity being less than half that of controls. No
explanation is apparent for the discrepancy between this
result and that of Harrington and Lavik.

(2) Passonneau and Totter (1955) found no inhibition of
incorporation of [8-i*C]adenine into purines of DNA in
chick embryos in vitro. Doses were from 1,000 to 20,000 r of
gamma-rays. The incorporation of i*C-formate and ^*C-
glycine was reduced after doses of 5,000 r or more: 1,000 r had
a variable effect. The chick embryos, which were suspended in
saline for the two-hour period of the experiment, showed a
rate of DNA labelling with formate and glycine that was prob-
ably less than that expected from the increase in amount of
DNA in vivo in the same period. Embryos whose hearts had
stopped had the same performance as survivors of the 20,000 r
dose. Very large doses were required to have any clear effect
on DNA metabohsm, although Lavik and Buckaloo (1954)
found an approximately 50 per cent inhibition of ^^C-formate
or i*C-cytidine uptake into DNA of chick embryos after
400-450 r. These facts suggest that this experimental
system cannot be regarded as showing normal biosynthesis
of DNA. If they are accepted as doing so, it could be argued
that adenine is incorporated by exchange, since Brown (1950)
found that the renewal rates for both RNA and DNA were
higher with adenine than with ^sp or i^N-glycine. (Payne,
Kelly and Jones (1952), however, did not observe a higher
incorporation with [4:6-i4C]adenine than with i*C-formate,
[2-i*C]glycine, or ^ap).

Taking all the evidence together, it appears that there is
no very clear indication that irradiation interferes with any
particular step in DNA synthesis. The blockage seems rather
to be a general one. This suggests that the inhibition of DNA
synthesis may not be a primary effect of radiation, but is the
result of blockage in some other event in the development of
the cell.

198 Alma Howard

Effect of irradiation on DNA metabolism in some
mammalian tissues

An examination of the literature shows that very wide
differences exist between tissues in their response to irradia-
tion with regard to its effect on DNA metabolism. This is
especially clearly shown in the work of Kelly and co-workers
(1955), who measured the incorporation of ^^P into DNA of
mouse small intestine, spleen, liver, bone marrow ("carcass"),
and two transplanted tumours after X-irradiation at four
dose-levels. Informative time curves were obtained by
sacrificing animals from 2 hours to 5 days after irradiation,
the isotope being injected at fixed short times before sacrifice.
The responses of the tissues were so different, both in dose and
time-response, that no general statement can be made about
them other than that a depression was always seen, and that it
was apparent at the earliest times after irradiation that were
studied. In the small intestine, for example, the maximal
effect of 800 r was seen at 3 hours, with full recovery at about
1 day and nearly three times the normal rate of incorporation
at 2 days. Bone marrow incorporation, on the other hand,
showed a maximal effect only at about 3 days after 800 r, and
was still very low at 5 days. The different response of the two
tumours, a mammary carcinoma and a lymphosarcoma, was
especially striking. They had approximately the same growth-
rate, mitotic index, and short-term incorporation of ^^P before
irradiation. After 800 r the mammary carcinoma showed a
reduction in incorporation to about one half, but no change in
weight or histological appearance beyond a decrease in mitotic
index. The lymphosarcoma showed reduction in incorpora-.
tion to 4 per cent of normal at one day, with later apparent
recovery; there was marked involution, and a large amount
of cell death. It is very clear that there had been important
changes in the cell population, and the authors point out
that most of the effects they observed in this and other tissues
could be explained by such changes. The reduction in
amount of various phosphorus compounds, including DNA,
in rabbit bone marrow after gamma-ray doses in the mean

Influence of Radiation on DNA Metabolism 199

lethal range has been related to alterations in the number
and type of cells present (Thomson et al., 1953). The instances
cited are only two of numerous experiments in which, as well
as the changes in DNA metabolism that were being studied,
alterations in the numbers and types of cells in the tissue were
clearly being produced by the irradiation at the same time.

Radiosensitivity of DNA metabolism

In some tissues (Holmes and Mee, 1955; Harrington and
Lavik, 1955) the efPect of 100 to 150 r in reducing DNA turn-
over can be clearly seen. While these doses are much lower
than those required to alter measurably many other processes
of cell metabolism, they are well above the minimum for
causing delays in the mitotic cycle. It is natural to ask
whether two so very radiosensitive effects may not be causally
related, and Hevesy many years ago suggested that cells may
be delayed by irradiation in entering mitosis because they
have been prevented from synthesizing their normal amount of
DNA. In several tissues, however, the synthesis of DNA
appears to be completed some time before prophase begins
(Howard and Pelc, 1953; Lajtha, Oliver and Ellis, 1954) and
it is not easy to see how stoppage of DNA synthesis can be
directly responsible for delay in cells already on the brink of
prophase, and containing their full double quantity of DNA.
There seems no doubt that these cells are sensitive to delay
since, in a great many tissues that have been studied, irradi-
ation is followed very quickly by a fall in the number of cells
entering mitosis.

Hevesy also pointed out that the delay in mitosis caused by
irradiation would result in interference in DNA synthesis.
In 1945 he said: "Since the ionizing radiation blocks cell
division, it will influence the said cycle of changes [DNA
synthesis], and a reduction in the number of desoxyribose
nucleic acids built up during a given period of time can be
expected to take place." It seems important to examine the
implications of this statement in the light of knowledge of
DNA metabolism that has accumulated over the past ten

200 Alma Howard

years. It is now clear that synthesis of DNA is a function of the
mitotic cycle. This is inherent in the fact that in any given
species, a fixed amount of DNA is associated with each chromo-
some set, so that, with due allowance for differences in ploidy
and for periods of synthesis, each nucleus contains a constant
amount of DNA. This means that each cell must double, but
no more than double, its content of chromosomal DNA during
every interphase that is to be followed by a mitosis. Further-
more, the time period in interphase occupied by this synthesis
appears to be fixed for any given cell type. As far as we
know, no other component of the cell behaves in this fashion
as regards amount per cell or dependence on the mitotic
cycle, so that DNA synthesis might be expected to be unique
in its response to radiation-induced changes in that cycle.

Radiation -Induced Changes in Cell Populations

The changes in the cell population that result from irradia-
tion of growing tissues arise in the following ways :

(1) Delay in entry of cells into and progress through mitosis,
expressing itself as a shift in the proportion of cells in various
stages of the mitotic cycle. Larger doses cause longer delays.
The sensitive period for delay is just before visible prophase
(in the grasshopper neuroblast, during prophase). Recovery
after moderate doses is characterized by a temporary increase
in the number of cells in mitosis due to the release of those
delayed. In the most favourable material, the delaying
effect of 4 r can be observed (Carlson, 1948). In many other
tissues, delays are known to result from very moderate
doses. In some such cases, protein synthesis, RNA turnover,
and increase in cell volume and dry weight all appear to be
unaffected (Klein and Forssberg, 1954).

(2) Death of cells.

(a) Due to physiological or morphological changes in the
chromosomes. Such changes may result in death of cells at
metaphase or anaphase of the mitosis following irradiation or
later, usually during the following interphase, due presumably
to loss of genetic material. The rate at which cells die in this

Influence of Radiation on DNA Metabolism 201

manner depends on the rate at which they reach mitosis. The
extent of tissue damage is greater at higher doses, dose rates,
and ion density of the radiation.

(b) In a manner not known to be associated with physio-
logical or morphological changes in the chromosomes, and
not due to loss of genetic material. Cells may die upon
attempting division (Laznitski, 1943a; Oakberg, 1955); in this
case, the rate of cell death again depends on the rate at which
cells reach mitosis, as in (a). They may, on the other hand, die
during the interphase in which they were irradiated, independ-
ently of any recovery of mitotic activity. The sensitivity of
cells to this kind of interphase death is enormously varied.
Lymphocytes in the lymph nodes are rapidly destroyed by
100 r (Trowell, 1952): in mouse ascites tumour there is no
evidence of cell death after 1250 r, at least until mitosis
reappears (Klein and Forssberg, 1954); chick fibroblasts in
culture exhibit interphase death at 2,500 r (Laznitski, 19436);
and some differentiated tissues having no measurable mitotic
activity are histologically unaffected by even higher doses
(Bloom, 1948a). In this respect, the lymphocyte appears to be
very exceptional, and it seems reasonable to regard interphase
death, of a kind unrelated to mitosis, as very unlikely in most
tissues except after doses of well over 1,000 r.

Results of Changes in Cell Populations

After moderate doses of radiation, i.e. less than about
1,000 r, the shifts described would be expected to affect the
amount of DNA being synthesized in a tissue as follows :

(1) The time at which the normal supply of cells entering
synthesis is reduced will depend upon the time in the cell cycle
at which DNA is normally synthesized. If synthesis begins
immediately after telophase, irradiation will have an early
effect in reducing the number of synthesizing cells. If there is
a time lag between telophase and synthesis, the effect of
irradiation will be deferred until this time has elapsed. Fig. 1
shows the time of uptake of labelled precursors into DNA, and
the estimated lengths of other periods, in the mitotic cycles

202

Alma Howard

of three tissues which have been studied by means of auto-
radiographs. In the bean root meristem, (Fig. 1 A), there is a
Gi period of up to 12 hours; the number of cells whose DNA
becomes labelled with ^^P remains normal for at least 6 hours
after irradiation (Howard and Pelc, 1953). In the Ehrlich

TIMING OF MITOTIC CYCLES

BEAN ROOT MERISTEM
T-30

HUMAN BONE
MARROW IN

CULTURE.
T- 40-45

MOUSE EHRLICH
ASCITES TUMOUR
T»I8

Fig. 1. Mitotic cycles deduced from autoradiograph studies. Time in hours.
M = metaphase ; D = mitotic division ; S = period of uptake of isotope into
DNA; Gj and G2 = periods in early and late interphase during which DNA
does not become labelled; T = total length of mitotic cycle.

A. Bean root meristem. ^^p
(Howard and Pelc, 1953.)

B. Human bone marrow. ^^P or [8-^^C]adenine
(Lajtha, Oliver and Ellis, 1954.)

C. Mouse Ehrlich ascites tumour. [8-^*C]adenine
(Hornsey and Howard, 1956.)

mouse ascites tumour (Fig. 1 C), no G^ is observed ; within
2 hours after irradiation there is a decrease in the num-
ber of cells taking up [8-^^C]adenine into DNA (Hornsey
and Howard, unpublished). If measured biochemically, the
mitotic delay in the tumour would appear as a reduction in
DNA turnover and specific activity, compared with controls,
although the amount of DNA per cell would increase for a
time equal to Gg (see Fig. 1), and remain slightly greater than
controls until mitosis reappeared. Such effects are not

Influence of Radiation on DNA Metabolism 203

incompatible with the pubhshed results of irradiation experi-
ments in this material (Klein and Forssberg, 1954; Forssberg
and Klein, 1954), and are in agreement with some recent work
of Kelly (1955).

(2) The degree to which DNA synthesis is affected will be
determined largely by the length of S (see Fig. 1) in relation to
the time length of the premitotic block. Thus the synthesis of
DNA in a tissue will be reduced to zero if the mitotic block
(plus Gj if it exists) is longer than S. In the Ehrlich ascites
tumour, a dose of 400 r stops mitosis for 9 to 12 hours. Since
S is about 12 hours, we would not expect to observe a period
when there was no synthesis, and after 12 hours there would
be a larger than normal number of synthesizing cells, as has in
fact been observed (Hornsey and Howard, unpublished).

(3) The recovery of DNA synthesis in a tissue will depend
upon the factors discussed under (2), i.e. the degree of depres-
sion, and also on the rate of cell death, the degree of tissue
disturbance caused by it, and any other effect which the
presence of dead or dying cells may have on the metabolism
of the survivors. These last two points we know little about.
The effect on specific activity will further depend on the rate
at which dead cells are removed from the population, either
by phagocytosis, migration, or some concomitant of differen-
tiation. The rate of return of the tissue to normal will be
influenced by its normal rate of cell replacement: thus in
the small intestine, where epithelial cells normally have a life-
time of approximately 2 days (Leblond, Stevens and Bogoroch,
1948; Knowlton and Widner, 1950), the regeneration of the
epithelium is very rapid (Bloom, 1948Z?) and DNA synthesis
has recovered by 1 day (Kelly et a/., 1955). The mitotic index
in the rat intestinal mucosa recovers by 3 days after 1,000 r
(Webber, Craig and Friedman, 1951).

Conclusions

In view of these considerations, it is plain that a purely
biochemical analysis of a growing tissue containing cells at all
mitotic stages cannot tell us whether the inhibition of DNA

204 Alma Howard

synthesis is due only to changes in the cell population, or
whether we may infer that irradiation is also having a primary
biochemical effect. For this information we must look to
experiments of the following kinds :

(1) Biochemical analysis of growing tissues supplemented
by studies on mitotic delay and cell death. The few published
studies on inaterial for which such information, however
fragmentary, is at hand, are compatible with the view that
inhibition of DNA synthesis is a result of radiation-induced
changes in the mitotic cycle and in the cell population. On
the other hand, it must be recognized that there are in the
literature some experimental results which can be explained
as due entirely to cell population changes only by assuming
characteristics of the normal mitotic cycle that may appear
unlikely. Thus Hevesy (1945) observed that in Jensen rat
sarcoma, a dose between 335 and 1,500 r reduced ^^P uptake
into DNA to less than half of normal within one hour of irradi-
ation. Unless this is an interphase effect, one must suppose
that synthesis follows telophase directly and occupies less
than 2 hours.

(2) Observations on individual cells. This has been done
with autoradiographs in experiments discussed previously.
In the case of bean roots, Howard and Pelc (1953) concluded
that the most probable length of G^ was 12 hours, and there-
fore that DNA synthesis was inhibited in cells irradiated
earlier in the ^ame interphase, i.e. 6-12 hours before the
beginning of synthesis. Since, however, the length of G^ can-
not be rigorously fixed from the information available, it is
not excluded that the sensitive period for inhibition of DNA
synthesis may coincide with that for delay in mitosis. The
results of irradiating the ascites tumour (Hornsey and
Howard, unpublished) are, as already stated, those to be
expected from mitotic delay. Lajtha, Oliver and Ellis (1954)
observed an immediate effect on synthesis of DNA in human
bone marrow cells in culture. The dose used was, however, so
large (5,000 r) that interphase death is to be suspected. No
cell was observed to enter mitosis after this dose.

Influence of Radiation on DNA Metabolism 205

A second method of observing irradiation efTects on DNA
synthesis in single cells is that of photometric measurement of
the amount of Feulgen stain. Grundmann's (1953) results on
bean root meristems at 2 or 4 hours after 200 r, or 4 hours
after 800 r, suggest that the changes in DNA classes could
be explained by mitotic delay and cell death.

(3) Analysis of tissue which is synchronized with regard to
the mitotic cycle. Such a situation is approached by mam-
malian liver regenerating after gross damage such as partial
hepatectomy or CCI4 poisoning. Since DNA synthesis begins
before mitosis appears, its interruption by moderate doses of
radiation cannot be due simply to mitotic inhibition. This
tissue thus stands as one for which this simple hypothesis is
definitely untenable. Since it is to be discussed in another
paper at this meeting, no comments need be made here.

In conclusion, it appears that in regenerating liver there is
some reason to think that DNA synthesis may be specifically
interrupted by moderate doses of ionizing radiation. In other
tissues, although it is possible that this is so, much more
needs to be known about the changes in cell population that
result from irradiation before a primary biochemical inter-
ference can be established with certainty. Meanwhile, we
must recognize that most experimental results can be ex-
plained as due simply to mitotic delay and cell death, and do
not require us to invoke a biochemical action of radiation on
DNA synthesis jper se.

REFERENCES

Bennett, E. L., and Krueckel, B. J. (1955). Univ. of Calif. Radn.

Lab. Reports 2827-2828.
Bloom, W., ed. (1948a). Histopathology of Irradiation from External

and Internal Sources, Chaps. 11, 18, 19. New York: McGraw Hill.
Bloom, W., ed. (19486). Histopathology of Irradiation from External

and Internal Sources, Chap. 10. New York: McGraw Hill.
Brown, G. B. (1950). Fed. Proc, 9, 517.

Carlson, J. G. (1948). J. cell. comp. Physiol., 35, Suppl. 1, 89.
FoRSSBERG, A., and Klein, G. (1954). Exp. Cell. Res., 7, 480.
Grundmann, E. (1953). Zweites Freiburger Symp. iiber Grundlagen

und Praxis chemischer Tumorbehandelung, p. 187.

206 Alma Howard

Harrington, H., and Lavik, P. S. (1955). Arch. Biochem. Biophys.

54, 6.
Hevesy, G. (1945). Rev. Mod. Phys., 17, 102.
Hevesy, G. (1948). Radioactive Indicators. New York and London:

Interscience Publishers.
Hevesy, G. (1949). Nature, Lond., 163, 869.
Holmes, B. E., and Mee, L. K. (1955). Radiobiology Symposium 1954.

London: Butterworth.
HoRNSEY, S., and Howard, A. (1956). Ann. N.Y. Acad. Sci., 63, 915.
Howard, A., and Pelc, S. R. (1953). Heredity, 6, SuppL, 261.
Kelly, L. S. (1955). Univ. of Calif. Radn. Lab. Report 3268, p. 50.
Kelly, L. S., Hirsch, J. D., Beach, G., and Payne, A. H. (1955).

Radiation Res., 2, 490.
Klein, G., and Forssberg, A. (1954). Exp. Cell. Res., 6, 211.
Knowlton, N. p., and Widner, W. R. (1950). Cancer Res., 10, 59.
Lajtha, L. G., Oliver, R., and Ellis, F. (1954). Brit. J. Cancer, 8, 367.
Lavik, P. S., and Buckaloo, G. W. (1954). Radiation Res., Abstracts, 1,

221.
Laznitski, I. (1943a). Brit. J. Radiol., 16, 61.
Laznitski, I. (19436). Brit. J. Radiol., 16, 138.
Leblond, C. p., Stevens, G. E., and Bogoroch, R. (1948). Science, 108,

531.
Oakberg, E. (1955). Radiation Res., 2, 369.

Passonneau, J. v., and Totter, R. R. (1955). Radiation Res., 3, 304.
Payne, A. H., Kelly, L. S., and Jones, H. B. (1952). Cancer Res., 12,

666.
Thomson, J. F., Tourtellotte, W. W., Carttar, M. S., and Storer.

J. B. (1953). Arch. Biochem. Biophys., 42, 185.
Trowell, O. a. (1952). J. Path. Bad., 64, 687.
Webber, B., Craig, B. R., and Friedman, N. B. (1951). Cancer, 4,

1250.

DISCUSSION

Hollaender: The work of Dr. Gaulden which Dr. Howard referred to
was done on the grasshopper neuroblast. Dr. Gaulden has shown that
one can counteract the effects of radiation on the rate of mitosis by
placing the neuroblast in a hypertonic salt solution immediately after
irradiation (see p. 303).

Van Bekkum : How long does the irradiation take ?

Hollaender : It takes about 1 minute.

Alexander: Could you tell us the experimental details of this treat-
ment with hypertonic salt?

Hollaender: It contains 1 -2 times the concentrations of inorganic salts
in the medium isotonic to the grasshopper neuroblast. In other words,
it is only slightly hypertonic to the cells.

Alexander: Could you tell us the time of immersion in the more con-
centrated salt solution?

Hollaender: Throughout observations (264 minutes).

Discussion 207

Spiegelman : Why was that tried ?

Hollaender: There were several reasons: (1) Harrington and Koza
(1951, Biol. Bull., 101, 138) working with grasshopper neuroblasts,
found that the cells swelled almost immediately after treatment with
100 r or more of X-rays. This suggested a radiation-induced change in
osmotic-pressure relationships in the cells. The cells looked as they
would do had they been placed in medium hypotonic to them.

(2) The radiation-induced "reversion" of middle and late prophase
neuroblast chromatin to an interphase condition, a primary cause of
mitotic inhibition in this cell, resembles the "disappearance" of chromo-
somes produced when cells are placed in solutions hypotonic to them.

(3) Gaulden found that the chromatin of telophase, interphase and
early and middle prophase cells could be made to resemble chromatin of
late prophase by placing the cells in culture medium hypertonic to them.
This change occurred within seconds and was accompanied by an
accelerated mitotic rate (Gaulden, M. E. (1956), "Visible characteristics
of living interphase and mitotic chromatin in the grasshopper neuroblast
and the effects of abnormal toxicity on them." In manuscript).

These observations together with those of Sugiura (1937, Radiology,
29, 352) who found growth capacity of irradiated tumour fragments to
be increased when placed in hypertonic solutions, led Gaulden to test
the efficacy of hypertonic medium in counteracting radiation- induced
reversion of chromosomes, which results in mitotic inhibition.

Lajtha: Dr. Howard mentioned the possible interphase killing effect of
5000 r, which I think is a very important point. It undoubtedly kills
some cells, you can see them dying in certain cultures. However, the
numbers are relatively low. We repeated the experiments with 1000 r
and I think we have indication for an interphase effect. The G^ period is
very long, or relatively long in the bone marrow cells, of the order of 20
hours or more. If, therefore, the cells would be damaged only during
mitosis, then for a considerable time afterwards undamaged G^ cells
would enter and go through their synthetic period making the normal
amount of DNA. We find on the other hand that even after 1000 r all
the cells which enter the synthetic period produce only a fraction of the
normal amount of DNA. The grain counts instead of the normal 60-80
are of the order of 10 or less, and we don't see any appreciable number of
dying cells after 1000 r. This rather suggests that the whole G^ period is
damaged by radiation. The one difficulty is that 1000 r has a direct
effect on the synthetic period as well, i.e. it will stop DNA synthesis then
and there. I think we must repeat these experiments with 300 r or less,
as you did with 150 r.

Howard: These effects ought to be separable since the dose effect is so
widely separated. One can have a big mitotic delay with 100 r.

Lajtha: A dose of 150 r did not inhibit the synthetic period in the bone
marrow cells, just as in your experiments with bean roots it did not
inhibit the S period.

Swanson : May I ask your opinion about this first effect of radiation
in terms of DNAse, i.e. where the chromosomes become sticky; is there
any clue as to what is actually happening there ?

208 Discussion

Howard: It has been proposed that this was due to the depolymeri-
zation of DNA on the chromosomes. I don't think there is any proof for
that or perhaps any disproof either. I don't know what physical
chemists think about this idea, but the stickiness can be produced by
very low doses compared with what is necessary to depolymerize DNA
in most experiments in the test-tube. There has been no clear histo-
chemical evidence that there is a change in polymerization of DNA in
cells.

Swanson: However, there is a pronounced oxygen effect here.

de Hevesy: You and Dr. Pelc have stated that 35 r has an effect.

Howard: Yes, we got a reduction in the number of cells synthesizing
DNA in a 12-hour period, certainly after a dose of 50 r. One can see a
maximal effect there. But we know that this dose has a big effect also
on the division, and while we have not done the appropriate timing
experiment after 35 or 50 r, it seems quite possible that delays in
division would explain that effect also.

Latarjet: I should like to add in answer to Swanson's question that
Dr. Ephrussi-Taylor and I have some data on a purified DNA, according
to which its inactivation by X-rays acting mainly through direct effect
is not influenced by the presence of oxygen. In these experiments,
protection against indirect effect was secured by 10 per cent yeast
extract. The protection within the cell cytoplasm is certainly higher.
Therefore, if we consider those lesions, such as chromosome breaks,
which are oxygen-sensitive, we may say either (a) that they do not
result from a primary effect of the radiation on DNA ; or (b) that oxygen
does not act at the level of a primary radiochemical change on DNA.

Swanson: This would be a metabolic event of some sort, and fits in
with what we believe.

Howard: It is certain that in some cells, at any rate, division of the
nucleus does not always determine division of the cell. These two things
are separable in many cells, and perhaps the synthesis of DNA is also
separable from the division of the nucleus.

Lajtha: I think that DNA synthesis and division are clearly separable.
One can inhibit mitosis with colchicine and certain concentrations of
heparin, and neither of them will inhibit DNA synthesis. The result will
be polyploid cells and arrest of metaphase.

Spiegelman: I would like to suggest that one of the most useful
systems that might be employed to study this phenomenon is a phased
thymine-less mutant where you can control DNA synthesis, nuclear
division, and examine for sensitivity. Here, many of the important
parameters would be more or less under fairly precise control. In point
of fact you can quite easily phase a thymine-less nucleus by controlling
the thymine.

Alper: Dr. Howard, would you be prepared to apply the same
reasoning to u.v. effects?

Howard: I understand that DNA synthesis can be immediately and
finally stopped by u.v. irradiation, and in this respect it seems to act
rather differently from ionizing radiation. Perhaps the reason is that the
nucleic acid itself has such a high absorption.

Discussion 209

Alper: It is known from Stapleton's work that the sensitivity of
bacteria is quite different if you irradiate them in the stationary phase
before they have started synthesizing anything at all, and just at the
end of that when they are about to go into the log-phase. It certainly is
tempting to feel that somehow this lack of sensitivity is due to the fact
that those about to enter the log-phase have already got their DNA
synthesized.

Hollaender : However, the story is different with u.v., where you have
the opposite effect to that obtained with X-rays. You have a very high
sensitivity immediately before they go into the log-phase. This may be a
question of absorption which has never been determined.

Alper: I have found that if you take bacteria in the stationary phase
and irradiate them, you get prolongation of the lag-phase, but if you
irradiate the bacteria which are just about to go into log-phase and plot
the growth curve after that, you get an increased lag-phase, and then
you get the catching-up effect which I mentioned and very much less
cell death.

Gale: May I ask if those cells are really not growing or are they just
producing morphologically odd forms ?

Alper: The experiments I have just mentioned were all done on viable
counts. But I have also been doing some morphological work and get
quite distinct dose-dependence curves, whether I am looking at the
added lag, at the number of long forms produced, or the number of long
forms that will go on and produce colonies.

Bracket: Dr. Howard, are you completely satisfied that ^^p^ or any
other precursor you are using in the autographic method, is really an
indicator of the time of DNA synthesis ? Can you rule out any turnover
of DNA? There is also the problem of the constancy of the DNA
content of the nucleus ; I am quite willing to think that it is approxi-
mately constant. I am willing, also, to think that DNA is relatively
stable, but I am not absolutely convinced that DNA is completely inert,
and that it is always entirely constant.

Howard: In answer to your first question, I think that from autoradio-
graphic work which Dr. Pelc and I did with the bean root, the period
of uptake of ^^p into DNA is reasonably in agreement with the period
during which the DNA is increased. But as regards the adenine labelling
in the ascites tumour the situation is much less clear, and there is a good
possibility there that the time at which the DNA is labelled with
adenine does not coincide with what one can observe biochemically, i.e.
an increased amount of DNA in the cell. This is still an open question,
because the biochemical results seem to be in serious conflict with each
other.

With regard to the constancy of DNA per chromosome set (I think we
should say that, rather than per nucleus), there seem to be a few
exceptional cases in which too much or too little DNA is found, to be
consistent with this theory. But the exceptions are rather few, and I
feel fairly satisfied that it is a general rule that the chromosome carries
an amount of chromosomal DNA which is fixed for that chromosome.
It would take a good deal more evidence than now exists to overthrow

210 Discussion

that idea. Some cells may be producing DNA as a sort of secretion
product, and in that case one might find more DNA or perhaps DNA
of a different kind, or a different degree of polymerization, in such cells ;
but these would also be exceptional cases.

Bracket: It appears that some workers do not quite agree with this
view, which is held by most American workers. Dr. Fautrez came to the
conclusion that, with the same apparatus, he can get different results
under different physiological conditions. .

Howard: I know of Fautrez' work, and I agree entirely that one should
not be dogmatic, but still I think it is up to him to prove his point,
because at least in some of his work insufficient allowance has been made
for synthesis due to preparation for mitosis. One has to have a pretty
complete knowledge of the changes in cell population that are going on
over a period of time to exclude this reason for different DNA values,
and this has not been sufficiently allowed for.

Davidson: I think it is true that in those cases where there are devia-
tions from what one might call the Boivin-Vendrely rule, if I may use
the term, the cells have been put under quite abnormal conditions,
and that if you stick to physiological conditions the rule does follow
fairly well. It is impossible to generalize completely, but on the whole I
think the amount of DNA per chromosome set remains unchanged under
ordinary physiological conditions.

There is one point I would like to make in relation to Prof. Brachet's
earlier remark about ^^P incorporation into DNA. We have recently
been doing a lot of work on incorporation of various precursors into
ascites cells in vitro, and the situation there is that under the conditions
employed there is excellent incorporation of ^^p jnto DNA, good
incorporation of [8-^*C]adenine, but next to no incorporation of labelled
formate or labelled glycine; this suggests that purine synthesis just does
not occur, and presumably DNA synthesis does not occur to any
appreciable extent either, although it is very difficult to measure the
total amount of DNA because the increase one would expect would be so
small as to be within the experimental error of the estimation. There is
no doubt whatever that ^^p jg in fact incorporated into the DNA,
because we have degraded the DNA to the individual deoxy nucleotides
and separated them and found incorporation into each individual
nucleotide, just as into the whole DNA.

Spiegelman: Some very careful experiments have been done recently
by Siminovitch, using cultures of bacteria as well as tissue, in an attempt
to detect such a turnover, and he finds none.

Gray: Prof. Davidson, I infer from what you say that you thought
that there was no cell growth going on in your in vitro preparations. Is
this the point of your remark ?

Davidson: It would appear that there is no de novo purine synthesis
going on, as indicated by lack of incorporation of formate and lack of
incorporation of glycine. There is good incorporation of formate into the
thymine of the DNA, as Totter found with marrow cells in vitro (Totter,
J. R. (1955), J. Amer. chem. Soc, 76, 2196). Incorporation into the
methyl group of thymine is excellent.

Discussion 211

Holmes: Could it be that you have got enough adenine present and that
the cells are simply using that ; some cells use it preferentially, and don't
synthesize de novol

Davidson: We consider this unlikely since the pool of acid-soluble
adenine compounds in the ascites cells seems to be small. Indeed we are
inclined to regard these cells as parasites on the purine-synthesizing
mechanisms of their hosts. They can, of course, utilize intact purines ;
that does happen.

Lajtha: It must happen, because we gave some aminopterin in low
concentrations to cultures, and this prevents the i*C-formate incorpora-
tion into thymine, but it did not prevent the i*C-adenine incorporation
into DNA. Now since we were not prepared to believe that these cells
synthesize a thymine-less DNA, we thought that there must be a pool of
thymine and that there must be a pool of adenine as well, so that if we
prevent the incorporation of labelled formate the cells can still use their
preformed pool substances.

Davidson: There is one interesting point here, and that is that in the
ascites cells which normally do not incorporate formate in vitro to any
appreciable extent into the DNA purines, the addition of a particle-free
saline extract of liver cytoplasm will very markedly stimulate the incor-
poration of formate into the purines of both RNA and DNA.

Howard: We all agree that the usefulness of the autoradiographic
method depends on biochemical analysis and biochemical identification
of the compounds, and this is not a very easy matter on which organic
chemists can at once agree. On the other hand, the autoradiographic
method is the only tracer method of looking at individual cells and this
seems to be a very important thing to do. Therefore, the autoradio-
grapher is in the biochemist's hands for advice on the identity of the
compound.

Alexander: Is there sufficient data to make a clear distinction between
interphase cell death and cell death following division? Can the possi-
bility be completely excluded that cell death occurs on average at a time
after irradiation which depends on the size of the dose ?

Howard: We can be fairly certain that there are these two kinds of cell
death.

Lajtha: With regard to Prof. Brachet's remark about the possibility
of exchange, we calculated the number of molecules getting into the cell,
into DNA, and both with i'*C-adenine and with i*C-formate we got
identical numbers as far as the technique allows: of the order of 20
million per cell DNA. Since this is valid both for thymine and for
adenine, if that had been exchange that would imply exchange of 20
million adenine-thymine pairs, and I find it very difficult to believe that
such an extent of exchange can happen if we accept the helical structure
of DNA.

THE INFLUENCE OF RADIATION ON THE
METABOLISM OF ASCITES TUMOUR CELLS*

Arne Forssberg

Institute of Radiophysics , Stockholm

For some years we have been concerned with studies on
effects of X-rays on Ehrhch ascites tumours. Some of that
work is inchided in the present survey.

When an intraperitoneally growing Ehrhch ascites tumour
in a stage of rapid growth is irradiated in vivo with a dose of
1250 r, comparatively moderate cell lesions arise (Klein and
forssberg, 1954). From analyses during a 48-hour observa-
tion period following that dose it appears that the number of
tumour cells is not significantly increased nor does the per-
centage of non-tumourous exudate cells increase. The first
property is due to the fact that cell division is completely
inhibited and that the slow reappearance of mitotic activity
which occurs some 20 hours after irradiation does not ap-
preciably increase the total number of tumour cells in the
sample. The second property is at variance with the findings
when doses of the order of LD50 or thereabouts are adminis-
tered. A relative increase in exudate cells is inter alia pro-
voked by the presence of disintegrating tumour cells. The
constant cellular composition of the sample is a prerequisite
for the present series of investigations because it enables
biochemical studies to be made on a cell population at various
times in the interval 0-48 hours after irradiation, under well
defined conditions. Furthermore, judging from the method of
supravital staining as well as from biochemical properties,
irradiation does not significantly increase the number of dead
cells. Although the cellular composition is fairly constant,
cytological changes do occur. Mitosis disappears during the

* Review based on work in co-operation with Drs. G. Klein and L. R6vesz,
Institute of Cell Research, Karolinska Institutet, Stockholm.

212

Radiation and Ascites Tumour Metabolism 213

first two hours following irradiation, and renewed cell division
which appears at 20 hours must be preceded by a period of
preparation well before that time.

Determinations of DNA, calculated per single cell, indicated
a slight increase only during the 48-hour postirradiation
period, whereas total N and RNA increased considerably.
Almost as a consequence of the rather unimpaired synthesis
of the cellular constituents (with the exception of DNA), the
average cell volume also showed a progressive increase. It is
interesting to compare the rate of cellular enlargement and
synthesis of N and RNA of the irradiated cells with the rate of
cell multiplication in the non-irradiated tumour. It appears
that all these measures are the same within the hmits of
error of determination. In other words, this impUes that the
production of cell mass in an irradiated sample occurs through
an increase in the mass of single cells while in the non-
irradiated population the cell mass production during the
same period is due to division, producing cells of ordinary size.

A survey of the current literature reveals a number of
difficulties with regard to the quantitative interpretation of
radiation effects when doses of the order of LD50 are given.
An example of this is provided by growth rate studies on Ehrl-
ich ascites cells irradiated in vitro : when mice were inoculated
with the irradiated cells and the cell multiplication assayed
(Revesz, 1955), results were obtained which suggested that
decay products from X-ray-killed and lysed cells may serve
the survivors as an additional substrate, thus enhancing the
growth rate. Furthermore, growth rate studies of artificial
mixtures of X-ray-killed and living cells gave similar results.
To arrive at more precise information on the effects of lethal
doses in the present case, model experiments were designed
and preliminary results may be mentioned here. The stimula-
tion to enhanced growth at certain dose levels could be due to
a general increase in the pool of metabolites, arising from the
disintegration of dead cells; alternatively some particular pro-
ducts may be more effective in this respect. For several reasons,
nucleic acids are the most interesting in this connection.

214 Arne Forssberg

When DNA suspensions, isolated from Ehrlich ascites and
purified from proteins, were injected intraperitoneally in
quantities of about 0 • 5 mg. in Ehrlich ascites of mice which
had been irradiated in the manner described, mitotic activity
reappeared in the tumour cell population somewhat earlier
and the number of cells in mitosis was higher than in samples
from animals treated by irradiation and saline injection only;
whether the DNA injection was given 2 hours before or 2
hours after irradiation, the effect was approximately the same.
In similar experiments with DNA isolated from calf thymus or
mouse liver no stimulating effect has so far been observed.

Since the ascites fluid contains DNAse, injected DNA can
be expected to be enzymatically degraded at a rapid rate,
and a species specific character of the DNA should be lost.
Gale (1955) has shown that incorporation of amino acids in
Staphylococcus aureus is inhibited in cells disrupted with
ultrasonic treatment and deprived of their nucleic acids, but
that this faculty can be restored by adding either homologous
DNA or RNA. If the nucleic acids were enzymatically de-
graded, reactivation of amino acid incorporation took place
even after addition of heterologous nucleic acid degradation
products, e.g. from yeast. In view of these results, the mechan-
ism of the DNA effect in our experiments is so far obscure.

One would expect the stimulation caused by Ehrlich DNA
to be rather transient as the depot is probably used up within
a short time, in contrast to what happens when X-ray-killed
cells are mixed with living cells. It can be assumed that the
lysis of dead cells is protracted over a fairly long period and the
material furnished — not only DNA but also other cellular con-
stituents— is available during correspondingly longer intervals
of time.

It can be readily demonstrated by isotope-labelhng methods
that DNA from X-ray-killed cells, or at least important parts
of the DNA molecule, can be transferred to living cells of the
same sample during growth. We used cells which were labelled
in vivo with i*C-adenine, and harvested the cells some days
after injection when 97 per cent or more of the adenine had

Radiation and Ascites Tumour Metabolism 215

been incorporated into DNA and RNA. ^*C-labelled cells
were X-ray-killed and mixed in various proportions with
imlabelled living cells, and mice were inoculated with these
mixtures. Analyses made on the fifth day after inoculation,
when control experiments showed that all X-ray-killed cells
were completely lysed, showed consistently high incorporation
of activity into the living cells, e.g. when a mixture with a
ratio of living : dead cells =1:1 was used, about 40 per cent
of the activity was incorporated into the living cells, and the
activitv was found to be distributed between DNA and RNA
in the same proportions as in the inoculated sample.

This transfer of activity and, thus, of metabolites from dead
to living cells is not unexpected but, nevertheless, it does not
seem to have received much consideration in radiobiological
work. The extent to which, and in particular, how soon after
administration of the dose such a transfer takes place, is still
uncertain. It would seem, however, that in studies with labelled
compounds, carried out several days after administration of
an LD50 dose, such secondary reactions cannot be ruled out.

In recent years, considerable work has been devoted to the
question of whether compounds like DNA, RNA and proteins
are metabolically stable and do not undergo concentration
changes during cell division and growth. Several observations
indicate that DNA from unicellular organisms is equally dis-
tributed between the daughter cells by mitosis without
previous degradation and that no replacement occurs in the
resting cells; this seems to be the case e.g. in bacteria (Her-
shey, 1954). In higher organisms the stability of DNA is
more controversial (Hevesy, 1948; Stevens, Daoust and
Leblond, 1953; Barnum, Huseby and Vermund, 1953), but
the evidence of some investigators favours this concept
(Barton, 1954; Fujisawa and Sibatani, 1954). The stability
of RNA as well as of DNA was advocated by Nygaard
(Nygaard and Rusch, 1955) from experiments on regenerating
liver. The proteins of ^-galactosidase from Esch. coli were
found by Hogness, Cohn and Monod (1955) to be static.
Evidence to the contrary has been put forward even in the

216

Arne Forssberg

case of RNA and proteins. Since the total activity of Land-
schiitz ascites cells, labelled with [2-i4C]glycine, is diluted by
cell multiplication more than could be expected from increase
ill cell mass, Greenlees and LePage (1955), for instance,
deduced an exchange (loss) of protein-bound activity amount-
ing to about 9 per cent per day. Using the same material.

O UJ

q:/^100-

UJ O
CO o

> 50-

-J >

-J —

UJ H-

-:^

UJ U

^ UJ

-J

UJ Lk 1

oco '

-6

TUMOR CELL NUMBER- V-

P DNA^ O
SPECIFIC ACTIVITY 0F< RNA

-o

• •

PROTEIN: A-

-5?

1-4 =

4 5 6

DAYS AFTER INOCULATION

Fig. 1. Total number of free tumour cells and inverted specific
activity of DNA, RNA and proteins as a function of time after
inoculation. All values were brought to a common scale (left)
by taking the values corresponding to the inoculum as equal to
one. Curves are drawn through the geometrical mean of the
values from the different series, each value representing a pool
of ascites from 4 to 15 mice. The right-hand scale shows the

number of generations.

Ledoux and Revell (1955) made the observation that the RNA
concentration per cell decreased considerably with the age of
the inoculated cell culture.

Ehrlich tumour cells, with [8-i^C]adenine or [2-'^^C]g[ycme
as precursors, were used in our studies on similar stability
problems (Revesz, Forssberg and Klein, unpublished). In
parallel analyses of specific activity and cell multiplication
during the week following inoculation of the cells labelled
in vivo, we obtained the results shown in Fig. 1. There is a

Radiation and Ascites Tumour Metabolism 217

close agreement between the growth curve and the dilution
of DNA activity. The corresponding data for RNA and
proteins show appreciable deviations from expectation
assuming stability. However, an important difference is that
in the case of proteins a progressive loss of activity through

PROTEIN

100-

zi-

Oll.

GLYCINE I:x
GLYCINE n: A

-50

cc
a.

T 1 \ 1 T 1 T-

12 3 4 5 6 7

DAYS AFTER INOCULATION

Fig. 2. Products of total number of free tumour cells and specific

activity of the protein fraction. The initial value is taken as

equal to 100. Two series of experiments.

exchange and release from the once incorporated i*C-glycme
occurs (Fig. 2), as compared to an initial deviation only in the
RNA curve taking place between the collection of labelled
cells and the second day of growth of the inoculate. The
continuous loss of protein-bound ^^C is in agreement with the
observation of Greenlees. The activity released from cell
proteins is, however, not in a chemical form which can be
used for further synthesis of nucleic acids. We are led to this

218

Arne Forssberg

conclusion by the fact that the specific activity curves of RNA
and DNA agree in the series labelled with ^^C-glycine and ^^C-
adenine. In the latter case no marking of the proteins or the
tissues of the host takes place.

The ratio of the specific activities of RNA : DNA is compara-
tively constant during the whole period of observation of the
transferred inoculates, and the mean value of 0 • 72 is seemingly
independent of whether adenine or glycine is used as precursor
(Table I).

Table I

Ratios of the Specific Activities of RNA : DNA in the Inoculum and at

Various Times after Inoculation

Inoculum

Days
after
inocu-
lation

Glycine I

Glycine II

Adenine I

Adenine II

Glycine I
Glycine II
Adenine I
Adenine II

111
0-97
0-95
101

2
3

4
5

0-60
0-81

0-79
0-87
0-73

0-69
0-69

0-80
0-91

0-56
0-58
0-64
0-59
0-52
0-60

0-72
0-69
0-93
0-78
0-81

Mean 1 • 01

0-75

Mean: 0-76 0-77 0-58 0-78

Mean and standard error of all four experiments :

0-72 =0- 025

Furthermore, in the inoculum sample at zero day the specific
activity ratio is 1, and earlier still, i.e. shortly after injection
of the labelled compound, values ranging from 1 • 5-4 are
found.

This decrease in the RNA: DNA specific activity ratio and
the eventual attainment of a steady state calls to mind the
similar results of Ledoux and Revell which, however, were
arrived at from determinations of the total amounts per cell
in the related Landschiitz tumour. Assuming that similar
conditions prevail in the Ehrlich cell, the initial loss in RNA

Radiation and Ascites Tumour Metabolism 219

activity (Fig. 1) might be due to a loss of highly active RNA
molecules.

During the first two hours after irradiation, the incorpora-
tion of [2-^*C]glycine into both DNA and RNA was depressed
to the same degree, averaging about 70 per cent of that of the
non-irradiated tumour cells in our experiments. This was
established through analyses in which glycine was injected
immediately after radiation and the tumour cells were
assayed at various times in the interval 0-120 minutes
(Forssberg and Klein, 1954). Intraperitoneally injected
glycine is very rapidly taken up by the cells. Five minutes
after injection, the uptake was found to be the same in irradi-
ated and control cells ; the decreased incorporation of glycine
in the nucleic acids is therefore not caused by any changes in
permeability.

On comparison of the purely chemical analysis of RNA and
DNA with isotope determinations, it appears that total RNA
synthesis proceeds roughly linearly with the production of cell
mass during the 48-hour period, whereas isotope incorpora-
tion, as stated, is reduced to 70 per cent of the normal value
during the first two hours. DNA incorporation of ^^C is also
reduced to the 70 per cent level as contrasted to the increase
in the total amount of DNA which proceeds at an average of
33 per cent of that of the unirradiated controls.

In the amitotic period which follows (time interval 2-20
hours), marked changes in the incorporation rate of both
RNA and DNA take place and specific activity values equal
to or even higher than those of the controls occur. These
results were obtained in experiments where i*C-glycine was
injected over a period of two hours at different times during
the amitotic period. As far as incorpt)ration rate is equivalent
to synthesis, this ought to imply that the initial depression of
synthesis is followed by a period of increased synthesis.

In the case of RNA, the increased incorporation rate
subsequent to the initial depression shifts the balance, so
that the isotope measurement can be made compatible with
the finding that the total RNA is synthesized for the most

220

Arne Forssberg

part at a normal rate. The fact that isotope incorporation
into DNA during the same period is somewhat increased as
compared with the initial rate, and occasionally also indicates
activities higher than those of the controls, still more em-
phasizes the discrepancy between total determinations and
isotope measurements. Similar indications of an overcom-
pensation in the incorporation of isotopes into DNA have

ACETO ACETIC ACID = •
CITRIC " ^

LACTIC " o

^ PYRUVIC • %

in 180

ai6Qj CELLS

^140

It) -

^120

Fig. 3. Changes in the cellular concentration of acetoacetic, citric,
lactic and pyruvic acid in Ehrlich ascites cells. Dose 1250 r. The
concentration in non-irradiated cells is taken as equal to 100 (left).
Corresponding changes in the ascites fluid ("supernatant", right).
Determinations during the first two hours after irradiation.

been reported also when lethal doses were administered (Kelly
et al., 1955).

Protein synthesis in our material is never appreciably
influenced by irradiation, although isotope measurements
indicate a slight depression of synthesis during the first hours
after irradiation, but a slight increase at approximately the
same time as the measurements indicates that DNA and RNA
activity is high. A period of general recovery is indicated by
all these findings. Simultaneously also, the metabolic changes
discussed below (Fig. 3) seem to level out.

When the mitotic activity is brought to a stop, a number of

Radiation and Ascites Tumour Metabolism 221

cellular reactions other than those already mentioned are
proceeding at an irregular rate; e.g. studies in progress of the
intermediate carbohydrate metabolism indicate reversible
changes in the concentration of various acids (Fig. 3). It
appears as if the citric acid consumption is blocked for a short
period of time, leading to increased cellular concentration.
If so, the blocking is a temporary one, as indicated by the slope
of the citric acid curve as well as by occasional analyses which
showed almost normal citric acid values some hours later.
The changes in acetoacetic acid concentration during these
first two hours after irradiation proceed in a similar fashion.

Simultaneously, the concentration of pyruvic and lactic
acid decreases. This may be due in part to a blocking of
carbohydrate metabolism. Determinations in the ascites
fluid (Fig. 3, ''supernatant") demonstrate that the latter
acids are also partly released from the cells into the peritoneal
fluid and thence from the entire ascites. Similarly, also, the
fluid from irradiated samples is richer in acetoacetic and
citric acid (values for the latter are not included in the curve).
This leakage renders quantitative determination in the cells
rather difficult. Two to four hours after administration of the
dose, i^C-labelled lactic acid was found to be incorporated into
liver glycogen ; thus, at least part of the lactic acid is stored in
the irradiated liver. Increased incorporation of ^*C from
labelled glucose into the liver glycogen from ordinary irradi-
ated mice has been reported by Lourau (1955).

As a result of in vivo irradiation the host animals also
receive the same dose as the ascites cells. It is of interest to
note that neither the intestines nor the liver show any signifi-
cant deviation from their normal state with respect to the
concentration of acetoacetic, lactic ^nd pyruvic acid during
this period of observation. Citric acid determinations were
highly variable but, on an average, were slightly increased in
the irradiated liver. Changes in the citric acid metabolism
have been reported by DuBois, Cochran and Douall (1951),
but on animals which had been both fluoroacetate poisoned and
irradiated.

222 Arne Forssberg

REFERENCES

Barnum, C. p., Huseby, R. A., and Vermund, H. (1953). Cancer Res.,

13, 880.
Barton, A. D. (1954). Fed. Proc., 13, 422.
Dubois, K. P., Cochran, K. W., and Douall, J. (1951). Proc. Soc.

exp. Biol., N.Y., 76, 422.
Forssberg, A., and Klein, G. (1954). Exp. Cell Res., 7, 480.
FuJiSAWA, Y., and Sibatani, A. (1954). Experieniia, 10, 178.
Gale, E. F. (1955). Ill Int. Congr. Biochem. p. 71, Liege: Vaillant-

Carmanne.
Greenlees, J., and LePage, G. A. (1955). Cancer Res., 15, 256.
Hershey, a. D. (1954). J. gen. Physiol, 38, 145.
Hevesy, G. (1948). Advances in Biological and Medical Physics, 1, 409,

New York : Academic Press.
Hogness, D. S., Cohn, M., and Monod, J. (1955). Biochim. hiophys.

acta, 16, 99.
Kelly, L. S., Hirsch, J. D., Beach, G., and Payne, A. H. (1955).

Radiation Res., 2, 490.
Klein, G., and Forssberg, A. (1954). Exp. Cell Res., 6, 211.
Ledoux, L., and Revell, S. H. (1955). Biochim. hiophys. acta, 18, 416.
LouRAU, M. (1955). Radiobiology Symposium, p. 225, London: Butter-
worth.
Nygaard, O., and Rusch, H. P. (1955). Cancer Res., 15, 240.
Revesz, L. (1955). J. nat. Cancer Inst., 15, 1691.
Stevens, C. E., Daoust, R., and Leblond, C. P. (1953). J. biol. Chem.,

202, 177.

DISCUSSION

Poj)jak: I would like to make some comments regarding the rate of
metabolism of ascites tumour cells, and the deductions one might make
when measuring metabolic events in vivo compared to in vitro. Prof.
Davidson mentioned that in vitro he gets excellent labelling with ^ap or
with adenine, but no evidence for purine synthesis. In vivo, on the other
hand, you get labelling from glycine and from formate. I feel that the
in vivo labelling from glycine and from formate may not necessarily
mean that purine has been synthesized within the ascites cell. We
thought the ascites cell was a very convenient preparation for measuring
certain problems of fat metabolism. We carried out some in vitro
incubations, and found only minute traces of synthetic ability of these
ascites tumour cells, e.g. in vitro they cannot synthesize fatty acids
from acetate under conditions of, say, liver slices or mammary gland
slices ; that relates to your figures for acetoacetic acid. Dr. Forssberg.
They can hardly oxidize acetate to CO 2, from which I must assume that
the citric acid cycle is working at a very poor rate indeed. I wonder
whether some of the in vivo incorporation data that one observes with
the ascites tumour might not be due to the fact that the compounds are
synthesized elsewhere and then transferred, because in the case of lipids,

Discussion 223

for example, we observed that when we had labelled lipids in the form of
egg-yolk then it all appeared very nicely in the ascites tumour.

Forssberg: May I ask if you are doing these experiments in an atmos-
phere of oxygen, because our experiments were carried out under rather
anaerobic conditions.

Popjak: These were aerobic, not in pure oxygen but in air.

Forssberg: According to Christensen and Riggs (1952, J. biol. Chem.,
194, 57) there is a rapid uptake of amino acids in ascites cells against a
strong concentration gradient. We ourselves found that 5 minutes
after an intraperitoneal injection the uptake was already very high and
was quantitatively similar in X-rayed and control cells. The glycine
seems to enter the cells directly from the intraperitoneal injection.

Popjak: I am not suggesting that this might be the case for all cell
constituents, but it might be that in certain cases the preformed sub-
stances from plasma are taken out through the ascitic fluid.

Krebs: Your last slide rather suggests that the conversion of lactic and
pyruvic acid into acetic acid is increased, and the two products of
acetyl coenzyme A, acetoacetate and citrate, are present in higher con-
centration. I wonder whether any other experiments would be in agree-
ment with this conclusion, that you get a more rapid oxidation.

Forssberg: Oxygen tension in the ascites fluid is rather low. On a
molar basis the concentration of acetoacetic acid seems to be higher
than that which corresponds to a condensation of pyruvic acid? It
appears, however, from the analysis of the fluid that these substances
begin to leak from the cells almost immediately after irradiation ; there-
fore, quantitative determinations are uncertain. Mouse liver and
intestines which are irradiated at the same time as the ascites cells do
not show these changes, or at least show them only to a minor degree.
Whether any other similar experiments have been done, I do not know.

Krebs: The decrease is not in arithmetical proportion to the increase.

Lajtha: I think that the ascites cell cannot synthesize many things de
novo. On the other hand, the transformation and the transport can be
extremely quick. We gave ^-^C-formate in vivo to mice, and within 45
minutes the ascites cells became so heavily labelled that they were use-
less for autoradiography. Not only DNA but also RNA and proteins
were labelled. However, the same ascites cells in the same ascites fluid
in vitro in 3- to 6-hour cultures did not show any uptake of i*C-formate,
except small amounts in DNA thymine. With regard to the very
curious discrepancy between the depression in DNA synthesis and the
labelling of the DNA, I wonder whether that could be explained by
cells dying only in mitosis, and therefore no increase of DNA would be
observed in total of mass, but nevertheless the same cells were not
being inhibited in the synthetic period which is fairly long in these cells.

Forssberg: The cells are carrying out some vital functions, e.g.
production of total cell mass (i.e. proteins and RNA), at a fairly normal
rate during the first 48 hours after irradiation and are "living" as judged
from vital staining; so I do not think there can be much cell death in
mitosis during this period.

Lajtha: Is the cycle time constant in these cells?

224 Discussion

Forssberg: Cycle time may be taken as approximately constant for a
48-hour observation period in controls. In irradiated samples mitosis
disappears for about 15-20 hours. What the cycle time may be when
mitosis appears, I cannot say.

Howard: Dr. Forssberg, since you have done experiments showing
that labelled nucleic acid of dead cells can appear in the nucleic acids of
growing cells in the tumour, and apparently can also stimulate division,
do you regard this as something which might happen in a tumour which
is given heavy doses of irradiation in which there must be a great deal
of cell death, and do you think this would be a factor in the treatment
of tumours with radiation ? These dead cells are doing something to the
metabolism of the living ones, in the way of stimulating growth; what
significance has this in therapeutic treatment of tumours ?

Forssberg: I think that in therapeutic treatment doses should be
kept as high as possible in order to kill as many tumour cells as possible.
In ascites cells these effects appear with fairly low doses. It seems to me
that the increased labelling of DNA which Dr. Kelly found 2-3 days
after irradiation could be caused by transfer from irradiation-killed cells.

Howard: This seems quite a possibility in many irradiated tissues.

Latarjet: Dr. Howard's question impels me to say a few words about
the mysterious effect which Dr. Delaporte observed in 1949 after u.v.
irradiation and which she has recently investigated after X-irradiation
in my laboratory. She irradiated Esch. coli bacteria with about 80 kr., a
dose which apparently leaves about 10'* colony-forming cells when a
heavy inoculum is plated on agar. If one looks at the plate under the
microscope during incubation, one observes at the early origin of a
colony not a single growing cell but a rather large number of enlarged
growing cells undergoing a few divisions. Dr. Delaporte's idea is that
once a surviving cell has started growing, surrounding "dead" cells, at a
short distance of less than 100 microns, are induced into growth.
Restorability would last a short time, thus limiting the process which
otherwise would expand to the whole plate. This restoration would be
very effective between clumped cells. As a matter of fact, after u.v. at
least, once a cell starts growing within a clump, all the cells of this
clump start growing too.

I must add that what is to me the fundamental question has not yet
been cleared up, i.e. whether there is induction to cell enlargement
followed by a few divisions (delayed death instead of immediate death),
or to true restoration i.e. to indefinite cell multiplication. This question
may be answered by distinguishing the restoring cell from the eventually
restored ones. I am now using two mutants of Esch. coli for this purpose.
The experiments have just begun. The only thing I can say at the
moment is that, under my experimental conditions, using a high dose of
80 kr, the phenomenon of "neighbourhood restoration", if it does exist,
is rare.

INFLUENCE OF RADIATION ON METABOLISM OF
REGENERATING RAT LIVER

Barbara E. Holmes

Department of Radiotherapeutics , University of Cambridge

Any attempt to discover something of the biochemical
reactions concerned in cell division or the effects of irradiation
on the mitotic processes, is liable to lead to the necessity of
dealing with single cells or cells dividing synchronously. The
use of growing tissues is limited by the fact that all stages of
the mitotic cycle are here present together and any estimation
can only give an average value. Histochemical techniques
have been evolved to make possible the examination of a
single cell and the elegant autoradiographic methods of Pelc
and Howard are among the most successful. For work on a
larger scale the synchronously dividing tissue is useful and,
among mammalian tissues, the regenerating liver is a con-
venient example.

Price and Laird (1953) and also Abercrombie and Harkness
(1951), Stowell (1949) and others who introduced the experi-
mental use of regenerating liver tissue, found that a large
synthesis of DNA had taken place in the remaining lobes of a
rat liver 24 hours after hepatectomy, whereas cell division
had not yet begun. An opportunity of studying the chemical
events leading up to mitosis was thus available. This tissue
was used in our laboratory to examine the radiosensitivity of
different stages of the mitotic cycle. Mrs. Kelly of the Donner
Laboratory used carbon tetrachloride poisoning to cause
partial destruction of liver cells, which was followed by
regeneration, and estimated the effect of whole body irradi-
ation on this. She found a very large increase in DNA synthesis
in the liver, beginning at 30 hours, and reaching its peak at
36 hours after administration of the drug; a high rate of

RAD. 225 9

226

Barbara E. Holmes

mitosis was not seen until 12 hours later. The DNA synthesis
could be inhibited by irradiation (800 r whole body) at 12
hours (not later than 24 hours) after poisoning, but even
2000 r could not inhibit the synthesis while it was actively in
progress. Another sensitive period for this inhibition was

lOOO

E
Q.800

O4OO

o
U

200 —

Few

4 A Irradiated before operotion

X X Irradiated at 12 hours

Control

3.2(25«/^/x

Figures represent mitotic ,
0 count /lOOO

] /o Mitos«s showing

chromosome fragments

19 6(66%!

^ y A 6 [some]

Xr-^'

J \ L_

6 12 15 18 21 24 27 30 33 36 39 42

HOURS AFTER KERATECTOMY.
Fig. 1. Rate of DNA synthesis and mitosis in regenerating liver.

found by Mrs. Kelly to occur later than 60 hours after poison-
ing (Kelly et al, 1955).

The curve of regeneration we obtained after hepatectomy
(Fig. 1) has some different time relationships. The peak of the
DNA synthesis rate seems to be shortly before the first large
outburst of mitosis. As soon as large numbers of mitoses are
present the rate is diminished.

A dose of 2000 r inhibits DNA synthesis by about 50 per
cent at any time during the cycle and the effect is immediately
apparent. Here our results differ from those obtained by

Radiation and Regenerating Rat Liver 227

Mrs. Kelly. Small doses of irradiation, in our case 450 r, do
not have any obvious immediate inhibitory action on DNA
formation if given during synthesis, although they delay
mitosis and cause chromosome breaks.

As it had been shown by the work of Pelc and Howard
(1953) and of Kelly and co-workers (1955) that small doses
given before the beginning of synthesis have a marked delay-
ing action on DNA formation, we carried out irradiations with
450 r at 12 hours after hepatectomy. It can be seen from
Fig. 1 that the increased rate of synthesis normally begins by
15 hours. The irradiation delayed the increased DNA forma-
tion and the onset of mitosis for about 10 hours; at the time
when the control liver showed a very high rate of synthesis
(at about 24 hours after hepatectomy) the difference between
the control and the irradiated tissue was very great. We
found that we could demonstrate the effect of 450 r of X-ray
irradiation at any stage of liver regeneration if we waited
9-12 hours for the difference between the control and irradi-
ated liver to become obvious. This gave direct confirmation of
the work carried out by Pelc and Howard on the bean root
with radioautographic techniques.

Recovery (or partial recovery), as measured by DNA
synthesis and mitosis, comes at about 36 hours after hepa-
tectomy. It is obviously difficult to say whether the cells con-
cerned in synthesis and division are the same cells which were
forced to delay their activity, or whether another group of
cells has taken their place. It has been supposed that cells not
yet preparing for DNA synthesis might be unaffected by the
irradiation and that these undamaged cells could enter into
DNA synthesis and mitosis in their turn, causing the apparent
recovery. We have been able to show that this is not the case.
Irradiation with 450 r before hepatectomy shows the same
inhibition and recovery at the same time (Fig. 1) as are shown
by irradiation 12 hours after, so that it is not possible to
picture an early interphase stage which is unaffected by the
irradiation. In three cases irradiation was carried out 24
hours before hepatectomy; here there is some recovery in the

228 Barbara E. Holmes

sense that mitoses occur at 27-28 hours after the hepatectomy.
These are fewer than in the control and show many chromo-
some breaks and the DNA synthesis rate is lower. It is
plain that this question of the irradiation of the resting liver
tissue should be further investigated.

It is worth mentioning that Dr. Koller, who carried out
cytological investigations on squashes of the irradiated liver
material, found a very high chromosome breakage rate
(percentage numbers in Fig. 1) in the belated mitoses after
irradiation at 12 hours after hepatectomy. This early stage
is not usually supposed to be a sensitive stage as regards the
production of chromosome breaks. (All the mitotic counts
were done by Dr. D. Cater.)

In much of the work on regenerating liver, described in the
literature, whole body irradiation is used. For our experiments,
irradiation was given immediately over the area of the right
hand liver lobe and the rest of the abdomen was screened with
lead rubber. In early experiments we removed food from the
cages and gave both control and irradiated animals injections
of glucose saline. More recently we have found that the
irradiated animals will eat quite well and we have not taken
any such special steps. Animals showing any adverse symp-
toms, or lack of muscular tone, or which have lost an unusual
amount of blood at operation or have subsequently injured
the muscle scar by struggling, cannot be used. These conditions
cause delay in regeneration.

In experiments with regenerating rat liver, as with Jensen
rat sarcoma, it is found that irradiation inhibits the ^^P
uptake into DNA but not into RNA. Ord and Stocken (1956)
agree with this, but point out that in some tissues RNA
synthesis is also affected. Abrams (1951) found some inhibit-
tion of 22P uptake into the RNA fraction of some mouse
tissues after whole body irradiation. In normal as well as
regenerating liver Kelly (1952) has found that whole body
irradiation will cause a depression of ^^P uptake into nuclear
RNA but an increase in cytoplasmic RNA.

Kelly and Payne (1953) also studied the effect of whole

Radiation and Regenerating Rat Liver 229

body irradiation on the incorporation of adenine in the
nucleic acids of various tissues. About GO per cent depression
of incorporation into DNA was found 1-3 hours after irradia-
tion while the effect on RNA was slight. In most tissues, the
depression of DNA synthesis after 48 hours was very large,
whereas in the intestine the synthesis is actually much above
normal at this time.

On the whole, the DNA synthesis is most affected in all
tissues, which made it reasonable to consider the possibility
that the synthesis of thymine or the thymine nucleotides of
DNA might be particularly sensitive to irradiation. An
attempt to show this was made by Mee (1956), who used
[ i^C] formate to follow the synthesis of the bases in RNA and
DNA and obtained a regeneration curve which followed the
32P uptake curve almost exactly. The bases were separated
and the activity was estimated. Three hours after a dose of
2000 r, the specific activity of the DNA was depressed to
half of the control value; the adenine, guanine and thymine
were, however, equally affected. The possibility still remains, of
course, that lack of thymine has prevented the appearance of
half the control amount of new DNA. Ord and Stocken (1956),
however, comparing the separated nucleotides of DNA in
tissues irradiated in vitro, have given data which suggest that
the addition of phosphorus to the purine nucleotides is more
easily inhibited than the uptake of pyrimidine nucleotide
phosphorus.

So far, this work had been concerned with changes in the
nucleic acid metabolism only, but it was plainly of interest to
relate changes in the general metabolism of the liver to these
special ones. An investigation of the changes in metabolism
accompanying regeneration and the effect of irradiation upon
them has been carried out by Dr. Itzhaki of our laboratory and
will be described in the following pages.

It has been known for some time (Ludwig, 1939; Gursch,
Vars and Rardin, 1948) that the neutral fat content of
regenerating liver was considerably above the normal level,
but this rise in neutral fat had not been correlated with any

230

Barbara E. Holmes

particular stage of regeneration. Table I shows that a large
increase in fat content occurs soon after hepatectomy and
persists for 2 days. It is not particularly connected with any
stage of the mitotic cycle and may merely be a sign of altered
or curtailed activity of the liver. The fat was extracted and
estimated by the hydroxylamine and ferric chloride method of
Stern and Shapiro (1953).

Table I

Fat Content o

f the Liver

Liver

Time after
hepatectomy

Number of
animals

Fat
{Per cent)

Normal

Regenerating

15 hours

20 hours

2Gl hours

2 days

5 days

9 davs

4
3
10
5
4
1

3-64
7-42
5-86
7-30
5-96
401
3-50

The respiration of the liver was known to be higher after
hepatectomy, and Schw^artz and Barker (1954) had measured
it at different times after the operation. Table II shows
Itzhaki's data, which can be considered in connection with
the synthesis and mitosis curves. The respiration is already
very high at the beginning of synthesis, is unaltered during
the period of high mitosis and continues to be high for 3 days.
The respiration was measured in a Warburg apparatus in the
presence of an excess of glucose.

The oxidation of glucose itself was estimated by measuring
the specific activity of COg derived from the oxidation of
glucose uniformly labelled with ^^C. The COg was trapped in
potassium hydroxide, sodium carbonate was added as carrier
and the carbonate precipitated as the barium salt, the weight
of the barium salt being always about 100 mg. The factor for
converting the counts, estimated to the total count, was known.

Radiation and Regenerating Rat Liver

231

The total activity of the CO2 is given as a percentage of the
total activity of the glucose of the medium. The oxidation of
glucose, as measured by this method, includes the direct
oxidation of C^ and the oxidation of the derived three-carbon
molecules through the Krebs cycle.

Fig. 2 shows an increase in total glucose oxidation after
hepatectomy. This can be seen here related to the other
metabolic changes and to the mitotic cycle.

Table II

Oxygen Uptake by Liver Slices

Liver

Time after
hepatectomy

Number of
animals

Oxygen uptake

{[il.OJmg. fat-free

dry tissue)

Normal

Regenerating

14 hours

20 hours

26^ hours

46 hours

3 davs

10
3
5
8
3
1

6-67 ± 018

915

903 + 0-81 *

900 ± 0-51 *

9-51

9-40

The slices were incubated in Krebs-Ringer phosphate solution containing
0*4 per cent glucose.

* Statistical comparison shows that these figures are signiflcantly higher than normal.

The comparison of samples of glucose specifically labelled
in the 1- and in the 6- position shows that both the direct
oxidation of glucose which results in the liberation of COg from
Ci and the oxidation through the Krebs cycle, which results in
liberation of COg from Cg as well as from C^, are very definitely
increased after hepatectomy. These figures will be published
later by Dr. Itzhaki.

A large number of measurements of respiration and of fat
content were made at 26 hours after hepatectomy and the
mitotic rates were measured by Dr. Cater. No connection
between the mitotic count and the respiration or fat content
could be demonstrated.

232

Barbara E. Holmes

Although it is well known that X-ray irradiation tends to
have very little immediate effect on the general metabolism
of a tissue, it did seem possible that an irradiation dose cap-
able of delaying the regeneration of the liver might also

+IIO

Fat

Oj uptake
D — Glucose Oxidation
® Mitosis

r-35

O
-30 o

■25 g

o
o

-20

-si

-lO

-i — I — T 1 r

12 16 20 26, 36
Hours

44 48
Period alter Hepatectomy

-I — I — r
Days

-5

\ ^O

Fig. 2. Fat content, oxygen uptake and glucose oxidation of regenerating

liver.

delay the appearance of increased respiration and increased fat.
This, however, was not the case, as Table III shows, even when
the rat was irradiated before hepatectomy.

The glucose oxidation, as measured by COg output in the
manner already described, gave somewhat different results,
which are shown in Table IV. The oxidation of glucose by
normal liver was unchanged by irradiation, but the increase in
glucose oxidation found during early regeneration could be

Radiation and Regenerating Rat Liver

233

inhibited by 450 r X-rays given before hepatectomy. The
glucose oxidation has, in fact, been prevented from rising
above the normal level in animals which are killed at 15 or 16
hours after hepatectomy, which is the only time interval so
far used. The glucose oxidation at the time of recovery from
irradiation must obviously be measured as soon as possible.

Table III

Effect of Radiation on Oxygen Uptake and Fat Content of

Regenerating Liver

Time after hepatectomy

Oxygen
uptake

([il. O^lnig.

fat-free dry
tissue)

Fat
(Per cent)

Mitotic

counts

per 1000

Irradiation

Killing

450 r
0

Preoperation*

20 hours
20 hours

8-43
8-20

5-42
6-00

450 r

6 hours

23 hours

12-55

5-36

450 r
0

12 hours

26 hours
26 hours

10 00
10 00

7-33
7-00

0
63

2200 r
0

23J hours

26^ hours
2Q^ hours

11-20
10-80

603
11-90

2200 r
0

20i hours

26| hours
26J hours

9-56
10-90

7-95
9-50

0
34

Values for normal liver: Og uptake 6 • 67, fat content 3 • 64.

* This rat was irradiated immediately before hepatectomy.

All these measurements were calculated on the basis of the
weight of fat-free dry tissue, since the fat content was
sometimes very high in the regenerating tissue. The effects of
X-rays on glucose oxidation will be published in more detail
later.

In the course of the irradiation experiments just described,
it could always be shown that 450 r given during or at the end
of DNA synthesis, although too late to prevent the synthesis,
still had a marked delaying and damaging effect upon mitosis.
We hoped to demonstrate, by another type of experiment, a

234

Barbara E. Holmes

Table IV

Effect of Radiation on Glucose Oxidation by Slices of Regenerating

AND Normal Liver

Yield of i^COg/lOO mg. fat-free dry tissue
{Per cent of total radioactivity of glucose)

Experiment No.

Regenerating liver

Normal liver

Non-
irradiated

Irradiated

Non-
irradiated

Irradiated

2-82
2 14
306
2-44
2-27
214
216

3-42

2-60
1-46
1-63
1-77
1-82
1-85
2-33

2-27

1-84
1-29
212
1-83
1-69
206
1-84
1-86

1-99

2 07
1-40
1-49
1-93
212
2-08

Mean ± S.E.
No. of animals
Group

2-56 ± 017*
8
A

1-97 ± 014
8
B

1-84 ± 008
9
C

1-85 ± 013
6
D

* statistical comparison shows that the mean of group A is significantly higher than that of
group B (P = 0-02) and that of C (P < 0 01).
Rats were killed 15^ to 16 hours after X-irradiation.

Irradiation dose was 600 r except in experiments 1 and 2 where the dose was 450 r. Partial
hepatectomy was carried out immediately after irradiation.

more direct connection between inhibition of growth and
inhibition of DNA synthesis by irradiation.

Dr. Dittrich, of Dr. Schubert's laboratory in Hamburg,
was kind enough to send a strain of Ehrhch mouse carcinoma
which had been made radioresistant by irradiation at a num-
ber of successive passages, as described by Dittrich, Hohne
and Schubert (1956). This tumour was grown as a solid
tumour in the leg of an inbred strain of white mice and was
compared with a normally sensitive strain of the same tumour
grown in the same strain of mice. Tumours inoculated on the
same day and grown to the same size were used, and ^^P was

Radiation and Regenerating Rat Liver 235

used as a tracer to estimate nucleic acid synthesis. Table V
shows the results. At 2000 r, which was the dose used by
Dittrich and Hohne to demonstrate the decrease in sensitivity,

Table V

X-Ray Irradiation of Ehrlich Mouse Tumour

Specific activity of DNA phosphorus as per cent of specific activity of inor-
ganic phosphorus.

Dose

Time after
irradiation

Usual strain
DNA RNA

Radioresistant strain
DNA RNA

2000 r
0

1\ hours
1^ hours

10
1-6

3-6
60

0-65
1-7

50
60

2000 r
0

\\ hours

0-5
1-65

3-2
50

0-6
1-25

30
4-5

2000 r
0

\h hours

0-83
1-85

4-6
6-5

0-85
1-7

4-65
6-2

2000 r
0

1| hours

1-9
2-35

6-5
91

2000 r
0

2 days

019
0-85

2-5
3-7

0-2
1-34

40
5-4

1650 r
0

1| hours

1-5
1-75

100
6-5

1650 r
0

1| hours

0-8
215

50
6-4

1-25

2-7

8-15

7-2

1500 r
0

2 days

0-25
0-9

41
4-35

0-33
0-75

4-8
3-65

1350 r
0

1| hours

1-26
1-5

7-6
60

0-89
0-55

60
3-3

we could see no difference in the immediate or delayed effect
in DNA and RNA synthesis. In this tissue the uptake of ^^F
into the RNA is somewhat decreased by irradiation. At the
lower irradiation doses of 1650 r and 1500 r there is a sugges-
tion that the decrease of RNA synthesis is no longer found in
the "resistant" strain and there is, perhaps, a chance that we

236 Barbara E. Holmes

may be able to demonstrate a slight strain difference here.
At 1350 r there is very little effect on RNA or DNA synthesis
in either strain.

It remains to be proved that the radioresistance still
persists in the Dittrich strain, and experiments are in progress
to test this point. At present, it seems that the effects on
DNA synthesis have very little to do with the radiosensitivity
of the tumours. These experiments are being repeated on
new strains sent from Germany.

REFERENCES

Abercrombie, M., and Harkness, R. D. (1951). Proc. roy. Soc. B., 138,
544.

Abrams, R. (1951). Arch. Biochem., 30, 90.

Bennett, L., Kelly, L., and Krueckel, B. (1954). Fed. Proc, 13, 181.

Dittrich, W., Hohne, G., and Schubert, G. (1956). In Progress in
Radiobiology p. 381. Edinburgh : Oliver & Boyd.

GuRSCH, F. N., Vars, H. M., and Rardin, I. S. (1948). Amer. J.
Physiol., 152, 11.

Kelly, L. S. (1952). Proc. Soc. exp. Biol., N.Y., 81, 698.

Kelly, L. S. (1953). Acta radiol., Stockh., Suppl. 116.

Kelly, L. S. (1954). Proc. Amer. Ass. Cancer Res., 1, 24.

Kelly, L. S., Hirsch, D., Beach, G., and Page, A. H. (1955). Radia-
tion Res., 2, 490.

Kelly, L. S., and Payne, A. H. (1953). Fed. Proc, 12, 76.

LuDWiG, S. (1939). Proc. Soc. exp. Biol., N.Y., 42, 158.

Mee, L. (1956). In Progress in Radiobiology p. 12. Edinburgh: Oliver
& Boyd.

Ord, M. G., and Stocken, L. A. (1956). Biochem. J., 63, 3.

Pelc, S. R., and Howard, A. (1933). Acta radiol., Stockh., Suppl. 116.

Price, J. M., and Laird, A. K. (1953). Cancer Res., 10, 650.

Schwartz, H. C, and Barker, S. B. (1954). Fed. Proc, 13, 131.

Stern, L, and Shapiro, B. (1953). J. din. Path., 6, 158.

Stowell, R. (1949). Arch. Path., 46, 164.

DISCUSSION

Roller: I cannot add anything more to Dr. Holmes' data except to
emphasize the fact that after irradiation there are chromosome breaks
in dividing cells of the regenerating liver. I may mention, however, the
very interesting fact that the number of mitoses which appear in re-
generating liver, treated with colchicine, is extremely high. We found
that 28-33 hours after hepatectomy, 85 per cent of the cells are under-
going mitosis. It seems that division must be extremely rapid and that
the duration of the mitosis is very short.

Discussion 237

Holmes: I didn't mention the colchicine experiments. We put the
colchicine in 3 hours before killing the animal because we had the strong
conviction that over a long period it was rather poisonous and decreased
the rate of mitosis. That certainly does not happen when it is only in
for 3 hours. As regards the induction of radiation injuries, I think I am
right in saying that usually one does not get many chromosome breaks
if one irradiates cells of Vicia or Tradescantia early in interphase before
DNA synthesis, but that was not true in the case of regenerating liver.
Many breaks were found when irradiation with 450 r was given before
the beginning of the period of DNA synthesis, i.e. at 12 hours after
hepatectomy and 13 or 15 hours before mitosis would be expected.
This would normally be described as irradiation during interphase.

Roller : Yes, that was the case.

Howard: How long was the colchicine present?

Holmes: Only the 3 hours. The ^^p was also injected 3 hours before
killing; we tried to collect all the mitoses that happened in that period.
There are no colchicine counts shown in Fig. 1.

Lajtha: This was surgical hepatectomy?

Holmes: Yes, ours was surgical hepatectomy.

Lajtha: The difference between your results and those of Dr. Kelly
may be due to the different means of producing hepatectomy. She pro-
duces it by chemical means, and that does not destroy the nuclei com-
pletely. Perhaps she has an experimental condition similar to that which
Dr. Forssberg has, with some DNA present in dying cells which may
serve as a pool for the regenerating cells.

Holmes: That is a very interesting suggestion.

Gray: Do I understand correctly that the wave of mitosis is not delayed
if you irradiate just before hepatectomy, but that it is if you irradiate
after hepatectomy?

Holmes: No, if you irradiate just before there is just as much delay
as if you irradiate in the "sensitive" period. If you irradiate it 24 hours
before hepatectomy there is some recovery. The rate of DNA synthesis
is low and some chromosome breakage is seen, but some mitoses do
appear at 27 hours.

de Hevesy: Do you know the actual growth rate, in the course of
1| hours, of those tumours in your last table? What percentage would
that be ? They can't grow without formation of DNA.

Holmes: I do not know.

de Hevesy: The ratio of the specific activity of DNAP at the end of
your experiment and the mean value of the specific activity of cellular
orthophosphate P during your experiment is supposed to give the per-
centage of DNA molecules formed in the course of 1| hours. If you
follow up the growth of your turnover for a longer time and extrapolate
from these data the amount of DNA formed during 1^ hours you will
presumably find half of the value supplied by the radioactive data only.
We and others interpreted such findings in the old days as indicating
that with the formation of two new DNA goes hand in hand the dis-
appearance of one. Recent work carried out by you and others indicate
however that no appreciable amount of DNA molecules disappears in

238 Discussion

the course of growth. A possible explanation of the above-mentioned
discrepancy is that the orthophosphate we isolate from the tumour is
an artifact and has a lower specific activity than that utilized in the
building up of the DNA molecule. If the genuine orthophosphate has
about twice the specific activity of the isolated one, the above-mentioned
discrepancy clearly disappears. We must furthermore consider the
possibility that the precursor of DNAP is not orthophosphate P but
the phosphorus of a compound formed in the cell boundary under
participation of highly active extracellular P. In experiments carried
out on composite homogenates made up from isolated nuclei and isolated
cytoplasm fractions from rabbit liver tissue Davidson observed recently
that the acid-soluble fraction of the cell sap contains a more effective
precursor of DNA than inorganic phosphate.

Holmes: In one particular series of regenerating livers Dr. Richards
found that a number of cells had formed an amount of DNA which
brought the content up to an octoploid level. If many cells synthesize
this large quantity before they divide, the earlier calculations of the
amount of new DNA required for each mitosis must be altered.

It has been found that the octoploid content of DNA is the usual
content in the Ehrlich ascites tumour cell immediately before mitosis
(Kelly, L. S., and Jones, H. B. (1956). Fed. Proc, 15, 108). Furthermore,
the same has been found to be true of the Krebs ascites tumour cells
(Richards, B. M., Walker, P.' M. B., and Deeley, E. M. (1956). Ann.
N.Y. Acad. Sci., 63, 831).

THE INDUCTION OF CHROMOSOMAL
ABERRATIONS BY IONIZING RADIATIONS
AND CHEMICAL MUTAGENS*

C. P. SWANSON
Department of Biology, The Johns Hopkins University, Baltimore

AND

BeNGT KlHLMANf
Institute of Physiological Botany, University of Uppsala

A DECADE ago, when the late D. E. Lea's (1946) book on
radiobiology appeared, a physical explanation of the events
leading to the production of chromosomal aberrations seemed
eminently satisfactory. The dosage and intensity relationships,
the results of fractionation experiments, the spacing of ions
along known paths by different types of radiations, and the
ideas revolving around the target theory and the "breakage-
first" concept, fitted together sufficiently well to give a good
measure of confidence in a strictly physical interpretation of
the available data. Seven years later, however, it was possible
to state that "The main features of the biological experiments
(with ionizing radiations) make very good sense when viewed
from the standpoint of radiation chemistry" (Gray, 1953).
As it applied to aberrations induced by ionizing radiations,
this enlarged concept — for it was an expansion of earlier
ideas rather than a shift in perspective — stemmed from the
initial studies of Thoday and Read (1947, 1949) and their

* Acknowledgement of financial support of the work reported here is
gratefully made to the U.S. Atomic Energy Commission [Contract AT (30-1)
1695] and to the National Science Foundation (Research Grant NSF-G2233).
We wish also to acknowledge our indebtedness to Dr. A. V. Beatty for permis-
sion to cite from his unpublished studies.

t On leave of absence from the University of Uppsala to The Johns Hopkins
University under the Exchange Visitor Programme of the U.S. Information and
Educational Act of 1948.

239

240 C. P. SwANSON AND Bengt Kihlman

later extension by Giles and his co-workers (reviewed by
Giles, 1954) which demonstrated the central role which
oxygen plays in governing the degree of chromosomal damage.
The radiochemical aspects of radiation and their relation to
chromosomal studies have been adequately treated elsewhere,
and it needs only to be recalled that the effects of radiation on
biological systems can be modified by a variety of experi-
mental conditions such that the chromosomal damage may be
amplified or diminished. Any initial complacency generated
by the knowledge of radiochemical events was of short dura-
tion, however, and it is now evident that the radiochemical
events are but a link which, in the living cell, connect the
physical events of radiation with the observable effects such
as aberrations. Latarjet and Gray (1954) have expressed
this in the following way:

I II III IV

Absorption Primary Chemical Observable

of radiant — > radiochemical — > reaction — > lesions
energy reactions chains

Step III constitutes the greatest unknown in the above
chain of events, and is the one on which our attention will be
largely focused. The inadequacy of the first two steps to
account for all of the parameters encountered in chromosomal
studies with ionizing radiations has been made evident by a
variety of observations: among others, the fact that oxygen
alone is capable of inducing aberrations (Conger and Fair-
child, 1952), the discovery of diff'erential rates of breakage and
rejoinability during the course of cell division (Sparrow and
Maldawer, 1950; Deschner and Sparrow, 1955) and the role
of metabolic inhibitors in modifying the final frequency of
aberrations (King, Schneiderman, and Sax, 1952; Wolff and
Luippold, 1955). These latter studies strongly suggest the
involvement of oxidative metabolism in the ultimate extent
and expression of radiation damage.

Although the radiobiological experiments may make "good

Induction of Chromosomal Aberrations 241

sense " when viewed from the point of view of radiochemistry,
certain pieces of data indicate that the radiochemical events
in the cell can be magnified without appreciably altering the
final frequency of aberrations. Gray, on several occasions
(1953, 1954a and b), has pointed out that there exists a close
parallel between the number of ion pairs per unit of path
length and HgOg production on the one hand and the frequency
of aberrations on the other. It is a well known fact, however,
that the frequency of aberrations is proportional to the oxygen
concentration only at low levels; when the concentration of
oxygen rises above 20 per cent little increase in aberration
frequency is found even though HgOg production continues to
increase. Allen (1954) and Ebert (1955) have also shown that
the addition of small amounts of hydrogen to oxygenated
water leads to a striking increase in HgOg production, pre-
sumably by promoting the reaction: 2 HOg + Hg -^ 2 H2O2.
If it is presumed that the cell behaves as an aqueous system,
then increases in aberration frequency are to be expected
when the cells are exposed to oxygen-hydrogen mixtures.
Mr. T. Merz has carried out these experiments in our labora-
tory, using a variety of hydrogen-oxygen mixtures, and finds
that while in Tradescantia microspore chromosomes chromatid
deletions are somewhat increased whenever hydrogen is
added to oxygen, exchanges and isochromatid deletions
remain relatively unaffected. Dominant lethals in Drosophila
are also greater in a mixture of 20 per cent Og : 80 per cent Hg
than they are in air, but there is no obvious relationship
which suggests that the amount of radiation damage is
proportional to H2O2 production. These results are also
supported by the data of Kimball (1^55) which indicate that
H2O2 is not involved in the induction of genetic damage in
Paramecium. There is danger, of course, in forcing too close a
comparison between what is known to occur in oxygenated
water and what is expected to occur in a cell which would be
buff'ered against environmental change. Several interpre-
tations are possible here, but more important is the fact that
the data force us to look beyond the radiochemical events for

242 C. P. SWANSON AND BeNGT KiHLMAN

some of the answers, and it is obvious that the comphcations
are many.

The studies of Wolff and Luippold (1955; see also Wolff and
Atwood, 1954) have been particularly instructive in focusing
attention on the involvement of metabolic systems in the
final expression of radiation damage in terms of chromosomal
aberrations. WolfP and his co-workers have demonstrated,
as have others before him, that breakage is oxygen-dependent,
but in addition his data also support the idea that the time
period of rejoining is similarly governed by the amount of
cellular oxygen. The older arguments concerned with the
"breakage versus restitution" controversy can now be dis-
pensed with since both are shown to be oxygen-dependent.
Based on fractionation techniques, and the use of inhibitors
of oxidative metabolism, these studies have been interpreted
as a demonstration of the fact that the rejoining of broken
ends is an energy-requiring event, and Wolff has proposed
that the radiation injures the metabolic system upon which
the repair of broken chromosomes depends. The greater the
dose of radiation, the greater the damage to this system, and
the longer the delay before rejoining can take place. The
breaks initially induced remain open during this period.
Treatment of root-tip cells between radiations with low
temperatures, KCN, CO in the dark, and DNP inhibit the
rejoining system, and so prolong the period between breakage
and rejoining; ATP, but not AMP, shorten the period. The
role of externally apphed ATP in effecting the rejoining system
must remain questionable for the time being since it is unlikely
that it penetrates the cell to act as such. However, the fact
that it is the time period of rejoining that is affected rather
than the rejoinability of broken ends itself is shown by pro-
viding the cells with these same agents as a posttreatment
after only a single dose of radiation is given. The final
frequency of aberrations induced by single doses of radiation
remains unaffected by any posttreatment.

Wolff's conclusions are supported by the data of Beatty,
Beatty and Collins (1956). Using a total dose of 400 r of X-

Induction of Chromosomal Aberrations 243

rays, and with intensities ranging from 1 to 50 r/minute, it
has been shown that the frequency of chromosome inter-
changes increases as the intensity decreases when exposures
were carried out in the absence of oxygen (hehum was used to
replace the oxygen of the cell). The reverse trend, of course,
holds for irradiations in air or in oxygen. The frequency of
aberrations obtained with an intensity of 1 r/minute in helium
was approximately equal to that found at an intensity of
25 r/minute in pure oxygen. These results are somewhat
unexpected, and following the line of reasoning expressed by
Wolff and Luippold (1955), it would appear that quite dif-
ferent sets of conditions prevail in the oxygen as opposed to
the helium series. Radiation at a comparatively high inten-
sity in oxygen leads to a high rate of breakage, but this is
offset by the fact that a large proportion of the breaks restitute
during the period of exposure ; high frequencies of aberrations
are obtained in the low intensity helium series with a consider-
ably lower frequency of breaks but with negligible rejoin-
ability during the period of radiation. It has logically been
assumed by Beatty, Beatty and Collins (1956), in the 1
r/minute helium experiments, that the circumstances of
anoxia are greatly exaggerated by a continued depletion of
residual oxygen through respiration during the 400 minutes
of exposure to radiation. The energy sources of the cell
which might otherwise be available for rejoining purposes
would be sharply depressed by removal of the oxygen,
and all breaks induced would remain open and be available
for rejoining when oxygen was once again added to the cell.
This hypothesis can be tested further. If cells are pretreated
for 400 minutes in an oxygen-free atmosphere, and then
irradiated at various intensities, they should have their
energy reserves at a low ebb, rejoining should not take place,
and no intensity effect should be observed. Or, conversely,
pretreatments in an oxygen-free atmosphere for varying
periods of time, followed by radiation at a constant intensity,
should yield frequencies of aberrations which increase as the
duration of the pretreatment increases. The latter experiment

244 C. P. SwANSON AND Bengt Kihlman

has been carried out (Beatty, Beatty and Collins, 1956),
and the data are in conformity with expectations.

The results discussed above deal generally with the rejoining
process, and shed little or no light on the breakage of chromo-
somes by radiations. Except for centrifugation and infrared
radiation, breakage appears to be unmodifiable by posttreat-
ments, and there is no experimental evidence which suggests
that breakage by radiation is mediated through the normal
metabolic pathways of the cell. It is of interest here to recall
that there is a lack of intensity effect in the mature sperm of
Drosophila (Muller, 1940) and in the generative cells of de-
hydrated pollen grains of Tradescantia (J. C. Kirby-Smith,
unpublished). These are cells in which the chromatin is
densely packed, and where it is unlikely that metabolic
systems having a high yield of free energy would be operative
to provide the necessary requirements for rejoining. Metabolic
activity in these cells would increase with the uptake of water
which occurs at the time of fertilization for Drosophila sperm
and during the germination of Tradescantia pollen, and the
opportunities for rejoining would become available. The fact
that a reduction in aberrations in these two types of cells is
obtained when irradiation is carried out in the relative absence
of oxygen is added support for the concept that breakage
itself is determined only by the physical and radiochemical
events in the Latar jet-Gray reaction chain rather than by
the succeeding metabolic events (see later).

Before attempting to fit the above data into a model system
it will be well to consider, in a comparative way, the effects of
chemical mutagens on aberration induction. A large variety
of chemicals which differ appreciably in structure and reactiv-
ity can induce chromosomal aberrations, but it seems desirable
to confine our attention to some of those which have been
studied intensively. These are di(2: 3-epoxypropyl) ether
(DEPE), 8-ethoxycaffeine (EOC), and maleic hydrazide (MH).
Viciafaba root-tips have been used as experimental material.
It is impossible as yet to say how any of these chemicals affect
chromatin to bring about the induction of aberrations, but it

Induction of Chromosomal Aberrations 245

is apparent that their actions differ among themselves, and
that all differ in their action from the ionizing radiations.
Chemically, MH is more similar to EOC than it is to DEPE.
Both MH and EOC are relatively unreactive compounds
whereas DEPE has a high chemical reactivity. The biological
effect of MH or EOC may or may not be related to the fact
that both possess a structural resemblance to the bases in
nucleic acids: EOC is a purine derivative, and MH, as first
realized by Loveless, is a structural isomer of uracil. Of the
three mutagens discussed, MH is the only one having acidic
properties. This is reflected in the strong pH dependence of
this compound for effectiveness; it is a powerful mutagen at
a pH of 5, but at the concentrations used its effect dimin-
ishes rapidly at higher pH levels, and practically vanishes
at a pH of 7 or above. These facts suggest that the
unionized compound penetrates much faster than the ionized
form.

In spite of their structural similarity, the biological effect
of MH bears no closer resemblance to that of EOC than it does
to that of DEPE. Both MH and DEPE act in early inter-
phase with the first aberrations appearing between 8 and 12
hours after treatment (Revell, 1953; McLeish, 1953). Aber-
rations induced by EOC appear within 2 hours after treatment,
indicating that it, like X-rays, acts in late interphase or early
prophase (Kihlman, 1955). MH and DEPE also show the
same relationship to temperatures ; in both cases the effective-
ness of the compound increases rapidly with increasing
temperature. The effectiveness of EOC increases in the range
of temperatures from 0° to 10° C, but then decreases at higher
temperatures. Few aberrations are obtained with EOC at
temperatures above 25° C.

On the other hand, the biological effectiveness of EOC and
MH, but not that of DEPE, is greatly reduced when oxidative
phosphorylation is inhibited during the period of treatment.
Their differences are also emphasized when one considers
their specificity of action which is reflected in a non-random
distribution of aberrations. In Vicia root-tip cells, DEPE

246 C. P. SwANSON AND Bengt Kihlman

produces a heavy concentration of aberrations in hetero-
chromatic segments in the middle of the long arm of S-
chromosomes (Loveless and Revell, 1949). MH-induced
breaks are also localized in heterochromatin, but in this
instance, the heterochromatin lying close to the centromere
in the nucleolar arm of the L-chromosomes is most frequently
involved (McLeish, 1953). EOC confines its major effect to the
same arm, but the aberrations involve principally the nucle-
olar constriction (Kihlman and Levan, 1951). The distri-
bution of breaks is more strikingly non-random the lower the
concentration of the mutagen, indicating that there are
preferential sites of action which may become obscured, how-
ever, if the concentration of mutagen is increased.

Two other observations are of interest in this respect. The
localization of breaks differs also with time after treatment as
well as with concentration of mutagen. DEPE seems to
induce a lowered percentage of localized breaks at 48 hours
after treatment than it does at 24 hours, while EOC-induced
breaks are more sharply localized at 22 hours than they are
either earher (6 hours) or later (48 hours). What these data
mean in terms of a mechanism of action is not entirely clear.
The suggestion has often been made that chemicals and ioniz-
ing radiations induce breaks in interphase chromosomes
because of their interference with DNA synthesis. No critical
evidence bears on this point, but the differential times and
sites of action, as well as the differential effectiveness of
mutagens and radiations under conditions of anoxia and in
the presence of inhibitors of oxidative metabolism, would
indicate that an induced disturbance of DNA synthesis cannot
provide a complete and satisfactory answer as to their mode
of action. The seeming correspondence of many of these
results with those obtained by Wolff on the rejoining process
is striking, and suggests a common metabolic pathway some-
where in the chain of events leading to the observable aber-
rations induced by ionizing radiations and chemical mutagens,
but it remains to be determined whether these common path-
ways are at comparable stages in the chain of events.

Induction of Chromosomal Aberrations 247

Discussion

An attempt can be made to formalize our notions of inter-
actions at the chromosomal and physiochemical levels within
the cell during and after exposure to ionizing radiation or
chemical mutagens. At the chromosomal level the two major
categories of events are breakage and rejoining, but each is
subdivisible. As to the breakage category, it is unlikely that
all breaks consist of fully broken chromatids or chromosomes.
Although difficult to assess, the concept of potential as dis-
tinguished from primary breakage (Thoday, 1953) gains some
credence from the infrared studies done in these laboratories.
Probably the ion density of the radiation would be the
principal factor involved in determining the spectrum of
chromosomal damage (Swanson, 1955a), i.e., the greater the
ion density the greater the portion of primary breaks as
opposed to potential breaks, and it seem likely, although no
proof is at hand, that the oxygen level of the cell would also be
a contributing factor (Swanson, 19556). The rejoining system
can be operationally divided into restitution and recombina-
tion, these being competitive actions for the disposal of broken
ends of chromosomes in the nucleus. The great majority of
broken ends do not, of course, contribute to observable
aberrations, but if we adhere to the idea of the existence of
potential breaks then a reduction in the number of damaged
sites could come about through the repair of potential breaks
or the restitution of primary breaks. There is no possibility of
distinguishing between them at present.

The experimental facts permit us to modify the Latar jet-
Gray scheme in the following way:

I II III IV V

Primary Secondary

Physical radio- radio- Metabolic Observable

events chemical chemical events effects

events events

The distinction between stages II and III is made on the basis
of time, with II covering the short-lived radicals and III

248 C. P. SwANSON AND Bengt Kihlman

those which may persist for some time. The studies of Wolff
and Luippold (1955) and Beatty, Beatty and CoUins (1956)
with ionizing radiations and those of Kihlman (1955, and
unpublished) with chemical mutagens make stage IV a neces-
sary part of the chain of events. There is no need here to dis-
cuss the physical and radiochemical aspects of this system
since they have been adequately covered by Gray elsewhere
(1953; 1954a and b), but it is of interest to assess our present
knowledge of the relationship between the Latar jet-Gray
chain of events and those taking place at the chromosomal
level. Stages I-III are obviously related to breakage, but to
weigh the contribution of each stage to breakage would re-
quire that one determine the relative importance of direct
versus indirect effects of ionizing radiations. At present it
would appear that we can only state that the indirect effects
seem to outweigh the direct effects if only because we realize
that a small amount of radiant energy introduced into a cell
can lead to an inordinate amount of damage. No direct and
convincing evidence is yet at hand which permits us to state
that metabolic systems in the cell have anything to do with
the breakage of chromosomes by ionizing radiations. It is
true, of course, that the sensitivity of the chromosomes
changes with the stages of cell division, but this variable
sensitivity may well be due to the state of the chromosome
rather than to any metabolic system which contributes
directly or indirectly to breakage. However, the fact that
spontaneous breakage is a variable phenomenon from species
to species, and often within species, suggests that it may be
due to altered physiological conditions such as those brought
on by nutritional deficiencies (Steffensen, 1953). On the other
hand, it seems quite certain that the metabolism of the cell
governs the action of certain chemical mutagens. Thus, as
indicated earlier, MH and EOC are both dependent upon the
oxygen of the cell for effective action, and the effects of both
are suppressed by inhibitors of oxidative phosphorylation.
The chemical mutagens, to be sure, would not involve stages
I-III in acting on chromosomes, but in their place it is prob-

Induction of Chromosomal Aberrations 249

able that chemical (non-enzymatic) and biochemical (enzy-
matic) events of comparable importance can be substituted.
It would appear, for example, that the initial action of DEPE
is chemical in nature since it is highly reactive, its effective-
ness is governed by the law of mass action (Revell, 1953), and
its action is not modified by pretreatment with DNP (Love-
less, 1953). EOC and MH, on the other hand, are modified in
their effectiveness by agents which inhibit oxidative phos-
phorylation, and it is likely that their initial action is through
enzymatically controlled steps in cellular metabolism.

Wolff's data indicate that the restitution and recombination
of ends of chromosomes broken by events taking place in
stages I-III require energy supplied through oxidative meta-
bolism, and that the rejoining system is comparatively sensi-
tive to small amounts of radiant energy. Considerable pro-
tection is afforded it by anoxic conditions. It is suggested, as
a consequence, that the principal damage to the rejoining
system is inflicted by active radicals and their derivatives
which have been induced by radiation. It is further tempting
to see a connection between the results described above which
indicate a possible role of oxidative phosphorylation in the
production of chromosomal aberrations and the recent
findings of Allfrey, Mirsky and Osawa (1955) which point to
the oxidative generation of energy-rich phosphate within the
nucleus, a process heretofore considered to be confined to the
mitochondria. It would appear, therefore, that the nucleus
does not necessarily exist and function in an anaerobic environ-
ment as suggested by Stern (1955), but much remains to be
done before the links in the chain are connected.

It has not been possible to separate breakage and rejoining
following exposure to chemical mutagens as Wolff seems to
have done in his radiation experiments. One cannot, therefore,
fully assess their effects on either system other than to point
out that with some chemicals oxidative phosphorylation
obviously determines their mutagenic effectiveness or in-
effectiveness. With other chemicals such a point of view
cannot be expressed with the same degree of surety. It is our

250 C. P. SWANSON AND BeNGT KiHLMAN

impression, however, that oxidative phosphorylation is
always involved in some of the steps in the chain of events
leading to chromosomal aberrations. The experimental data
so far available suggest that this step is toward the end of the
chain, and that it may be one normally occurring in the
chromosome. The metabolic events (stage IV) are probably
several in number, and the larger the number before the actual
induction of aberrations the less obvious will be the influence
of inhibitors of oxidative metabolism on chemical mutagenesis.
Admittedly we are treading on somewhat uncertain ground
in the above discussions. We have, for example, spoken of
breakage and rejoining as separable events in the induction of
chromosomal aberrations. This may or may not be so. At
the present time, the radiation experiments seem most
satisfactorily explained by making this distinction, but both
Revell (1953) and Loveless (1953), although differing in their
interpretations, agree in considering these two events insepar-
able so far as the induction of aberrations by chemicals is
concerned. The chemical experiments described above have
not yielded critical evidence on this point and further dis-
cussion of them will be reserved for subsequent papers where
a more detailed examination of the data can be made.

REFERENCES

Allen, A. O, (1954). Radiation Res., 1, 85.

Allfrey, V. G., MiRSKY, A. E., and Osawa, S. (1955). Nature, Lond.,

176, 1042.
Beatty, a. v., Beatty, J. W., and Collins, C. (195G). Amer. J. Bot.,

43, 328.
Conger, A. D., and Fairchild, L. M. (1952). Proc. nat. Acad. Sci.,

Wash., 38, 289.
Deschner, E., and Sparrow, A. H. (1955). Genetics, 40, 460.
Ebert, M. (1955). Radiobiology Symposium (Liege), p. 30. New York:

Academic Press.
Giles, N. H. (1954). Radiation Biology, Vol. 1, part 2, p. 713. New

York: McGraw Hill.
Gray, L. H. (1953). Brit. J. Radiol., 26, 609.
Gray, L. H. (1954a). Radiation Res., 1, 189.
Gray, L. H. (1954fo). Acta Radiol, 4:1, 6S.
Kihlman, B. (1955). Hereditas, Lund, 41, 384.
KiHLMAN, B., and Levan, A. (1951). Hereditas, Lund, 37, 382.

Discussion 251

Kimball, R. F. (1955). Ann. N.Y. Acad. Set.. 59, 638.

King, E. D., Schneiderman, H. A., and Sax,JK. (1952). Proc. nat.

Acad. Sci., Wash., 38, 34.
Latarjet, R., and Gray, L. H. (1954). Acta Radiol., 41, 61.
Lea, D. E. (1946). Actions of Radiations on Living Cells. Cambridge

University Press.
Loveless, A. (1953). Heredity (SuppL), 6, 293.
Loveless, A., and Revell, S. (1949). Nature, Lond., 164, 938.
McLeish, J. (1953). Heredity (SuppL), 6, 125.
MuLLER, H. J. (1940). J. Genet., 40, 1.
Revell, S. H. (1953). Heredity (SuppL), 6, 107.
Sparrow, A. H., and Maldawer, M. (1950). Proc. nat. Acad. Sci.,

Wash., 36, 636.
Steffensen, D. (1953). Proc. nat. Acad. Sci., Wash., 39, 613.
Stern, H. (1955). Science, 121, 144.
SwANSON, C. P. (1955a). Genetics, 40, 193.
SwANSON, C. p. (19556). J. cell. comp. Physiol., 45, 285.
TiioDAY, J. M. (1953). Heredity (SuppL), 6, 299.
Thoday, J. M., and Read, J. (1947). Nature, Lond., 160, 608.
Thoday, J. M., and Read, J. (1949). Nature, Lond., 163, 133.
Wolff, S., and Atwood, K. C. (1954). Proc. nat. Acad., Sci., Wash., 40,

187.
Wolff, S., and Luippold, H. E. (1955). Science, 122, 231.

DISCUSSION

Hollaender : I wonder whether the effect of oxygen alone would pro-
duce as many breaks in Tradescantia as a metabolic system?

Swanson: This is one reason for assuming that a metabolic system is
involved. With Conger's work on oxygen alone it seems to me that it is
much more likely that a metabolic system is involved than it is to relate
the effect to hydrogen peroxide or some reactive system of that sort.
One can, for instance, follow Ebert's lead in adding hydrogen to an
oxygenated system which, if we consider the cell to be an aqueous
medium, would lead to an increase in hydrogen peroxide concentration.
This will induce a larger proportion of breaks, but our data bear no
simple relationship to Ebert's curve. Added hydrogen increases the
frequency of aberrations. There must be some saturation phenomenon
involved; we have gone through the whole range of hydrogen- oxygen
mixtures, but we just get a slight increase and it doesn't matter what
the hydrogen oxygen mixture is.

Alper: In fact Ebert's curve for hydrogen peroxide yield with this
mixture is very flat, particularly for low doses.

Swanson: Then I have misinterpreted Ebert's curves because I
assumed from them that a mixture of 25 per cent hydrogen and 75 per
cent oxygen gave a very large increase in hydrogen peroxide formation.

Alper: The hydrogen peroxide determinations were made on the basis
of total dose. If you irradiate water in the presence of oxygen alone,

252 Discussion

without hydrogen, you get a family of curves for hydrogen peroxide
yield against oxygen concentration. With low doses you get the equilib-
rium yield at low oxygen concentrations, whereas with a high dose you
don't get the equilibrium yield until the oxygen concentration is high.
If you start with 100 per cent oxygen, then add hydrogen, the equilib-
rium yield rises sharply, for low doses, and is independent of hydrogen
concentration over a wide range of hydrogen-oxygen mixtures. As you
decrease the oxygen to zero and increase hydrogen to 100 per cent the
yield stays constant and falls sharply to zero as oxygen concentration
falls to zero. With higher doses the range of mixtures over which you
get constant yield becomes much smaller, and the relative proportions
of hydrogen and oxygen become critical. With low doses, however, you
get a very wide range of mixtures with which yield is unaltered.

Swanson : What do you mean by low doses ? What range ?

Alper: 10-20,000 r with the methods he has used up to now, but
presumably this range would be extended if one could use lower doses.

Swanson: In the hundreds?

Alper: It depends on how low he has gone with the oxygen. I don't
know what the actual range was. This doesn't mean that hydrogen
peroxide is constant.

Howard: Prof. Swanson, you say that if you add hydrogen to an
oxygenated system the frequency of aberrations is increased. This is
different to what we found with bean roots in which we got less damage
in presence of hj^drogen, even when oxj'^gen was also present.

Swanson : I am aware of that. The study done by Mr. Merz was on the
microspores of Tradescantia. I think that the gases were accurately
controlled. There is some danger, of course, of a flash-back, but we
managed to avoid it. We got practically the same results regardless of
what we added in the way of hydrogen, making certain at all times that
the oxygen was at least 20 per cent.

Howard: Did you have hydrogen under pressure?

Swanson: No, this was just the flowing gas, under no pressure.

Haddow: Am I right in believing that maleic hydrazide has no effect
on animal cells ? I think maleic hydrazide certainly has no effect what-
ever of these kinds on animal cells.

Swanson : I only know that McLeish reported that it was ineffective on
onion chromosomes where no visible heterochromatin is demonstrable,
but this is not entirely correct because it will break them very effectively
if you adjust the temperature and the pH. Maleic hydrazide, therefore,
is not a mutagen restricted in its activity to heterochromatin, although
that is the preferred site of action.

Haddow: We have come across this in a practical way in connection
with maleic hydrazide which, it was believed, might possibly be car-
cinogenic. But it is certainly not a strong carcinogen.

Swanson : I wonder whether this would be true if the data were taken
over a period of time, as with the diepoxide. We assumed that there was
no oxygen effect with the diepoxides, but if one examines the cells at a
later time after treatment, at least with the metabolic inhibitors, one
does observe a decided effect.

Discussion 253

Koller: I wish to mention that Dr. Aiierbach would Hke to repeat her
early experiments, because she does not think tha£ an oxygen effect in
the case of mustards really exists. The experimental conditions were
not sufficiently stringent.

Spiegelman : Do these Tradescantia microspores which you treat have
an^^ metabolism which is detectable ? Do they respire or glycolize ?

Swanson: Tradescantia is treated in the bud stage, and it is going
through very active division, so I presume it is an actively metabolizing
cell.

Alper: You quoted some data where after seven hours exposure to
oxygen you had 73 breaks /1 00 cells at a dose rate of 1 r/min. How
many would that be with just seven hours of oxygen?

Swanson : These data are from a paper by Beatty, Beatty and Collins
(Amer. J. Botany, in press). They graciously loaned me the data for this
symposium. There are no control data, however, which disturbed us a
little, and that is why we have become interested in the problem. Root-
tip cells seemed to be the easiest material for us to study at the moment
and we obtained a rather high frequency of aberration with long periods
of anoxia alone.

Howard: Is it possible that in your work on Vicia root- tips, where you
use all these different treatments — the temperature treatment, blockage
of the metabolism of the cell, the chemicals you are using, and radiation—
you can really take into account the fact that you must be altering the
mitotic cycle, and therefore the moment at which you look at the treat-
ment is highly relevant? Would you not need rather complete time-
curves to control this?

Swanson: I expect that you are right here. The only thing I can say is
that it is the conviction of Dr. Kihlman, who has done the chemical
mutagen work, that this is not a factor. He had worked this out in
terms of time relationships and he feels that the mitotic activity is not a
controlling factor, at least not to the extent that it throws the data all
otit of proportion.

Koller: On one of your slides you showed two curves, one concerning
the interchanges and the other the isochromatid breaks. You pointed
out that irradiation and chemical mutagens interfere with the chrom-
osome rejoining process which has two components, restitution and re-
combination. Did you find a decrease in the interchanges and a similar
decrease in the isochromatid or in the open breaks ?

Swanson: With the mutagens there are relatively few breaks that do
not undergo recombination. We don't find many incomplete exchanges
or the non-sister reunion tyge of isochromatid deletions. The propor-
tions of the two do not change with treatment, although they do change
with time. This, of course, is a function of the spatial relationships of
the chromatids, but there is no appreciable change in the proportions of
the two tj^es with any particular treatment. So I think that they are
comparable types of aberration.

Van Bekkum: With regard to the metabolic inhibitors such as dinitro-
phenol, have you any data on the effect of ATP, for instance ?

Swanson : I can quote only the data of Wolff, to the effect that ATP

254 Discussion

externally applied, shortens the rejoining period. He also has unpub-
lished data which indicate that adenine, added to the solution in which
the roots are immersed, does it even more rapidly.

Bracket: Is there any cytological effect of the inhibitors alone, dinitro-
phenol for instance?

Swanson: They don't appreciably affect the mitotic cycle or the
appearance of the cells at the concentrations and times employed.

Brachet: In Acetabularia, where the nucleolus is very conspicuous, it
changes quickly and considerably in shape and RNA content in the
presence of dinitrophenol and similar substances. Regarding the part of
the cell where these poisons may be acting, it is interesting that, in
Acetabularia, Stich got the same transformation as with dinitrophenol
by leaving the algae in the dark for several weeks. Of course, since the
chloroplasts are only present in the cytoplasm, the effect is primarily the
reaction of the nucleolus to metabolic events taking place in the cyto-
plasm. In eggs also, dinitrophenol produces big cytological changes and
mitotic inhibition ; but it seems that it induces disturbances of the
nuclear RNA, rather than changes in the chromosomes.

Swanson: We have looked at them when stained with Feulgen and
there appears to be no difference between them.

Pirie: You said that dinitrophenol reduced this effect of mutagenic
chemicals. Yet it is not a blocking agent to metabolism, it is an upset-
ting agent, an uncoupling agent which is surely very different to blocking;
respiration will increase, and your ATP will go down.

Swanson: It should be emphasized that we have only reached the
point of asking biochemical questions. With EOC, for example, the
toxic effects are not interfered with by dinitrophenol ; if the temperature
is raised up 28°, the major action of EOC is one of reducing root growth.
The roots actually turn black. This is not interfered with by dinitro-
phenol, so that the mutagenic effect on the chromosomes must be
quite different.

Holmes: Did Kihlman find any change in mitotic index with dinitro-
phenol ?

Swanson: There is no change in mitotic index with dinitrophenol.
This has been checked very carefully.

PRIMARY SITES OF ENERGY DEPOSITION
ASSOCIATED WITH RADIOBIOLOGICAL

LESIONS

L. H. Gray

British Empire Cancer Campaign Research Unit in Radiobiology,
Mount Vernon Hospital, Northwood

The concern of this symposium is with metaboUc pathways
in the development of radiobiological damage. Direct ob-
servation of the metabolic activity of the irradiated cell may
hit the trail early or late. It may possibly be a help in inter-
preting observations of metabolic activity to consider whether
there is any independent evidence as to where any of the
pathways begin. The evidence might in principle be either
chemical or anatomical.

In the study of biological damage induced by u.v. radiation,
useful information has been obtained by comparing "action
spectra" with absorption spectra. Mutation, chromosome
fragmentation, inhibition of colony formation, are among
the effects which are usually associated with the nucleotide
type of absorption, while spheration of the nucleolus and
spindle damage are among those in which the primary energy
absorption appears to be in protein. The study of biological
damage induced by u.v. radiation and radiomimetics, how-
ever, gives us no reliable evidence concerning damage induced
by ionizing radiations, since many cases are known in which
the damage induced by these several agents proceeds, at
least in part, by different pathways (Gray, 1954).

Ionizing radiation delivers energy to atoms in a highly
localized, but unselective, manner, almost regardless of the
molecular configurations of which they form a part. The
types of chemical change which follow in small and large
molecules have been described by Dale and Butler (this

255

256 L. H. Gray

symposium), but direct observation of the living cell im-
mediately after irradiation has not so far yielded any definitive
information as to the relative importance of these changes for
the initiation of radiobiological damage. Burns (1954) has
set limits of —1-7 cc./mole to +2-4 cc./mole to the early
chemical changes associated with lethality in haploid yeast,
which are probably recessive lethal mutations. This figure
may be compared, for example, with changes of — 20 to
— 70 cc./mole for the volume change associated with each
peptide bond broken in the enzymic hydrolysis of protein.
Moreover, positive results were obtained by McElroy (1952)
and McElroy and Swanson (1951) for u.v. and nitrogen
mustard mutations in Neurospora and Aspergillus. The inter-
pretation of the negative results with ionizing radiation should,
however, be accepted with caution as far as reactions which
take place along the track of the ionizing particle are concerned,
since they involve the application of thermodynamic consider-
ations to a highly transient and irreversible system.

The yield of information by the chemical approach is thus
very small. It may be that we have not yet examined radiation
response in chemical systems at the right level of organization
for the important physicochemical changes to be revealed.

Anatomical Evidence

The anatomical approach has proved more profitable. A
variety of experimental techniques have been brought to
bear on this problem.

1. Tiie use of radiations of limited penetrating power

Many years ago Zirkle (1932) took advantage of the hmited
range of the polonium alpha particle (up to 30 y.) and of the
fact that in the fern spore the nucleus (diameter 10 (j.) lies to
one side of the protoplast (diameter 38 pi), to compare the
results of irradiations which included or excluded the nucleus.
The biological criteria were inhibition of cell division. All
these effects could be brought about by irradiations from the

Pathways of Radiobiological Damage 257

side remote from the nucleus, but only by the use of doses at
least twenty times as great as when the nucleus was included
in the field of irradiation. Zirkle drew attention to the interest-
ing fact that the cracking of the cell wall, which is a function
related to water imbibition and to chlorophyll development,
which might not normally be thought of as under nuclear
control, was evidently initiated by moderate doses of radiation
through an injury originating in the nucleus.

About the same time Henshaw and Henshaw (1933)
exposed Drosophila eggs to polonium alpha particles at dif-
ferent times after the eggs were laid and found a strong
positive correlation between the proportion of eggs prevented
from hatching by a given exposure to alpha radiation and the
inclusion of nuclei in the irradiation field. The correlation was
the more striking because the stage of development which
brought the largest number of nuclei into the field of irradia-
tion happened to be one of minimum sensitivity to X-rays,
which, of course, irradiate the whole egg uniformly.

Within recent years this type of study has been extended
by Pollard (1955) and his colleagues to smaller cells by the use
of very slow electrons. In this case specimens have to be
irradiated in vacuo. This automatically excludes any form of
radiation damage which may proceed from ionization of the
aqueous phase and does not measure the inactivating effect
of radiation under physiological conditions. On the other
hand, the fact that the Yale Group have obtained results with
biologically active molecules and viruses under precisely these
conditions, which in general accord rather well with studies
of the same molecules and viruses irradiated under more
natural conditions, may be considered to justify a cautious
acceptance of the results obtained with slow electrons. These
indicate in the case oi Bacillus suhtilis spores (Hutchinson, 1955)
that from the standpoint of viability (colony-forming ability)
after irradiation, the spore has a completely insensitive coat
about 230 A thick, a body of intermediate sensitivity which is
of smaller size but approaches to within 20-30 A units of the
surface at one point, and a comparatively sensitive core.

RAD. 10

258 L. H. Gray

The structure of B. subtilis has not yet been estabhshed by
staining methods but in Bacillus megatherium cytological
structure has been described by several workers (Robinow,
1953; Yuasa, 1956) which corresponds rather strikingly to
Hutchinson's (1955) description.

Davis (1954) has studied the inactivation of Tl bacterio-
phage by slow electrons. She finds that the surface coat of the
phage particle, about 100 A units thick, is extremely insensi-
tive. If the surface coat is identified with protein in this case
and the core with DNA, then it is evident that when the
whole virus is exposed to radiation in the dry state the
inactivation is predominantly associated with energy de-
posited in the DNA. Energy deposition in the protein only
brings about inactivation at a dose level sixteen times higher
than that which suffices when energy is absorbed in the DNA.
The irradiation of oriented tobacco mosaic virus particles
leads to the same conclusion (Pollard and Whitmore, 1955).

2. Micro Beams

The irradiation of selected small regions of living cells by
protons and by pencils of u.v. radiation has been brought to
a high degree of technical perfection by Zirkle and Bloom
(1953). In the case of the proton beam 80 per cent of the
particles fall within a circle 2-5 [x in diameter and 96 per cent
within a circle 5 (jl in diameter. The convergent hetero-
chromatic u.v. beam intensely irradiates an approximately
isodiametric volume about 7 [x across. The cells principally
studied have been from adult newt heart cultured at 23° C
and observed by phase contrast illumination as they enter
and pass through division.

Irradiation of the centromere region of a chromosome by
either radiation may result in the loss by that chromosome
of directed movement, so that it never moves to the equatorial
plate. Similar irradiation of regions of the chromosome not
including the centromere were never observed to produce this
effect. Somewhat larger exposure to u.v. resulted in a change
in refractive index which was at first confined to the length of

Pathways of Radiobiological Damage 259

the chromosome actually irradiated but which spread in the
course of 30 minutes to three times the original length. This
effect was only produced by protons when the dose was in-
creased more than 100-fold. Micro-beam irradiation of pro-
phase chromosomes with a few dozen protons was found to be
very effective in producing chromosome stickiness, and chrom-
osomes lacking functional centromeres and chromosome frag-
ments. Irradiation of metaphase chromosomes was about as
effective in producing chromosome stickiness but very rarely
destroyed the function of the centromere or produced chromo-
some fragments. In contrast to the marked effects of a few
dozen protons delivered to small chromosomal regions,
relatively huge numbers were ineffective when delivered to
similarly small extrachromosomal regions. Thousands were
delivered to ends of spindles, and hundreds of thousands to
cytoplasm. No effects could be seen at the irradiation sites
or elsewhere. An occasional sticky chromosome which was
seen could be ascribed to protons which were scattered into
the chromosomes when very large numbers were aimed at
extrachromosomal targets.

In contrast to the protons, heretochromatic u.v. radiation
produced very striking effects when extrachromosomal regions
were given exposures of the same order as those which, given
to chromosomal regions, produced stickiness and loss of
centromere function. To destroy the spindle and induce a
deranged metaphase it is not necessary to include part of
the spindle in the u.v. irradiated region. A slightly greater
exposure of an equal volume of cytoplasm produces the same
effect, from which it was concluded that effects on the spindle
were quite probably due entirely to the absorption of u.v.
radiation by some cytoplasmic component. We may recall
here the observations of Carlson and McMaster (1951), who
used a variety of monochromatic u.v. radiations, that in
grasshopper neuroblasts the derangement of spindle mechan-
ism exhibited a protein-like action spectrum.

It had, of course, long been believed that the chromosome
fragments seen at metaphase following the irradiation of

260 L. H. Gray

many different types of cell in interphase or early prophase
were the result of the passage of a single ionizing particle
either through, or in the immediate vicinity of, the chromo-
some thread.

Zirkle and Bloom's experiments strongly support this view
but they do not, of course, prove that only one particle was
involved. They only irradiated a very small fraction of the
length of the chromosome thread in any one exposure, and in
order to secure a reasonable frequency of breakage had to use
10-12 protons, corresponding to an average dose within the
micro beam of perhaps 2,000 rads. The inference that breaks
are produced by single ionizing particles still rests on the linear
relation between breakage frequency and dose. Since this
relation was observed by Kotval and Gray (1947) to hold even
at such low doses (4 rads.) that few of the irradiated nuclei
were traversed at any time by more than one particle, there
can be little doubt about the validity of this inference in this
particular case. Similar considerations make it rather certain
that the induction of lysogenicity in bacteria observed by
Marcovich (1954) must be due to a single electron.

3. Nuclear Transfers

The fertilization of the ovum provides a natural means of
introducing an irradiated nucleus into unirradiated cytoplasm
and, in special cases, of the opportunity to study the converse,
namely the development of a cell containing an unirradiated
nucleus in irradiated cytoplasm.

As remarked earlier (p. 257) developing Drosophila eggs are
more readily killed by nuclear than by cytoplasmic damage.
Opinion is still divided as to whether the killing of these eggs
at their most radiosensitive stage can reasonably be ascribed j
entirely to nuclear damage.*

Direct evidence, however, has been provided by Whiting
(1949) through her studies of radiation damage in Habrohracon.

* Note added in proof: Rather conclusive evidence in favour of the import-'
ance of nuclear damage at this stage has been presented by Ulrich (1955), who
compared the lethality among eggs in which either the anterior half only or ^
the posterior half only had been exposed to X-rays.

Pathways of Radiobiological Damage 261

By appropriate matings, individuals could be obtained
derived either from irradiated cytoplasm and irradiated
(female) nucleus or irradiated cytoplasm and unirradiated
(male) nucleus. The dose required to inhibit development of
the latter was 54,000 rads., which was twenty-two times as
great as that required to inhibit the former. The author con-
cluded that in those animals which failed to develop after
irradiation of the cytoplasm only, the injured cytoplasm
acts in a direct manner in killing the embryo and not indirectly
through injury to the untreated chromosomes. However,
Nakao (1953) has recently presented evidence to show that
when fairly heavily irradiated silkworm eggs are fertilized by
unirradiated sperm within 2^ hours of irradiation, the eggs
which are laid show plenotypic changes which are character-
istic of the loss or mutation of certain genes located on the
paternal chromosomes.

One of the earliest observable effects of radiation in many
types of cell is what is commonly referred to as the inhibition
of mitosis or of cell division. In eggs it is observed as a delay
in first cleavage, in yeast as a delay in the second budding
after irradiation, and in dividing tissues as an immediate fall
in the mitotic index. Most, or all, cells of a population which
is at a uniform stage of development are affected, and to an
extent which increases with the dose. It is measurable in grass-
hopper neuroblasts after a dose of only 4 r, in many plant
and animal dividing tissues after doses of about 50 r, in yeast
after about 1,000 r and in sea urchin eggs after about 10,000 r.

Careful investigation of the phenomenon in several of the
classes of cell mentioned has revealed, as a common feature, a
prolongation of the time taken by the cell to pass from the
terminal stage of interphase to the end of prophase. The classi-
cal experiments of Henshaw (1940) on cleavage delay in the
eggs of the sea urchin (Arbacia punctilata), discussed quantita-
tively in detail by Lea (1946), have provided the following
information :

(a) Irradiation at the dose levels employed does not affect
fertilization, the approach of the two pronuclei, or

262 L. H. Gray

fusion, all of which proceed at the normal rate, but it
does result in a great prolongation of the first mitotic
prophase.

(b) Irradiation of either sperm or egg alone before fertiliza-
tion produces a comparable delay in cleavage.

(c) The injury to the egg is one which is repaired at a rate
of about 1 per cent /minute at 20-25° C and 0-3 per
cent /minute at 0° C. No detectable repair takes place
in the sperm.

(d) Irradiation of enucleated eggs prior to fertilization
causes no cleavage delay, though the time taken in
cleavage is in this case much longer than in the cleavage
of a normal egg.

(e) When the sperm and the egg are each irradiated prior to
fertilization, the injuries sustained by each exactly
summate in their effect on cleavage delay, if allowance
is made for repair.

The conclusion of Henshaw and of Lea that in this case the
prolongation of prophase is the result of a reparable injury
sustained by nuclear material seems inescapable. In view of
the period of the cell cycle which is critical in this phenomenon,
it is suggested that the injury is one connected with the con-
densation of the chromosomes. The further conclusion that
the nuclear material is directly injured is much less well
founded. Direct injury of nuclear material seems probable
since the delay is the same whether it is the sperm or the egg
which is irradiated despite the enormous disparity in the
respective volumes of cytoplasm and because of the absence
of effect in enucleated eggs. However, the sperm is not devoid
of cytoplasm, and nuclear injury could conceivably arise
through a disturbance in cytoplasmic metabolism. The dose
relations are not linear but those of an effect which varies with
the log of the dose. This suggests the existence of material,
essential to the passage of the cell through the critical phase,
which is inactivated at a rate which is some function of the
administered dose and repaired at a rate which depends on

Pathways of Radiobiological Damage 263

the general level of metabolic activity {cf. the "cumulative
dose" concept developed by Lea (1946), Friedenwald and
Sigelman's (1953) treatment of mitotic delay in corneal epithel-
ium and Burns' (1954) treatment of division delay in yeast).

Temporary arrest of cells about to enter division may be
caused by a great many agents, including hypoxia, depletion
of phosphate and glycogen. The possibility — indeed one may
even say the probability — must always be kept in mind that a
given result is achieved by different pathways under the
influence of chemical, u.v. and ionizing agents.

In one or two materials the transfer of nuclei by microdissec-
tion procedures has been used to study the respective roles of
nuclear and cytoplasmic damage. Duryee (1949) studied the
incidence of nuclear pyknosis which develops in the course of a
few days at 22° C in salamander oocytes after exposure to
doses of around 3,000 rads. The oocytes were in early meiosis
at the time of irradiation. This form of damage appears
within half an hour if the dose to the oocytes is raised to 50,000
rads. When nuclei were isolated from the egg by microdis-
section and washed free of cytoplasm, this dose of radiation
produced negligible damage. Since in the intact egg this dose
did not produce immediate visible damage it is clear that even
after such relatively large doses metabolic processes must
intervene before the injury is apparent, and it is therefore not
unexpected that isolated nuclei appear unaffected. That the
nuclei have not been rendered insensitive by the microdis-
section was shown by the fact that the injury developed if
they were exposed to irradiation in the presence of cytoplasmic
brei, prepared by grinding in a mortar the contents of three
eggs from which the nuclei had been^removed. The brei could
either have provided conditions essential for the metabolism
of the nucleus or injured the nucleus indirectly through a
toxic product formed in the brei as a result of irradiation.
Evidence in support of the latter view was contributed by
microdissection experiments in which cytoplasm from irradi-
ated eggs (3,000 rads.) was transferred to unirradiated eggs
and resulted in nuclear pyknosis. The microdissection

264 L. H. Gray

procedure itself occasionally resulted in pyknosis. Discrete
chromosome fragmentation, which arises from irradiation of
the intact oocyte, was not recorded as a result of the injection
of irradiated cytoplasm.

Ord and co-workers (1952) have used nuclear transfers
between amoebae to evaluate the site of action of nitrogen
mustard and X-radiation when these agents give rise to
division delay and cell death. It was not possible to irradiate
isolated nuclei. Inferences were based on comparisons be-
tween amoebae treated whole and amoebae reconstituted
from an untreated nucleus and cytoplasm treated either in the
presence or absence of a nucleus. Although both agents are
lethal to the cell at considerably lower doses when the nucleus
is treated than when only cytoplasm is treated, important
differences between the effects of the two agents were noted.
With regard to radiation damage, it was found that damage to
the cytoplasm was of two kinds, a reversible damage which is
maximal at 100,000 rads., and lethal damage which becomes
prominent at 290,000 rads. This is, of course, a very high
dose. Exposure of an aqueous solution to this dose could
completely transform reactants at the millimolar concentra-
tion level. It is not surprising that it should be lethal to
cytoplasm. The comparatively high mean lethal dose for
nuclear damage (120,000 rads.) may be due to the occurrence
of each genetic factor at a high multiplicity (high polyploidy
or polyteny) as is almost certainly the case with Paramecium,
which has a radiosensitivity comparable with that of Amoeba
(Kimball, 1949).

It was concluded that a dose of X-rays which is lethal to all
nuclei in the amoeba will not cause lethal damage to cytoplasm,
and that the nuclei were probably damaged independently of
the cytoplasm.

Polyploid and Multinucleate Cells

The study of polyploid and multinucleate micro-organisms
has thrown considerable light on the matter under discussion.

Pathways of Radiobiological Damage 265

The beautiful experiments of Latarjet and Ephrussi (1949)
with haploid and diploid yeast have become classical in this
connection. Within the last few years our knowledge of the
radiobiology of yeast has been greatly extended by the very
careful investigations of the Berkeley Group led by Tobias
(1952). Haploid yeast cells exposed to X-rays either form a
double, of which only one cell contains nuclear material, or
form a complete colony. Irradiated diploid cells give rise to
all intermediate forms between the double and the complete
colony. Correspondingly haploid cells which had not budded
at the time of irradiation show a strictly exponential relation
between survival fraction and dose, whether exposed to X- or
alpha-radiation, while diploid cells show a sigmoid relation
for both radiations, indicating that more than one particle is
responsible for the lethal damage. Budding cells, in which
mitosis is in progress, are much more resistant than the
interphase cells and even in haploid yeast do not show an
exponential relation between survival and dose.

An elaborate analysis of the radiosensitivity of haploid,
diploid, triploid and tetraploid cells, and of the sensitivity of
clones grown from survivors from a previous irradiation, led
the authors at one time to conclude that the lethality was
entirely explicable in terms of recessive lethal mutations.
This proved to be too simple an interpretation. At present,
death of haploid cells is ascribed to the induction of recessive
lethal mutations — some of which could be chromosomal —
and that of other ploidies to a mixture of recessive and
dominant lethality. Contributions from cytoplasmic damage
cannot be excluded in the case of the higher ploidies.

Finally, we may consider the very interesting experiments of
Atwood (1955, and personal communication), who studied the
radiosensitivity of binucleate and multinucleate Neurospora
conidia. Binucleate conidia were formed in which the two
nuclei carried, as markers, genes for nutritional deficiencies.
Such binucleate cells will grow on minimal medium. When
irradiated and grown on complete medium, sigmoid survival
curves were observed similar to those for diploid yeast, the

266 L. H. Gray

shape corresponding well with expectation for a two-hit type
of effect. When grown on minimal medium, however, log
survival curves were strictly linear and with the slope approxi-
mately double that of the asymptotic (low survival) portion
of the log survival curves of organisms grown on complete
medium (LD37 --^ 15,000 r). These relations are to be expected
between survival on complete and minimal medium if the
injury is of the nature of a dominant lethal mutation, since
on minimal medium inactivation of either nucleus would be
lethal to the spore. Atwood concluded that the injury which
was lethal to the spore was entirely nuclear in origin and of
the nature of a dominant lethal mutation. The experiment
was repeated using different combinations of the markers and
with trinucleate cells, each of which was doubly marked. All
were consistent with the hypothesis of dominant lethal
mutation as the cause of death. Whatever the precise nature
of the damage to the genetic material, its effects are obviously
very far-reaching since a nucleus so damaged is unable even
to synthesize the single amino acid or other growth factor
required by the second nucleus. It is remarkable that meta-
bolic activity can be so completely inhibited by the passage of
a single ionizing particle.

Summary

The results which have been discussed are summarily
classified in Table I. It is at once obvious that the forms of
damage which have been analysed are not representative of
radiobiology as a whole. Seven out of sixteen entries are con-
cerned with cell reproduction, and in these the criteria
adopted are tantamount to a test of the reproductive integrity
of the cell. Six other cases are concerned with nuclear
components or nuclear function. To destroy the reproductive
integrity of the cancer cell is the aim of radiotherapy and in
this connection the effects listed in Table I are of special
interest despite their limited range. The cell is a unit of
biological organization and, as might be expected, vital

Pathways of Radiobiological Damage

267

Q
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I— I

O O

o
o

H
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I— (

Davis (1954)

Burns (1954)
Latarjet and

Ephrussi (1949)
Atwood (personal

communication)
Ord et al. (1952)
Henshaw (1940)

Duryee (1949)

Nakao (1953)
Henshaw and Hen-
shaw (1933)
Whiting (1949)
Zirkle (1932)

Zirkle and Bloom
(1953)

Sheppard and
Stewart (1052)

Mito-
sis

inter-
venes

+ ++ + 1

1 1 + + 1 1

1 1 1

jvuajvtu oif9Ud3

fo ssoj p)jmvqD3Ui

fo njijiqissoj

1 + + 1

1 1 + + 1 1

1 1 1

Provisional
assignment of

site of
primary injury

Cyto-
plasm

lift f o^

in inm

ooo

+ +-• ooi
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6
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"<0500 o ceo

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in in in

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+ ++ ooo

AAA

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ooo

I-l r-l I-H
I I I

Not known

to involve

the nucleus

Chlorophyll

synthesis

Cracking of

Net loss of
potassium
(40,000 r)

Synthesis not

necessarily

essential

Cleavage

delay
Nuclear

pyknosis
Egg coloiir

Cell division

Chromosome

breaks
Chromosome

stickiness
Inactivation

of centro-

mere
Spindle
damage

Synthesis of
gen. mater-
ial essential

Rep. integ.
(Plaques)
Rep. integ.
Rep. integ.

Rep. integ.

Rep. integ.
(Hatching)
Rep. integ.

Doses

in
rods.

15,000
60,000

15,000

100,000
10,000

3,000

3,000
200

2,000
10,000 (a)
30,000 (a)

o
o

o_^

Tl (dry)

Haploid yeast
J Diploid yeast

i Neurospora

conidia
Amoeba
Arbacia sperm

Salamander

oocytes
Silkworm eggs
Dros. eggs ^

Habrabracon eggs
Fern spores

Human
erythrocyte

■

Virus

Micro-
organisms

Protozoa
Crustacea

Amphibia
Insect

Plant

Tn vitro
Culture

268 L. H. Gray

processes can be destroyed by a sufficiently large dose to either
nucleus or cytoplasm. The mammalian erythrocyte provides
positive evidence for cytoplasmic damage (Sheppard and
Stewart, 1952). In ten of the cases listed in Table I it has been
found, however, that over a certain range of doses a given
effect is produced if the nucleus is included in the field of
irradiation and not produced if the nucleus is either excluded
from the field of irradiation or introduced into the cell after
irradiation of the cytoplasm. In one or two cases the same
effect has been achieved by the irradiation of cytoplasm
without irradiation of nuclear material, but only by the use
of doses 3-20 times greater than those which are sufficient
when nuclear material is irradiated. If we admit also the less
direct evidence presented in the case of lethality in haploid
and diploid yeast and in Neurospora conidia, then in thirteen
cases out of sixteen it may be said that the cell owes its
sensitivity predominantly to the susceptibility of its nuclear
material to injury by ionizing radiation.

Account must be taken of the fact that in all the cases con-
sidered, the nucleus has been irradiated in the presence of
cytoplasm. It might be argued, therefore, that the injury
which makes itself apparent in the nucleus is in fact secondary
to a cytoplasmic injury.

It would appear that nuclear damage can be subdivided into :

(a) Forms in which the damage is seen in most or all cells
at a given stage of development, is graded with dose,
and for which formal dose relations are not character-
istic of a single particle initiation. Cleavage delay,
division delay and unspecific nuclear pyknosis are
typical examples.

(b) Effects seen in some cells and not at all in others, such
as the induction of mutations and chromosome struc-
tural damage, having dose relations characteristic of
individual particle effects.

With regard to Class (b) it would, in my view, be difficult to
sustain this hypothesis in the light of the experiments of

Pathways of Radiobiological Damage 269

Zirkle and Bloom and of the relations which have been
observed between survival and dose.

The evidence in the case of Class (a) damage is equivocal.
Some of the experiments of Henshaw and Whiting were
expressly designed to reveal nuclear injury resulting from
^-primary damage to the cytoplasm, but failed to do so even at
quite high dose levels. However, nuclear damage arising
from a transient disturbance in cytoplasmic metabolism or to
labile toxic products produced in the cytoplasm could have
escaped detection, despite the fact that eggs were fertilized
immediately after irradiation.

Kaufmann, McDonald and Bernstein (1955) have observed
that doses of 250-1,000 r delivered to Drosophila larvae
salivary gland cells in vivo affect the colloidal properties of the
cells, and that doses of 1,000 r delivered in vitro affect the
stability of gels of calf thymus nuclei. Somewhat analogous
experiments have been made by Anderson (personal com-
munication). Kaufmann and co-workers show further that
nucleoprotein is damaged by doses (1,000 r) which are without
effect on either the DNA or the protein components when
irradiated separately. These experiments appear to have an
important bearing on Class (a) nuclear damage. They may also
provide the clue to the outstanding sensitivity of lymphocytes
and thymocytes since the lethal effects of radiation on these
two classes of cell bear certain resemblances to biological
effects known to proceed from Class (a) nuclear damage.

The metabolic study of Class (b) nuclear damage, as induced
by ionizing radiations, appears to pose an extremely difficult
problem on account of the random nature of the primary
injuries. It would seem that there must be a period of time
during which a lethal injury is following hundreds of different
pathways in different cells. Mutations which ultimately prove
lethal may only be expressed a considerable time after irradi-
ation, either because the definitive mutation is delayed until
the time of gene reproduction or because nuclear control over
cellular metabolism is not immediate. During this interval of
time an opportunity is offered for the study of metabolic

270 L- H. Gray

disturbances induced at higher dose levels as a result of either
nuclear or cytoplasmic damage, but these may be quite
irrelevant to the metabolic pathways leading to loss of repro-
ductive integrity at low dose levels.

REFERENCES

Atwood, K. C. (1955). Amer. Naturalist, 88, 295.

Burns, V. W. (1954). U.C.R.L. Report 2812.

Carlson, J. G., and McMaster, R. D. (1951). Exp. Cell Res., 2, 434.

Davis, M. (1954). Physiol. Rev., 94, 293.

DuRYEE, W. R. (1949). J. nat. Cancer Inst., 10, 3.

Friedenwald, J. S., and Sigelman, S. (1953). Exp. Cell Res., 4, 1.

Gray, L. H. (1954). Radiation Res., 1, 189.

Henshaw, p. S. (1940). Amer. J. Roentgenol., 43, 899.

Henshaw, p. S., and Henshaw, C. T. (1933). Biol. Bull, Woods Hole,

64, 348.
Hutchinson, F. (1955). Ann. N.Y. Acad. Sci., 59, 494.
Kaufmann, B. p., McDonald, M. R., and Bernstein, M. H. (1955).

Ann. N.Y. Acad. Sci., 59, 553.
Kimball, R. F. (1949). Genetics, 34, 412.
KoTVAL, J. P., and Gray, L. H. (1947). J. Genet., 48, 135.
Latarjet, R., and Ephrussi, B. (1949). C.R. Acad. Sci., Paris, 229, 306.
Lea, D. E. (1946). Actions of Radiations on Living Cells. Cambridge

University Press.
Marcovich, H. (1954). Nature, Lond., 174, 796.
McElroy, W. D. (1952). Science, 115, 623.

McElroy, W. D., and Swanson, C. P. (1951). Quart. Rev. Biol, 26, 348.
Nakao, Y. (1953). Nature, Lond., 172, 625.
Ord, M. G., Danielli, J. F., Harris, E. B., and Lamerton, L. F.

(1952). Nature, Lond., 170, 921.
Pollard, E. C. (1955). Ann. N.Y. Acad. Sci., 59, 469.
Pollard, E. C, and Whitmore, G. F. (1955). Science, 122, 335.
RoBiNOW, C. F. (1953). J. Bad., 66, 302.
Sheppard, C. W., and Stewart, N. (1952). J. cell. comp. Physiol, 39,

Suppl. 2.
Tobias, C. A. (1952). Symposium on Radiobiology, p. 357. London:

Chapman & Hall.
Ulrich, H. (1955). Naturwissenschaften, 16, 468.
Whiting, A. R. (1949). Biol Bull, Woods Hole, 97, 210.
YuASA, A. (1956). Nature, Lond., Ill, 387.
ZiRKLE, R. E. (1932). J. cell. comp. Physiol, 2, 251.
ZiRKLE, R. E., and Bloom, W. (1953). Science, 111, No. 3045, 487.

DISCUSSION

Latarjet: I should like to say a few words about this problem of
ploidy and survival curves. The main fact, as Dr. Gray mentioned, is
that the number of hits which comes out of the survival curve fits with

Discussion 271

the ploidy. This has been found in all types of cells which have been
investigated so far. However, in spite of the fact that several full
papers have been written by others on this subject, a major difficulty
remains which Dr. B. Ephrussi and I encountered in our original work.
When ploidy increases, not only does the number of hits increase
accordingly, but also the radioresistance of the individual unit which
undergoes the hit. The slope of the straight part of the survival curve
decreases.

Let us consider the simplest situation, that, for example, of several
virus particles inside the same cell, which can multiply within the
irradiated cell as long as they are themselves active, and let us suppose
that the cell remains infective as long as it contains one active particle.
It has been found that the survival curve of the " infectivity " of such
multiply infected cells, early after infection, i.e. before the virus has
begun to multiply, fits in with the multiplicity of infection. The curve
agrees with the general equation :

survival = 1 — (1 — e - aD)ri

where n is the number of infecting particles, d the dose, and a the coeffic-
ient which characterizes the radiosensitivity of the individual particle.
When n increases, a does not change, all curves have parallel straight
parts.

In the case of ploidy, however, a decreases when n increases. At the
same time, the morphology of the lesions may differ. In haploid yeast,
one observes only immediate death and double giant lethals. In
polyploid yeast, one observes in addition the many classical figures of
delayed recovery. I think that in the latter, some kind of multiplicity
reactivation takes place, but I really do not know what this means for
chromosomes. As a matter of fact, no simple hypothesis has given a
satisfactory account for the experimental results.

Gray: With regard to Norman and At wood's results, I think these are
exactly the results to be expected because in a cell containing two
nuclei with nutritional deficiency you have two targets, the inactivation
of either of which is lethal if you plate on minimal medium. If you plate
on complete medium you must inactivate each of two targets. The
slope of the former inactivation curve being twice that of the latter is
thus in accordance with expectation. In fact, the slopes differ by a little
less than a factor of two, because there were recessive lethals to be taken
into consideration. I had not spotted this in the yeast.

Swanson: In Tradescantia one can irradiate cells which are either
haploid or diploid, and the chromosome sensitivity is different on a
chromosome basis. Yet these presumably are the same types of chromo-
somes. We have done this fairly extensively on diploid and tetraploid
individuals of the same species of Tradescantia, but we found a con-
siderable difference in sensitivity in microspore chromosomes.

Butler: Dr. Gray mentioned the effect of pressure; I don't think that
really works. The thermodynamic theory is based on equilibrium con-
siderations, there is an activated state through which the material is
passing. With ionizing particles you have got something equivalent

272 Discussion

to a very high temperature. If you wanted to work this out you would
have to introduce the equivalent temperature, in other words the
temperature which would produce that particle, so that your tempera-
ture would be very high and you would not expect any volume change.
The same applies to the effect of temperature. There should be no effect
of temperature on the initiating step, but if you do get an effect of
temperature it is an effect on the subsequent steps.

Lajtha : Does one get these ploidy effects only if one irradiates under
dry conditions, or do they occur if the organism is suspended in dilute
medium ?

Gray : They are ordinary living cells, they are not dry.

Lajtha: The opposite happens in mammalian cells and in living bean
roots where the actual synthetic stage during which the 2 n DNA in-
creases to 4 n seems to be less sensitive to small doses of radiation than
the interphase stage with its stationary 2 n amount of DNA.

Gray: If you score the amount of chromosome damage per cell, this
amount of damage increases with ploidy, but of course it may be less
lethal to the cell because damage to any one chromosome is more
likely to be covered by the other chromosome sets. Diploid yeast is less
sensitive than the haploid; the same applies to Neurospora, the higher
ploidy is less sensitive despite the fact that more actual chromosomal
damage is produced.

Koller: Miss Lamy, working with MuUer in Edinburgh, found no
difference in radiation sensitivity between the diploid and the triploid
Drosophila.

Gray: This was concluded in yeast in the higher ploidies also.

Alexander: I think an important point arises both out of Dr. Gray's
and Dr. Swanson's papers and that is : is the breakage of the chromo-
somes already a metabolic event or is this the primary chemical change
following directly on the absorption of the energy from the radiation ?
I believe that the amount of energy which Lee and others have calcu-
lated as necessary for giving a break is not sufficient to turn the nucleo-
protein from the chromosome from a gel into a sol. This would have to
be the case if the chromosome-break was produced by chemical action.
I think even for the break it may be necessary to postulate some
metabolic process. This view, I believe, is supported by the experiments
on the effect of radiation on the properties of the nucleoprotein gel
which I mentioned earlier (this symposium, p. 57). Irradiation of whole
cells produces much greater changes than irradiation of nuclei. A
possible interpretation is that the nucleoprotein is not damaged by a
few hundred r at the concentration at which it is present in the cell, but
that an enzyme acting on the nucleoprotein is released by radiation.
Dounce showed that there are enzymes in the cell which can liquify this
nucleoprotein gel extremely efficiently, and that only the most careful
preparations of nuclei which are absolutely clean from all adhering
matter can give nucleoproteins which remain as stable gels.

Swanson: If you are referring to chromosome breaks, wouldn't this
presuppose that there should be a posttreatment effect?

Alexander: Yes, but it might have to be extremely rapid. In this

Discussion 273

connection I was encouraged by the report from Dr. Hollaender (this
symposium p. 206) that mitotic suppression could be reduced by treat-
ment with hypertonic salt immediately after irradiation. A posttreat-
ment would have to be given quickly because if it is in fact a question of
allowing enzyme to get as a gel particle, then this may happen within
seconds after irradiation. The suggestion that the attack on the chromo-
somes does not follow directly from the uptake of energy certainly
raises the possibility that mutations may be prevented by a suitable
aftertreatment.

Swanson: In terms of breaks we have been able to use only two
devices to modify their frequency by posttreatment : one is mechanical,
i.e. centrifugation, the other is infrared, which is another irradiation.
There is no other means of which I am aware.

Alexander : One really wants a physical method, because chemicals
would have to diffuse in and it is obviously difficult for one diffusion
process to catch up with another one.

Gray: Dr. Alexander, I wonder whether, in the experiments to which
you refer, the number of nuclei w hich are affected is proportional to dose,
or whether all nuclei are affected but to a degree which varies with the
dose.

Alexander: I suspect that radiation affects many nuclei to some
extent, the effect is not confined to a few. These experiments are rather
difficult to do accurately because of the difficulty of isolating clean nuclei.
Some of the apparent discrepancies in the literature concerning the
effect of radiation on cellular nucleoproteins could be explained if a meta-
bolic process intervened since time factors then become all-important.
Butler: I think that would be explained if the nuclear damage caused
by radiation made the nucleoprotein more open to attack by enzymes.
Nucleoprotein preparations all slowly break down, and it appears to us
that that is due to the action of deoxyribonuclease, because we found
that the breakdown is accompanied by a degradation of the DNA.
If you examine the DNA from an intact gel and then examine the DNA
from the nucleoprotein gel which has been kept for some time, and has
become liquified, you find that it is the DNA that has become degraded.
Therefore we conclude that this is an action of deoxyribonuclease which
may well have come from the cytoplasm, so that your observation could
be explained as the action of radiation on the nucleus causing some
degree of damage to the nucleoprotein material which in some way
renders the entry of the deoxyribonuclease easier.

Alexander: That was the exact interpretation which I had placed on
it, that is, the irradiation is not sufficient to liquify the gel. All it did was
to enable a metabolic process to take place. Butler's suggestion that the
nucleoproteins from cells become more susceptible to attack by enzymes
after irradiation has already been established by Cole and Ellis. But
this effect by itself could not explain the formation of chromosome
breaks since any enzymes capable of attacking nucleoproteins must be
stored well away from the chromosomes if cells are to survive at all,

Butler: There would have to be initial radiation damage. You are
merely showing it up.

274 Discussion

Pirie: Does your weakening effect increase with time after radiation
or can you stop it, i.e. if you irradiate your whole cell and immediately
take out the nuclei, are they then stable?

Alexander: There is certainly a time effect with whole cells but for
technical reasons it is very difficult to reduce the time interval to less
than about five minutes.

Holmes: Do we know any useful DNAse inhibitors? You could not
apply them after irradiation, they would not act quickly enough.
Could they be used as protective substances ? In the case of a proteolytic
enzyme, for instance, we might imagine that cystine and cysteine would
interfere with it.

Alexander: This is an idea to which Prof. Bacq and I have given much
thought. We don't think that DNAse is the only enzyme concerned
and it is possible that there are some other enzymes which are capable
of de-geling the nucleoprotein in other ways.

Spiegelman : Has the effect of citrate been tried on this ?

Alexander: Yes, but citrate does not protect.

Gray: Where is the DNAse situated in the cells?

Bracket: It is mostly cytoplasmic.

Alexander: Is there evidence for any DNAse in the nucleus?

Bracket: There is always the possibility that some of the DNAse is
sticking to the nuclei. One finds a little DNAse in them, but it is dif-
ficult to say, when an enzyme is present only in small amounts in a given
cellular fraction, that it has not been adsorbed during the isolation pro-
cesses.

Hollaender: We would like very much to have a physical anti-ioniza-
tion agent. Hypertonic salt solution is probably something of a physical
agent; putting a new balance of ions in where this balance has been
upset. But if one could visualize an agent which would counteract the
ions, hold them in some form immediately following radiation, I think
the problem would be very much simpler.

Swanson: There is one possibility in the infrared. Some work has
been published recently by Moh and Withrow (1955, Plant Physiol.,
abstracts), where the 6200 A region is believed to be inhibitory in
terms of chromosome breaks, while the 7100-8200 A region adds to the
X-ray damage. These correspond to the regions of the spectrum that
were worked on by Hendricks in his seed germination studies, with the
shorter wavelengths being inhibitory, the longer ones capable of break-
ing dormancy.

I EFFECTS OF RADIATION AND PEROXIDES ON
VIRAL AND BACTERIAL FUNCTIONS LINKED TO

DNA SPECIFICITY

Raymond Latarjet

Laboratoire Pasteur du VInstitut du Radium, Paris

In the course of the last few years, attention has been drawn
to the production of organic peroxides within irradiated
Hving cells, tissues, and organisms. These substances are in
general very reactive, and, in the presence of oxygen, they can
elicit chain reactions of peroxidation. It has been thought
that they might play the role of intermediates in the produc-
tion of certain radiolesions, i.e., the role of true radiomimetics,
profoundly affecting cell metabolism. As a matter of fact,
peroxides have already been considered responsible for the
posteffect of radiation, and for the sensitizing influence of
oxygen ; mutagenic and lethal effects have been obtained with
these substances; conversely, certain radiolesions have been
prevented by posttreatment with peroxidases.

These considerations led me some time ago to undertake
quantitative experimental comparisons of some effects pro-
duced by organic peroxides and by radiation in simple biological
systems such as a bacterial transforming agent, bacteriophages
and bacteria, with special emphasis on some specific hereditary
characters carried by their DNA. The present paper groups
the first results obtained by my collaborators and myself.
It should be clearly understood;

(a) that these results are of preliminary character;

(b) that differences among biological systems and even
among peroxides forbid any generalization on these
results at the present time;

(c) that, in our opinion, peroxides are only some examples
(perhaps very important ones) among the many chemical
mediators which are brought into play in irradiated
living systems.

275

276 Raymond Latarjet

Material and Methods

1. Bacteria and bacteriophages. Escherichia coli, non-
lysogenic strain B, and the phages of the T series, lysogenic
strain Kl2, its temperate phage X, and the strain K12S
sensitive to X, were used according to the classical techniques
for growth and plaque formation.

2. The transforming agent TP Sr, which confers resistance
in Pneumococcus to 2 mg. of streptomycin per ml. without
inducing bacteria of intermediate resistance, was chosen.
TP of several stocks were used, containing about 0 • 6 mg. of
DNA per ml. The techniques for preparation and purification
of this nucleic acid, for producing the bacterial transforma-
ations, and for quantitative titration of the active agent have
already been described in detail (Ephrussi-Taylor and Latarjet,
1955).

3. The X-ray source was my usual molybdenum target tube
operating at 37 kv and up to 42 mA. Its radiation, filtered
through 0-05 mm. aluminium, delivered up to 1 krad./sec.
to the preparation, with an average wave-length of 0-9 A.
The samples were irradiated in plexiglas cups containing 0-4
ml. spread in a layer which absorbed about 10 per cent of the
incident radiation. In some experiments, the cups were
placed in vacuum chambers with aluminium windows.

4. Two organic peroxides have been used:

(i) Commercial cumene hydroperoxide (Hercules Powder
Co.), a viscous liquid which contained 40 per cent of
active product (Formula I). Its aqueous solution
reached saturation for a concentration by weight of
about 10-4.

CH,— C — CHj
O — OH

Formula I

DNA AND Effects of Radiation and Peroxides 277

(ii) Crystallized disuccinoyl monoperoxide (Formula II),
synthesized in this laboratory by R. Royer and B.
Ekert. This is in the form of prismatic colourless
crystals, which melt at 128° C, and explode weakly and
without danger in a flame. They are water-soluble at a
concentration by weight of 10"^, and are very stable in
the dark and in dry air. This compound is immediately
hydrolysed when dissolved in water, yielding one
molecule of succinic acid and one molecule of succinic
peracid, which is in fact the active principle of the
solution.

CO — CH,— CHf-COOH

oh'"'"o'

CO— CHj— CH^— COOH

Formula II

The solution, in the dark at room temperature, liberates
about 5 per cent of its peroxidic oxygen per day. All solutions
were titrated for peroxide activity by Mr. B. Ekert. The
cumene peroxide was titrated by the thiofluorescein method
(Dubouloz, Monge-Hedde and Fondarai, 1947); the succinic
peroxide was titrated by oxidation of ferrous iron and spec-
trophotometric dosage (302 my.) of the ferric iron. Precision
reached 2 pig. of peroxide per ml.

In some respects, the — C — O — OH function of the cumene

i •

compound differs from the — C — O — OH of the succinic com-
pound. While many similarities were observed, striking dif-
ferences sometimes appeared in their action on the biological
samples. Cumene peroxide does not render the solution acid,

278

Raymond Latarjet

but succinic peroxide introduces some acidity which must be
taken into account by suitable controls in certain experiments.
If, in order to keep the pH at a given value, one dilutes the
peroxide in an acetate buffer, the titre in peroxidic oxygen,
and consequently the activity, remains unchanged. However,
we did confirm that the results which are reported in this
paper were actually due to the chemical effect of the peroxide,
not to acidity.

Inactivation of bacteria by peroxide

Bacteria were washed, then resuspended in saline in the
presence of peroxide. At given times, aliquots were diluted

0 3 6 9 12 15

MINUTES OF CONTACT

Fig. 1. Inactivation of growing Esch. coli B by succinic

peracid.

DNA AND Effects of Radiation and Peroxides 279

(in order to eliminate the peroxide) and then plated on
nutrient agar for colony counts. All survival curves were of
the multiple-hit type (Fig. 1). The results can be summarized
as follows:

(1) Sensitivity to peroxide varies as sensitivity to radiation.

12 16 20 24

MINUTESof CONTACT

Fig. 2. Inactivation of (1) growing and (2) resting Esch.
coll K12S by cumene hydroperoxide.

All bacteria are more sensitive in the growing than in the
resting stage (Fig. 2). If one compares several strains, one
finds not only the same order of increasing sensitivity to
peroxide and to radiation (B/r -> K12 -> B), but even the same

280

Raymond Latarjet

ratios. For example, strains B/r and K12S are equally
sensitive both to radiation and to peroxide; B/r is about 6
times as resistant as B to peroxide, as it also is to radiation.

(2) There is no photorestoration after peroxide treatment.
On the contrary, Dr. C. C. Brinton observed that under certain
conditions doses of light, which are harmless when given
alone, may strikingly increase the lethal effect of pretreatment
by peroxide. This interesting fact, which will be published,
recalls a former observation by Latarjet and Miletic (1953).

(3) The rate of inactivation very much depends on experi-
mental conditions. It rapidly increases with temperature.
It decreases when the bacterial concentration increases,
soluble organic compounds of the washed suspension and
killed cells providing efficient protection to the survivors.

Inactivation of bacteriophage by peroxide

Naked phage is very sensitive to peroxide. Inactivation
proceeds at a rate which is either exponential or of low
multiplicity of hits (Fig. 3).

(1) Sensitivity of phage may be influenced by its concentra-
tion, but this effect may depend on whether or not the phage
has been purified. A T2 lysate in 56 synthetic medium
containing 5 X 10^^ particles per ml. was dialysed against
distilled water, then diluted. Each dilution was treated for
15 minutes at 18° by 2 X 10"^ succinic peroxide (Table I).

Table I

Log phage concentra-

T2 survival per cent

tion per ml.

normal lysate

dialysed lysate

012

2-4

0-22

2-3

005

1-2

001

4-7

DNA AND Effects of Radiation and Peroxides 281

In the dialysed lysate, sensitivity is independent of concen-
tration below 10^; but in the normal sample, it steadily
increases with dilution, and becomes much greater than in the
purified lysate. This fact (and others which we encountered in

MINUTES of CONTACT
Fig. 3. Inactivation of phage T? by succinic peracid.

other types of experiments) shows that toxicity of peroxide
may increase in the presence of some organic substances. We
believe that when oxygen is present these substances can be
peroxidized by chain reactions, thus increasing the titre of
peroxide groups. This point should be studied, for example by
removing oxygen before peroxide treatment. One should

282 Raymond Latarjet

also consider, in the case of T2, the possible inactivation of
a phage inhibitor (Sagik, 1954) which would influence the
apparent rate of inactivation of the phage itself.

(2) A large phage like T2 can be inactivated by damage to
the DNA, or to the tail, or both. If DNA only is damaged, the
phage still attaches itself to its bacterial host, and kills it,
without multiplying. If some specific site at the tip of the
tail is knocked out the phage does not attach, or attaches in
such a fashion that it loses simultaneously its infective and
bactericidal powers.

It is known that X-rays attack both sites at different rates
depending on whether the radiation acts through direct or
indirect effect. When it acts indirectly both sites have about
the same sensitivity, but when the direct effect becomes
more marked the relative resistance of the tail increases much
more than that of the DNA. For example, in T6 inactivated
by pure direct effect, the DNA is 28 times as sensitive as the
bactericidal activity (Latarjet and Fredericq, 1955).

By treating a concentrated suspension of T2 in buffer
(10^^ particles per ml.) with succinic peroxide. Dr. Maxwell
found that both sites are attacked, and that the DNA is
about 2-5 times as sensitive as the other site. This ratio is
about the same as that found by Watson (1950) on T2 irradi-
ated by X-rays in 0 • 8 per cent broth. It should be of interest
to examine this aspect of peroxide-treated phage. Arber and
Kellenberger (1955) have already observed some morphologi-
cal changes in T2 treated with hydrogen peroxide.

(3) The sensitivities of different phages have been compared
in numerous experiments. The temperate phage X, which has
the same size as the virulent phage T2, but a smaller content
of DNA, is much more resistant to peroxide than the latter,
as it is more resistant to radiation. Among the virulent
phages of the T-series, T5 is the more resistant ; then come the
three T-even phages with about the same sensitivity. But,
contrary to what happens with radiation, the smaller phages
Tl, T3, T7 are more sensitive, possibly because of the lack of
a thick membrane around their DNA; for example, 10"*

DNA AND Effects of Radiation and Peroxides 283

cumene peroxide after 15 minutes contact at 37° left 18 per
cent of X and 0-4 per cent of T2; 2 X 10"^ succinic peroxide
after 5 minutes contact at 18° left 24 per cent of lyophilized
T4, and 0-2 per cent of lyophilized Tl.

Inactivation of bacteria -bacteriophage
complexes by peroxide

Bacteria-bacteriophage complexes are very sensitive to
peroxide, especially during the first half of the latent period.
As soon as new intracellular mature phage is formed, the
infective power of the complex becomes more resistant. In
the following experiment, B-T2 monocomplexes were brought
into contact, at various times during the latent period, with
10 ~* cumene peroxide at 37° for 10 minutes, then diluted and
plated for survival (Table II).

Table II

Time at beginning of contact
{minutes after infection)

Survival per cent

0-21

0-18

0-20

016

This situation is similar to that observed in complexes treated
with strongly illuminated nutrient broth (Latarjet and
Miletic, 1953). In this last instance, it had been found that
complexes which survive either u.v.-irradiation or contact
with illuminated broth, are damaged in such a way that their
latent period is lengthened, and their yield in new phage
particles lowered. The same partial damage has been observed
by Dr. C. C. Brinton in B-T2 complexes treated with sub-
lethal doses of cumene peroxide.

The behaviour of the peroxide is so similar here to that of
radiation, that an even more specific similarity has been looked
for. Phage X and its indicator bacterial strain K12S are

284 Raymond Latarjet

respectively more radioresistant than phage T2 and its host B.
However, the K12S-X complex is as radiosensitive as the B-T2
complex. This is due to the fact that the "capacity" of K12S
to grow X is far more sensitive than the capacity of B to grow
T2. As a matter of fact, in both X- and u.v.-irradiation, if one
adds the cross-sections of X and of the capacity of Kl2S, one
finds roughly the cross-section of T2, the cross-section of the
capacity of B being negligible (Latarjet, unpublished).

A situation similar in all respects has been found with
cumene peroxide: Kl2S and X are respectively more resis-
tant to peroxide than B and T2. However, the infectivi-
ties of K12S-X and B-T2 complexes have about the same
sensitivities.

Such parallelism, concerning a very specific biological
situation, appears to be very significant for the radiomimetic
character of peroxide.

Posteffect after treatment with peroxide

A posteffect has been observed by Alper (1954) after X-ray
treatment of bacteriophage. In my laboratory, Miletic (1955)
found that under strong illumination, nutrient broth becomes
toxic for B-T2 complexes, and that this toxicity proceeds for a
certain time after the treated complexes have been washed
and resuspended in an inert medium. Catalase suppresses this
posteffect. A similar phenomenon has been obtained after
peroxide treatment of B-T2 complexes. When the treated
complexes are diluted and plated on a minimal synthetic
medium, the toxic effect initiated by the peroxide proceeds in
such a way that the same final number of survivors is obtained
independently of the time of plating. This number is the
result of the immediate effect plus the posteffect. On the other
hand, when the complexes are plated on broth agar supple-
mented with catalase, the toxic effect is stopped immediately;
the final number of survivors shows the actual figure at the
time of plating.

The following experiment, carried out in my laboratory by

DNA AND Effects of Radiation and Peroxides 285

Dr. C. C. Brinton, illustrates this finding: 7 minutes after
infection, B-T2 monocomplexes growing at 37° C in synthetic
56 medium, are mixed with 10* cumene peroxide. After
5 minutes of contact, the suspension is diluted 1000-fold in
buffer. From time to time 0-1 cc. is spread:

(a) on broth agar supplemented with catalase, in the
presence of an excess of bacteria grown in broth ;

(b) on 56 agar in the presence of the minimum number of
bacteria required to give plaques.

After incubation, the number of plaques gives the number of
surviving complexes plus a few free T2. The number of the
latter is determined in parallel controls which have been
plated after elimination of the complexes by chloroform
treatment (Table III).

Table III

Number of surviving complexes

Time elapsed after

(a) (b)

dilution of peroxide
{minutes)

on broth agar +
catalase

on 56 agar

165

102

100

One sees that the number of survivors in the (b) series is
almost constant and very soon reaches the minimum which,
in the (a) series, is obtained only after 100 minutes, during
which a constant posteffect shows up.

It appears likely that this posteffect is similar to that
observed after radiation treatment. This conclusion reinforces
the idea that the radiation posteffect is mediated by radio-
formed organic peroxides. However, the possibility is not

286

Raymond Latarjet

ruled out that peroxide immediately absorbed by the bacteria
could slowly diffuse through the bacterial body towards the
sensitive sites of the complex, a process which could also
account for the observed effect.

Part-inactivation of bacteriophage

Indications that ionizing radiations and some chemicals can
increase the sensitivity of biological systems to further toxic

NORMAL
T4

PRETREATEO
T4

60
TIME IN MINUTES

120

Fig. 4. Inactivation of normal and cumene-hydroperoxide
pretreated phage T4 by ascorbic acid.

effects have now been obtained by several workers. In
particular, Alper (1955) has shown that bacteriophage which
has been irradiated in dilute suspension is more sensitive to
inactivation by reducing agents. It seemed of interest to
investigate to what extent organic peroxides could replace
ionizing radiation in the production of part-inactivated phage.
This investigation is being carried out in my laboratory by
Dr. D. Maxwell who has already observed the following

results.

The work of Alper was done on phages S-13 and T3. Dr.

DNA AND Effects of Radiation and Peroxides 287

Maxwell found that X-irradiation also part-inactivates the
larger T-even phages of the T series. Working with purified
T4, he has studied the inactivation by ascorbic acid of phage
pretreated by peroxides.

Succinic peroxide, although inactivating the phage, failed
to produce part-inactivated particles. In contrast, however,
cumene peroxide was found to produce part-inactivated
phage. Concentrated phage T4 was treated for 5 minutes
with 10"^ cumene peroxide, which produces about 50 percent
inactivation. The phage was then diluted to stop further
action of the peroxide, and then treated with 10 ~^ M-ascorbic
acid. Fig. 4 shows that the rate of inactivation of the pre-
treated phage is greater than that of normal T4.

It is of interest that cumene peroxide produces (while
succinic peroxide does not) a very typical change; ionizing
radiation also produces this change when acting under con-
ditions of indirect efPect. Experiments currently being carried
out by Dr. Maxwell seem to indicate that most of the part-
inactivation is due to a change occurring in the DNA, although
there may be a slight effect on the tail.

Mutagenicity of succinic peroxide

Mutagenic effects by some organic peroxides have been
observed in Neurospora by Dickey, Cleland and Lotz (1949).
Moreover, mutagenicity of some irradiated organic media can
be attributed to long-lived radioformed organic peroxides
(Wyss et al., 1950). Preliminary experiments carried out with
succinic peroxide indicate that such mutagenic activity cannot
be observed with all peroxides nor necessarily in all biological
systems.

(a) Dr. R. F. Kimball (1955, personal communication), of
Oak Ridge, exposed Paramecia to peroxide for 10 minutes
at 26° C in the dark. Following exposure, individual Paramecia
were isolated and several days later, groups of 25 autogamous
animals were isolated from each treated animal. Those
exautogamous clones which reached a maximum population

288 Raymond Latarjet

in 4 days were considered normal. The percentage of normal
clones was inversely related to the number of mutations.

The peroxide proved to be very toxic. Very few animals
survived exposure to 12 [xg./ml., but almost all survived
10 [xg./ml. Altogether, Dr. Kimball had 25 autogamous clones
from each of 344 treated animals of which 224 were exposed
to 10 [Jig. /ml. and 40 each to 12, 8, and 6 (Jig./ml. The percent-
age of normal exautogamous descendants from these 344
treated animals was 96-0, against 97-5 in the controls. The
difference is not significant.

(b) Dr. Luzzati and M. R. Chevallier (1956, personal
communication), of Strasbourg, used resting Esch. coli strain
B. Full-grown bacteria in broth were washed and resuspended
in buffer in the presence of 1 X 10"^ to 2 X 10"* peroxide.
Contact was maintained during 30 minutes at 37°. The
survival ranged from 0-3 to 10-^. After contact, the bacteria
were washed and plated (1) for B/1 mutants, resistant to phage
Tl (end-point mutations); (2) for B/Sr mutants, resistant to
streptomycin. No induced B/1 mutant was ever observed,
whereas the treatment induced up to 4000 B/Sr mutants out
of 10^ survivors.

This is one more example of mutagenic specificity (Demerec
and Cahn, 1953).

Inactivation of pepsin by peroxide

Most biological DNA is usually combined with protein.
We have seen that the hereditary material of bacteriophage is
very sensitive to peroxide, and it will be seen (p. 289) that pure
DNA of bacterial origin is even more sensitive. In order to
get some idea of what might happen to the protein moiety of
nucleoprotein treated with peroxide, a first series of tests has
been carried out on pepsin in this laboratory by Dr. Monier.

CrystaUine Armour pepsin (P.M. 35,000) was dissolved, at a
concentration of 5 X 10-^ m in 0-1 M-acetate buffer. Succinic
peroxide was added, and after a certain time of contact was
removed by dilution. Proteolytic activity on casein was

DNA AND Effects of Radiation and Peroxides 289

determined by Anson's method (with a precision of 3 per cent)
before and after treatment. Table IV groups the main
results obtained so far:

Table IV

Concentration of peroxide (molar) pH 5 • 2

6x10-5

6x10-4

6x10-3

Time of contact

85-5°

35-5°

35 5°

0°

(minutes)

Proteolytic activity

100

117

123

122

125

115

122

111

101

115

120

109

105

240

360

20 hours

Inactivation is only slightly increased at pH 3-6. Two
main facts may be pointed out :

(a) the very great resistance to peroxide of the enzymatic
activity, when compared with that of DNA ;

(b) the slight enhancement of enzymatic activity by short
exposures.

Inactivation of a transforming agent by peroxide

Transforming agents (or transforming principles, TP) are
pure DNA of bacterial origin, endowed with specific biological
properties, which can be extracted and transferred without
losing their activity. When accepted by a suitable cell, a
transforming agent endows this cell with a character possessed
by the strain from which it has been extracted. The "trans-
formed " cell perpetuates this character. One unit of the agent
can be detected by the formation of one transformed bacterial
clone. Therefore, such agents provide a unique material for

RAD.

290 Raymond Lataiuet

quantitative experiments on the actions of radiations or .
other aggressors upon the specific biological activity of DNA.'

In collaboration with Miss N. Cherrier, we have recently
undertaken a first series of experiments. Our TP, extracted
from a streptomycin-resistant strain of Pneumococcus and then
purified, showed a titre of 10^ transforming units per [xg. of
DNA. A solution containing 6x10-^ [ig. of DNA per ml. of
distilled water was brought into contact with the peroxide
at various times at 37°. After contact, aliquots were diluted
a hundred-fold in the transforming medium in the presence of
sensitive bacteria. After the transformations were effected,
the bacteria were plated in triplicate on agar supplemented
with streptomycin. The transformed clones were enumerated
after 2 days of incubation. Controls were done in order to
ascertain that the hundred-fold diluted peroxide remaining
in the transforming medium did not curb the transformations.

The following results have been obtained so far. I

(1) Surprisingly, cumene peroxide at concentrations ranging
from 10"^ to saturation (about 10-^) produced no noticeable
inactivation after contacts of from 1 to 3 hours, either in the
dark, or in visible light.

(2) However, succinic peroxide proved to be extremely
toxic :

(a) control experiments showed that, at a concentration of
5 X 10-^ in the transforming medium, the peroxide
decreased the number of the transformations induced
by untreated TP to about 20 per cent of the normal
value. At a concentration of 1 X 10-^, there was no
further toxicity. On the contrary, the peroxide increased
the number of transformations by a factor of about
1 • 5 ; this recalls the previously mentioned increase in
the activity of pepsin (see p. 288) ;

(b) contact with TP in distilled water was done at peroxide
concentrations of from 3 X 10-^ to 1 X 10-^. In all
experiments, there was a very sharp exponential
inactivation during the first few minutes of contact,

DNA AND Effects of Radiation and Peroxides 291

leaving 10 per cent or less of active units, the inactivation
of which proceeded exponentially also, but at a much
lower rate (Fig. 5). This remarkable feature has been
constantly observed in the inactivation of the same TP
either by u.v. (Latarjet and Cherrier, unpubhshed) or
by X-rays. The parallelism between X-radiation and

$ .1

> 10

1 —

— r

T"'

•

-A

■\

*■

">>

•

"^^Vw*

^Vs.

^**^

,—L.

-J

40 50 60 70

MINUTES OF CONTACT
Fig. 5. Inactivation of TP/sr by succinic peracid.

peroxide is, here again, so striking that the results
obtained with the former acting by direct effect on the
same material should now be^ mentioned, with special
emphasis on the radiobiological aspect of this work.

Inactivation of a transforming agent by the direct

effect of X-radiation

This work has already disclosed a number of facts which
have been or will be published in detail (Ephrussi-Taylor and

292

Raymond Latarjet

Latarjet, 1955, 1956). Attention will be called here briefly to
four points.

(1) Inactivation. Non -aggregated DNA ("normal" TP),
obtained as the supernatant of a purified and centrifuged
preparation, when irradiated in frozen 10 per cent yeast
extract (direct effect), yields a typical broken inactivation

100
90

^? 40 -

I/)

X 10 RADS

Fig. 6. Direct X-ray inactivation of TP/sr.
1: Aggregated TP, preparation Bl ; 2: both preparations treated
with urea, and normal non-aggregated TP; 3: aggregated TP,

preparation B2.

curve (Fig. 6, curve 2). The majority of the units are inacti-
vated at a rate which corresponds to a target with a molecular
weight of about 5 X 10^. This is less than one-tenth the
weight of the whole DNA fibre, as measured by Dr. Doty on
the same preparation by light diff*usion. The other units
display such a high resistance that the corresponding target
would not include more than a few hundred nucleotides.

DNA AND Effects of Radiation and Peroxides 293

This dualism is not due to genetic heterogeneity, since DNA
extracted from bacteria transformed by the resistant units
gives the same broken curve. It is not due to aggregation, as
will be seen below, since urea, which breaks the aggregates,
does not change this curve.

Dr. Ephrussi-Taylor suggests that an explanation might be
looked for in two different mechanisms for inactivation. The
sensitive one would damage some structure of the molecule-
fibre which is needed in one step of the transformation process,
such as a successful integration in the bacterium. The
resistant one would damage the fundamental structure actually
endowed with the biological specificity. This structure should
thus be exceedingly small.

(2) Aggregation and energy transfer. We used two samples
prepared by Dr. Simmons, in which DNA was aggregated.
The first one (Bl) gave an inactivation curve (Fig. 6, curve 1)
with a long plateau and a slope similar to the initial one of
"normal" TP (curve 2). We can consider in this case that
each aggregate, although containing a number (10-20) of units,
acts as one transforming particle when brought into contact
with a sensitive bacterium. The type of aggregation is such
that each unit is individually inactivated by radiation, i.e., that
energy transfer, if it exists, is insufficient to spread throughout
the aggregate. When this preparation was treated with 5
M urea, its titre rose, its viscosity increased, and its inactiva-
tion curve became similar to curve 2 of normal TP.

The second sample (B2) was actually the centrifugation
residue of the preparation, the supernatant of which had
formerly been used as normal TP. It gave the inactivation
curve 3 of Fig. 6, similar in shape to that of normal TP, but
with a two- to threefold increased sensitivity. When treated
with urea, this sample too gave the normal curve 2. We may
consider that there exists a second type or degree of aggrega-
tion, of lower multiplicity than the first one (2-3 normal
units), which is also dissociated by urea, but within which
radiant energy migrates in such a fashion that the whole
cluster may be inactivated by a single hit occurring within

294 Raymond Latarjet

any one of its units. Although the concept of energy transfer
within a single fibre is familiar, that of transfer between
fibres, the links of which are still unknown, carries more
subtle implications. It recalls the transfer between enzyme
and substrate postulated by Setlow (1955) in order to explain
the increased radiosensitivity of hyaluronidase when combined
with hyaluronic acid.

However, it remains possible that, in such a cluster, a hit
results in the formation of a very stable cross-link between the
aggregated fibres, and that this cross-link prevents successful
incorporation by the asssay bacterium.

(3) Protection against direct and indirect effects. It has been
suggested recently (Alexander and Charlesby, 1954) that
radiant energy could be transferred from an absorbing macro-
molecule to surrounding solutes. This process would bring
some protection against the direct effect of radiation upon the
macromolecule. We have observed that:

(a) In the liquid state, TP is much more resistant in 10 per
cent than in 0 • 1 per cent yeast extract.

(b) It is still more resistant in frozen 10 per cent and 0-1
per cent yeast extract as well.

We are inclined to interpret these results as follows:

(a) In the liquid state, increasing the concentration of
yeast extract gives additional protection of TP against
the indirect effects of the radicals produced in water.
The form of the DNA fibre, with a great surface : volume
ratio, explains the high sensitivity to the indirect effect.

(b) In the solid state, there is no noticeable energy transfer
from DNA to the components of the extract. In the
present case, there is no protection against the direct
effect, as far as physical processes which take place in
the solid state are concerned.

(c) Freezing provides additional protection by preventing
migration of active distant radicals from water to DNA,
without preventing them from recombining before the
system is melted.

DNA AND Effects of Radiation and Peroxides 295

(4) Influence of dissolved oxygen and hydrogen. We have
observed that the X-ray sensitivity of TP in hquid 1 per cent
yeast extract, a condition where the indirect effect is predomi-
nant, is influenced neither by oxygen nor by hydrogen. The
rate of inactivation is the same (a) under normal conditions
(saturation with oxygen); (b) after oxygen has been removed;
and (c) after new saturation with hydrogen. Apparent lack
of eff'ect of hydrogen may be due to the fact that the high
doses used can themselves produce a lot of hydrogen in the
solution. We are dealing here with the specific biological
activity of a DNA molecule. Its inactivation may result from
some kind of structural change of a nature resembling those
involved in gene radiomutations, which, as a matter of fact,
are also oxygen-independent. It follows that radiobiological
effects sensitive to the presence of these gases should not be
considered as the end-results of primary injury to DNA. In
particular, those radiation-induced mutations which display
high oxygen dependence (chromosome breaks), are likely to
result from primary attack upon other material than DNA.

Summary and conclusions

Cumene hydroperoxide and succinic peracid have been
used on bacteria-bacteriophage, and on a transforming agent
of Pneumococcus (TP). The effects observed have been com-
pared to those produced by radiation under similar conditions.

1. Both peroxides inactivate bacteria. Sensitivities of dif-
ferent bacteria to peroxide are of the same order as their
sensitivities to radiation.

2. Bacteriophages are inactivated by peroxides. After
contact, damage is observed both on the abihty of the phage
to kill its host, and on its abihty to multiply after attach-
ment to the host has taken place.

3. A posteffect similar to that produced by X-radiation has
been observed in bacteria-bacteriophage complexes treated
with organic peroxide.

4. Phage T4 treated with a sublethal dose of cumene

296 Raymond Latarjet

peroxide is sensitized to the toxic effect of a reducing agent
such as ascorbic acid. This "part-inactivation" is similar to
that produced by X-radiation. Succinic peracid does not
part-inactive T4.

5. Some mutagenicity has been observed for succinic
peracid in Esch. coli B, but not all mutations are produced.

6. Pepsin is slowly inactivated by peroxide after short
exposures have slightly enhanced its enzymatic activity.

7. A TP (DNA) which endows Pneumococcus with resistance
to streptomycin, has displayed an extremely high sensitivity
to succinic peracid, but remained undamaged after treatment
with cumene hydroperoxide. The inactivation curve is broken,
as it is in the case of X-ray inactivation, the break being due
neither to aggregation of DNA fibres, nor to genetic hetero-
geneity. It is postulated that inactivation results from two
different mechanisms. A quantitative analysis of the X-ray
curve leads to the hypothesis that the particular structure
within the DNA fibre actually endowed with the transforming
activity is exceedingly small.

8. X-ray inactivation of several preparations of TP has
disclosed two types of aggregation of DNA fibres, which is
disrupted by urea.

9. There is no oxygen effect in the X-ray inactivation of
TP, and no protection against the direct effect of radiation by
organic solutes.

10. These results, of prehminary character, stress the radio-
mimetic activity of organic peroxides and, therefore, their
possible role as intermediates in some actions of radiation
in Uving material. They also disclose that the behaviour
of a peroxide and of a peracid sometimes displays striking
differences.

Acknowledgements

I hope that the preceding text has clearly underlined the paramount
part played by my collaborators in the present work. It is a great
pleasure for me to thank them here :

Dr. B. Ekert, who performed the peroxide titrations, and, with Dr. R.
Royer, synthesized the succinic peroxide; Drs. D. Maxwell, C. C.

DNA AND Effects of Radiation and Peroxides 297

Brinton and R. Monier, who obtained some of the most significant
results on bacteriophage, bacteria and pepsin; Dr. H. Ephrussi-Taylor
and Miss N. Cherrier, who participated in the experiments on the
transforming agent ; Mr. P. Morenne and Miss G. Hiernaux for their
excellent technical assistance; Dr. J, Jagger and Mrs. P. Monnot for
their help in preparing the English text.

REFERENCES

Alexander, P., and Charlesby, A. (1954). Nature, Lond., 173, 578.

Alper, T. (1954). Brit. J. Radiol, 27, 50.

Alper, T. (1955). Radiation Res., 2, 119.

Arber, W., and Kellenberger, E. (1955). Schweiz. Z. Path., 18, 1118.

Demerec, M., and Cahn, E. (1953). J. Bact., 65, 27.

Dickey, F. H., Cleland, G. H., and Lotz, C. (1949). Proc. nat. Acad.

Sci., Wash., 35, 581.
Dubouloz, p., Monge-Hedde, M. F., and Fondarai, J. (1947). Bull.

Soc. chim. Fr., 800, 900.
Ephrussi-Taylor, H., and Latarjet, R. (1955). Biochim. biophys.

acta, 16, 183.
Ephrussi-Taylor, H., and Latarjet, R. (1956). In press.
Latarjet, R., and Fredericq, P. (1955). Virology, 1, 100.
Latarjet, R., and Miletic, B. (1953). Ann. Inst. Pasteur, 84, 205.
MiLETic, B. (1955). These Universite, Paris.
Sagik, B. p. (1954). J. Bact., 68, 430.
Setlow, R. (1955). Ann. N.Y. Acad. Sci., 59, 471.
Watson, J. D. (1950). J. Bad., 60, 697.
Wyss, O., Haas, F., Clark, J. B., and Stone, W. S. (1950). J. cell.

comp. Physiol., 35, Suppl. 1, 133.

DISCUSSION

Butler: Do Stent's experiments, in which he produced a great many
disruptions of nucleotide threads without losing the activity, fit in well
with yours ?

Latarjet: Yes.

Spiegelman: It is remarkable that this estimation of the order of
magnitude of a tenth fits in with two quite independent experiments.
One is Benzer's whose gene size is down tb 10 nucleotide pairs, and the
other is Stent's experiment where with ^^p decay about one out of ten
leads to a lethal event.

Gray: Another way of putting what has been said is that in the
target the volume is not necessarily aggregated. A second point is that
the first part of the curve seems to me to indicate that one in ten of the
particles are not subject to this inhibition of penetration into the
bacillus. In some way they must be different from the other nine, since
they are not appreciably inactivated by the doses which you used.

Latarjet: They behave differently after irradiation.

298 Discussion

Spiegelman : So you really have a heterogeneous population with
respect to the sensitivity to attachment.

Latarjet: I wouldn't say attachment.

Spiegelman: That's just an analogy.

Alper: Whether or not radical reactions in the classical sense could
take part in events inside the cell, it seems that there is something
rather mysterious going on in the radiation chemistry of various in vitro
systems. Dr. Dale has told us that all the enzymes that he has investi-
gated in dilute solution showed no oxygen effect. This seems all right if
the changes are due to — OH radicals, because the presence of oxygen
does not affect the number of these radicals. Whether any of these
systems have also been examined in the presence of hydrogen, I don't
know; but certainly it does seem rather inysterious in Dr. Latarjet's
case because he has tried oxygen and he has also tried hydrogen. Now
if you are going to regard the agent responsible as the — OH radical
(and I should think on any sort of radiation chemical picture we must
still regard indirect action in in vitro systems as due to radicals), it is very
difficult to see how you can get indirect action which is not affected by
the hydrogen, which converts — OH radicals into hydrogen radicals.

Latarjet: May I say that perhaps we have an artifact in the experi-
ment carried out in the presence and in the absence of hydrogen, since
the material was very radiation-resistant. We worked with doses of up
to 500,000 r and, in the absence of gas, we did not prevent the formation
of a great amount of hydrogen radicals in the medium due to radiation.
Therefore, there was perhaps in this instance no experiment at all in the
absence of hydrogen; all were probably in the presence of hydrogen.
That might account for the fact that we did not find any difference with
and without hydrogen.

Alper: This means that your in vitro indirect effect could be due to
hydrogen radicals, and this is the only thing it could be that would fit
with your experiments.

Alexander: I don't think that interpretation is necessarily valid, if we
accept some of Dainton's latest work that the reaction between hydrogen
and — OH radicals to give H atoms is not very favourable. If there is
nothing else for the — OH to react with then this reaction will occur,
but in Latarjet's system where there are great quantities of proteins
and other organic matter, it is most unlikely that the — OH radicals will
interact with dissolved hydrogen. We found that as little as 0-02 per
cent of organic matter prevented the reaction of hydrogen at atmospheric
pressure with H atoms (Alexander, P., and Fox, M. (1954), Trans.
Faraday Sac, 50, 605).

Latarjet: Our preparation was highly active; we had one million
transforming units per [ig. of DNA. That is a great advantage and
therefore we worked with an amount of DNA whch was about 10" ^ [ig.
per g.

Spiegelman: But it was protected.

Latarjet: It was very much protected by 10 per cent yeast extract.

Alper: This means that the hydrogen cannot react with the — OH
because there is a lot of organic material present to grab the — OH. But

Discussion 299

then there are still enough — OH radicals to react with the active mater-
ial, and presumably the hydrogen concentration is quite a lot greater
still than the number of molecules of protected material.

Alexander: The two peroxides studied by Latarjet are chemically very
different, and a great deal is known about their reaction with proteins.
The organic peracid is the type of substance which has enabled Sanger
to do his insulin work, i.e. the type of material which reacts very rapidly
and very specifically with SH and with -S-S- groups to convert them to
sulphonic acid. Cuinene peroxide is quite different: it also reacts with
protein and also attacks the SH and -S-S- groups but it does not give
sulphonic acid in quantitative yield, it gives largely sulphate. Unlike
the peracid it attacks the peptide bond. Chemically these two substances
show such different behaviour that one would expect their biological
effects to be quite different.

Latarjet: It is a constant phenomenon.

Dale: Dr. Latarjet, in your first slide (on pretreatment), how did you
protect yourself against the aftereffect ? When you treated your material
and then exposed it to ascorbic acid or vice versa, couldn't there have
been an aftereffect, or what precautions could you have taken? The
time was roughly the same as on your aftereffects slide.

Latarjet: It was such a slight dose (we had 50 per cent survival) that
the aftereffect was not great, and it was the same in both the control
and the treated samples.

Popjak: Dr. Latarjet, with regard to the radiomimetic effect of these
peroxides in mice, you said that you gave the substance in oil?

Latarjet: We first injected cumene hydroperoxide dissolved in oil.
Then we had some trouble because the toxicity of the solution increased
with time. It seemed that cumene peroxide initiated some peroxidation
in the oil itself. Therefore, we turned to the water-soluble persuccinic
acid. We are now coming back to cumene, using a new organic solvent
which has been synthesized in our laboratory.

Popjak: I wonder whether the effect might be due to the peroxidating
action on the highly unsaturated fatty acids, and that in effect you might
be producing the essential fatty acid deficiency in view of the skin
lesions. The effect might be of that type.

GENERAL DISCUSSION

de Hevesy: I feel sure that you all share my view that we ex-
perienced a most profitable and exceedingly pleasant meeting. Our
thanks are due to all who addressed us and participated in the dis-
cussion, but first of all to our Chairman, Prof. Haddow, and to the
organizer of this meeting. Dr. Wolstenholme, to the assistant
secretary, Miss Bland, and to all members of the able and friendly
staff of the Director.

If I am permitted to add a personal remark, I wish to say that I
never experienced a more pleasant meeting.

We have traversed various territories and it is difficult to decide
which of the countries passed has the most beautiful scenery. One
may say that nothing was more fascinating than following the path
and fate of seeded marrow-cells as was done by Dr. Loutit which
revealed among others the powerful effect of radiation on immunity.
It really sounded like a fairytale. Some, however, may give pre-
ference to the discussion of the great variety of changes in the
enzymatic pattern produced by irradiation, in which various speakers
participated, and to the presentation of the philosophy of such
happenings put forward in such a fascinating way by Prof. Krebs.
Protection was one of the main fields of discussion. Formerly, a
geneticist as far as he was interested in the application of X-rays
was anxious to produce the maximum number of mutations. By
working on these lines very important results were obtained, among
others in the field of agriculture. Quantity and quality of crops were
improved. I doubt if any geneticist envisaged in those days that
the time might come when the main concern will not be to produce
mutations by irradiation but protect against them. Dr. HoUaender
reported results of his and his colleagues' endeavour to achieve
protection against mutative effects of radiation. This was followed
by an animated discussion on different aspects of genetic happenings.

That the basic problem of radiobiology, the site of primary bio-
chemical lesion, is yet unsolved was emphasized by different speakers.
Now in view of the great variety of enzymatic changes produced by
irradiation one may be inclined to consider such inactivation to be
the primary radiation damage. What makes one doubt the correct-
ness of this assumption is the fact that while the same enzyme when
present in a radiosensitive organ can easily be inactivated, when
located in a less radiosensitive one proves to be refractory to even
large doses. We were told by Dr. Van Bekkum that when the rat is

300

General Discussion 301

irradiated with 100 r the oxidative and phosphorylating power of its
mitochondria gets markedly reduced and that 50 r suffice to obtain
similar effects in the thymus. But if we expose rats to much larger
doses the mitochondria extracted from their liver go on to oxidize
and phosphorylate at a normal rate. Now you can of course say
that the composition of the liver differs from that of the spleen and
thymus; that the liver contains constituents having a powerful
protecting effect which prevent radiation energy reaching the
sensitive spots. You may also say that the enzymatic pattern
differs in the liver from that of the spleen or thymus, recalling the
considerations put forward by Prof. Krebs and that conditions for
enzyme inactivation are more favourable in the last-mentioned
organs. But the above-mentioned difference is not only shown when
comparing liver with spleen or thymus but also other moderately
sensitive organs with very sensitive ones. Thus we have to consider
the possibility that inactivation of enzymes is preceded by cell
lesion. As to the latter, a conspicuous parallelism is shown between
radiation sensitivity and rate of DNA formation in the organ con-
sidered. You can easily find exceptions to this regularity. Lym-
phocytes in which no DNA formation takes place are an example,
some plant seeds, some plants. In view of the very great number of
parameters involved it is very difficult to find any regularity which is
valid without exception. It has been known for many years that
cells exposed to irradiation often die when trying to divide. Li
emphasized this point in his classical book. Our knowledge of this
type of cell death was enlarged by investigations reported by Forss-
berg in his address. He has shown that by irradiating mice with
ascites tumour the formation of DNA in the tumour cells is blocked,
at the same time synthesis of several metabolites goes on. Such a
selective influence is bound to have serious consequences. DNA
formation being obstructed, the cell cannot divide and thus it has to
accommodate all additionally formed metabolites in the mother cell
which correspondingly swells. At a later stage, when the power of the
cell to synthesize DNA recovers, the cell can divide. But division of
such an abnormal cell is often fatal.

When considering the above-mentioned exceptions we must take
into account the fact that exposed cells may die unconnected with
division processes. Even unexposed erythrocytes die and their
death may be accelerated by interference with oxygen supply,
production of haemolysing substances and other agencies. It was
shown by Howard and Pelc that irradiation may interfere with a
very late phase of the mitotic cycle in which the full DNA complement
of the cell is already reached, and Dr. Howard told us that she con-
siders this type of interference to be the primary one, a view against

^02 General Discussion

which some evidence was brought by Dr. Lajtha. Recent results
obtained by Mazia when studying connection between RNA syn-
thesis and cell division suggest that this late radiation effect may
possibly be interference with RNA formation.

The mechanism responsible for DNA synthesis is not known but it
is quite probable that its formation necessitates the presence of
intact DNA protein molecules and that interference with these and
possibly also with the RNA protein molecule are the primary cell
lesions. In the mammalian organism unique size and length of a
DNA protein molecule much favour uptake of radiation energy.
We were told by Prof. Mitchell that he is inclined to consider a macro-
molecular lesion of DNA protein to lead to a healing of carcinoma.

As to the effect of radiation on the DNA protein present in the
tissue, it is just ten years since Errera irradiated nucleated red
corpuscles and determined the rigidity prior to irradiation and after
exposure to a very massive dose of 5000 r or more. He found the
rigidity to be reduced. Quite recently, in Dr. Hollaender's Institute,
Anderson carried out experiments with diluted homogenates of
thymus, determining their viscosity prior to and after exposure.
He succeeded in reducing the viscosity after exposure to only 25 r.
It may be purely fortuitous or not — it is difficult to tell — ^that the
dose that will interfere with DNA synthesis in the thymus, as
determined by Ord and Stocken, is about the same as that which is
necessary to depress its viscosity, thus to depolymerize thymus DNA.
Now I just wonder if it would be possible or profitable to carry out
with other tissues, experiments similar to those which Anderson did
with thymus. Thymus, of course, has the high DNA content which
makes it easier to work with, but it may be possible to take other
tissues and to investigate if there is any parallelism between the ease
with which deploymerization takes place and DNA formation, and
thus also between radiosensitivity. We come here very near to a
suggestion made by Prof. Mitchell. He remarked that it is possible
that the difference in behaviour of radiosensitive and refractory
tumours is due to the fact that the refractory tumours contain DNA
of low grade of polymerization ; correspondingly, irradiation cannot
easily produce further changes in these. But even if these conclusions
were not to be substantiated, it could be quite possible that depoly-
merization of the refractory tumour tissue would need a larger dose
than that of a sensitive tumour.

It is thus quite possible that enzymic processes are preceded by
cell lesion produced by interference with nucleoproteins. But even
if this interference with DNA protein and possibly also with RNA
protein should prove to be a very important early step, introducing
cell damage, it is not necessarily the first one.

General Discussion 303

Dr. Hollaender mentioned the very rapid irradiation effects pro-
duced by change in salt concentration of the surroundings. Perhaps
Dr. Hollaender is willing to give us some more details of how this
exciting experiment was carried out, and also to tell us if it is possible
and advisable to carry out experiments similar to those which
Anderson did in his laboratory with thymus tissue, with other types
of tissue, and if he expects to find marked differences.

Hollaender: Dr. Gaulden, in previous studies, had demonstrated
that if she subjects the neuroblast to hypertonic culture medium, the
chromatin of cells in middle prophase takes the appearance of late
prophase and the chromatin of late prophase assumes the appear-
ance of very late prophase. These changes occurred in 15 to 30
seconds after the cells were placed in a medium hypertonic to them.
This apparent advancement of stages of mitosis was shown to be
accompanied by an accelerated mitotic rate.

She then decided to set up some experiments to determine whether
the "reversing" action of X-rays on middle and late prophase prim-
arily responsible for mitotic inhibition at low doses could be prevented
by subjecting the neuroblast to a hypertonic medium which advances
these stages. The results indicated that this can be easily accomp-
lished. Whereas the mitotic rate of irradiated (3 r of X-rays)
neuroblast in isotonic medium was depressed down to about 25 per
cent of normal level, the mitotic rate of irradiated neuroblast in
hypertonic medium was depressed only down to about 75 per cent
of normal level. Thus, subjection to hypertonic medium immediately
following irradiation resulted in less radiation damage than if iso-
tonic solution is used. It was also found that if the cells were put in
a hypertonic medium more than a minute after irradiation had been
stopped, they responded to the irradiation in the same manner as
irradiated cells in isotonic solution. The most successful tests were
accomplished by irradiating these cells in a very slightly hypertonic
salt solution and then, immediately after irradiation, putting them
in a more definitely hypertonic medium. Under these conditions, one
could ehminate practically all the effect of mitotic inhibition pro-
duced by X-rays. It is not possible at this time to tell whether the
influence of hypertonic medium is due to removal of water from the
cell or to increased concentrations of certain inorganic salts or to both.
(See the forthcoming paper, " Prevention of X-ray induced mitotic in-
hibition in grasshopper neuroblasts by post-irradiation subjection to
hypertonic culture medium", which Dr. Gaulden is now preparing.)
I believe such an approach with hypertonic salt solution could be
used in connection with other radiation work. The important thing,
probably, is to get it into the cell very quickly after irradiation has
stopped so that the damage is still "reversible".

304 General Discussion

Now going back to Dr. Anderson's work, which is being extended
by Mr. Fisher, of course the original work at Oak Ridge was initiated
on the effects on the nucleic acids on the basis of the experiments we
had done many years ago in co-operation with Drs. Greenstein and
Taylor at the National Cancer Institute and National Institute of
Health, when we found that 10-20,000 or 50,000 rontgen were
necessary to depolymerize sodium thymonucleate, and it might
interest you that the background for the work on the nucleic acids at
Oak Ridge was this finding. But it was quickly found out by Dr.
Carter and later on by Dr. Cohn that the materials we were working
with were either very impure compounds or isolations of mixed
materials with which experiments could not be repeated from time to
time, and it was finally decided at that time to go more thoroughly
into the whole problem of nucleic acids, first of isolation and then
structure of the nucleic acids, and we are just starting again to study
the effects of radiation on these mixtures or compounds if we can get
them pure enough. I think that there are many possibilities, even
with the impure mixtures, to follow what radiation will do in them;
concentrating entirely on first getting the purest possible compound
may not tell us this story. I feel that even these crude extracts may
give us more information than the very pure compounds which we
finally will have to study; these crude mixtures may tell us much
more of what is happening inside the cell. I think Dr. Anderson
picked the easiest tissues to handle, as Prof, de Hevesy pointed out.
But he plans to go ahead on other tissues too and see if he gets the
same type of result.

de Hevesy: Errera needed 5,000 r, Anderson only 50. Errera
worked with nucleated red corpuscles of the hen, in which no DNA
turnover takes place. So it is not impossible that the tremendous
dose difference, 50-5,000 r, is not only due to Anderson's improved
technique but the fact that Anderson picked out a very radiosensi-
tive system while Errera worked with a very radioresistant one.

Alexander: I think this difference between Errera and Anderson is
largely one of concentrations. One can add as much water as one
likes to these nucleoprotein gels and when dilute they show dilution
effect typical of indirect action in a very pronounced manner.
Before concluding that one system is more sensitive than another we
must take concentration into account. Errera 's concentration was
governed by the physiology of the cell and was quite high. In the
test-tube one can handle it at much lower concentrations, and one
of the reasons why Anderson could detect such small doses must
have been that he worked at concentrations which were much lower
than those of Errera.

Butler: With regard to de Hevesy's point about the other tissues,

General Discussion 305

the rat thymus is rather particular in that the nucleoprotein is not
completely dissociated in this salt solution. If you take the same
thing with beef thymus you get a much greater degree of dissociation
and you don't get this remarkably high specificity.

Haddow: Dr. Howard has put forward a suggestion, namely that
Dr. Dale should tell us to what extent he believes his five questions
have been answered.

Dale: In my presentation, I deliberately did not express any
opinion, giving simply a background survey, and at the end of it I
put some questions (p. 33) which involved the two main manifesta-
tions of indirect action, the dilution effect and the protection effect,
and asked this audience whether there is scope for these two mani-
festations to explain the possible mode of action of enzymes. One of
the main questions, i.e. the fourth question, was answered to a cer-
tain extent in the way in which I put it, but Prof. Krebs in his con-
tribution modified this and answered it more or less in this way,
that he thought the determining factor is not so much the disturb-
ance of the enzyme itself, but of the substance which is concerned
with the enzymatic process, that is of the substrate ; and of course the
dilution effect will just as well act on that if it acts at all.

I understood from Prof. Kreb's answer to my .third question that
there is a possibility of a minute amount of available substrates being
interfered with at the steady-state, which may be low. The steady-
state concentration of the substrates may be a determining factor,
and may be responsible for disorganization of consecutive steps in
the enzymatic action.

I also asked the question "Are these intermicellar spaces in the
inhomogeneous cell structure filled with high concentrations of pro-
tective substances?" This is an objection that is usually made, that
there are plenty of protective substances which will obviate the
indirect action. Now there are model experiments by Stein and one
co-author in which he irradiated gelatin gel in which he had incorpor-
ated methylene blue, and there was little or no interference with the
action of degradation of this methylene blue. In this case one would
say, taking the gelatin as the cytoplasmic model for the interior of
the cell, that the micellar structure of this gel did not interfere with
the indirect action. Indirect action could still take place in the
solvent-filled spaces between those gelatin structures. Also, Gordon
and his co-worker used agar gels and did not find a protective effect
of these on substances distributed in these gels. In answer to my
fifth question, I would say quite emphatically that any experi-
ments on these lines are doomed from the start. They cannot
give any indication of what is happening, because all that this type
of experiment is concerned with is the total amount which is present.

306 General Discussion

and that is not what matters at all. If anything matters it is the
functional part and not the store, and the notoriously small amounts
of change by moderate radiation doses in any substance can, of
course, have nothing to do with the bulk from which they are
changed by radiation.

My first question is similar to that of the separation of substrate
and enzyme and — to extend it — the enzyme can diffuse or the en-
zyme is phase-bound and the substrate is diffusing to it. Evidence
has been brought forward by some workers that substrate and
enzyme, at least in certain cases, are localized separately and in
order to get to each other they have to be in transit of some form.

One more point that I should like to mention is the apparent and,
to a certain extent, neglected importance of chain reactions. Some,
of course, are known and we have a very interesting example of a
dose-rate dependent chain reaction with respect to the liberation of
sulphur from thiourea, which at a dose rate of 0 • 39 has G values of
17,000-20,000 and there is virtually no limit if one goes down still
further with the dose-rate. It is also known that for instance, the
oxidation of cysteine to the disulphide is a chain reaction, and
furthermore (which makes it so difficult to reconcile any scheme
devised by physical chemists for any reaction with radicals) that the
action of some of the radicals very often must cause changes which
lead to new radicals which are probably very difficult or nearly im-
possible to put into a reaction scheme. That refers to oxidations, to
dehydrogenations which are one-step reactions, leaving a radical
which again may do something, and the phenomenon I have shown,
namely the "changing quotient" (i.e. that the protective power per
unit mass of the substance added declines on increasing the concen-
tration) can be explained also by the formation of a radical from
protector molecules which again hands on the energy.

Holmes: Dr. Hug and co-worker (Hug, O., and Wolf, I. (1956),
Progress in Radiobiology, p. 23. Edinburgh: Oliver & Boyd)
made a very useful contribution to the knowledge of the effects of
irradiation of systems in a steady state. They irradiated with X-rays
luminescent bacteria which were emitting light of a steady intensity.
A diminution of light-intensity was shown as soon as the irradiation
was begun and the intensity fell continuously as long as the irradia-
tion continued. Directly irradiation ceased the light emission began
to recover and became steady at an intensity rather lower than that
originally found. The recoverable part of this system was un-
doubtedly an irradiation-sensitive unit of the light system which was
restored by the further activity of the bacteria.

Dale: That is quite possible. There is an American worker too,
who works with fireflies. It is a very sensitive reaction.

General Discussion 307

Popjak: May I contribute to that? I think the real significance of
those experiments might be that the phenomenon was observed
during irradiation, and Hug has observed the luminescence while the
bacteria were irradiated. Immediately on starting irradiation the
luminescence decreased, when he stopped irradiation it returned, and
so on, although I think that with higher doses the luminescence did
not quite return to the original starting value. The importance of
this is that it is well worth thinking of experimentation in which we
try to look at enzyme reactions while irradiation is going on.

Laser: I have mentioned before that the enzyme notatin is inacti-
vated by X-rays more strongly if irradiation takes place in the
presence than in the absence of its specific substrate (glucose). I
should add that I conclude that the enzyme is most sensitive to
X-ray damage at the stage of a semiquinone.

Dale: Which would fit in with what I mentioned.

Latarjet: Dr. Dale, may I ask you if your chain reaction of thiourea
takes place in the absence of oxygen?

Dale: On the contrary, it takes place in the presence of pure
oxygen. If you decrease the oxygen tension the dose-rate depend-
ence is not abolished, but the absolute effects are getting smaller and
smaller. In the absence of oxygen there is hardly any effect.

Mitchell: One point which we have not discussed at all at this
meeting, and which I think might be of interest, is the relative
biological efficiency of different radiations for metabolic effects,
particularly the comparison of effects of radiation with low and high
specific ionization, in biosynthesis of nucleic acids. There are the
experiments of K. G. Scott (1946, Radiology, 46, 173) and the more
recent experiments of A. Howard and S. R. Pelc (1953, Heredity^
Suppl. to Vol. 6, p. 261). I wonder if people consider that further
information might be obtained from a special study in this direction.

Howard: To follow up that suggestion with regard to effects of
different types of radiation on DNA synthesis in tissues would be
extremely useful. We have very little information on this point, and
it would give us one obvious means of sorting out the mitotic delay
and cell death on the one hand, and the effect on DNA synthesis on

the other hand.

Haddow: When Banting discovered insulin, or most probably
rediscovered it, about 1922, he was expected by the public to make a
great series of further discoveries along the same lines, including a
cure for cancer, the study of which he took up shortly thereafter. I
remember Sir Henry Dale telling a story of how, round about 1923,
he was phoned up in great excitement by a Press reporter to know
was it true that Banting had discovered a cure for metabolism. We
may not have discovered the cure for metabolism, but we may be on

308 General Discussion

the way to the control of the disorders of metabolism which are
brought about by ionizing radiations.

I think it has been very well worth while coming here for all sorts
of reasons, not least to hear Professor de Hevesy say what he said a
short while ago. I thought this was a very moving and masterly
summary. He was obviously impressed by what he called the fairy
tale which Dr. Loutit has told us, and I think we are specially grate-
ful to Dr. Loutit for recalling the early key observations, particularly
those of Jacobson, and the later work of Lorenz who is so well
remembered in the Ciba Foundation. I have a feeling that the pro-
gress of work depends rather on the making of such key biological |
observations. We must not detract from the skill or prowess of the
biochemist ; but from my own experience, with all respect to my
biochemical confreres, I find that very seldom are they able to direct
one to make the discovery. The discovery very often having been
made by the biologists, they can then explain it in many cases, or
endeavour to do so. And this leads me to another impression I have
had during this meeting: that we more and more approach the j
holistic view of the cell. As Dr. Zamecnik showed so graphically, ^
atoms and molecules tend to mean very little in themselves, and it is
the way in which they are put together that really matters. We will
all recall for a very long time to come the courage shown by Krebs in
throwing his paper and slides out of the window. Lastly I should
like, from myself and on your behalf, to tender thanks to Dr.
Hollaender, Professor Butler, Dr. Gray and Dr. Wolstenholme, for
all their help in the early arrangements of a profitable and memorable
Symposium.

AUTHOR INDEX TO PAPERS

Barnes, D. W. H.

PAGE

140

Van Bekkum, D. W. .

Brachet, J. .

Butler, J. A. V. .

Dale, W. M.

Forssberg, A.
Gale, E. F.

. 212
. 174

Gray, L. H.
Hollaender, A.

. 255
. 120

Holmes, B. E.

. 225

Howard, A.

. 196

Kihlman, B.

. 239

Krebs, H. A.

Laser, H.

. 106

Latarjet, R. .
Loutit, J. F.

. 275
. 140

Pirie, A.

Spiegelman, S.
Stapleton, G. E.
Swanson, C. P.

. 185
. 120
. 239

Zamecnik, P. C.

. 161

309

SUBJECT INDEX

Acetabularia, darkness on, 254
dinitrophenol on, 254
enucleation on, 3, 12, 13, 14, 15, 17,

23
phosphorus metabolism in, 12
ACTH {see Adrenoeorticotrophic hor-
mone)
Action spectra, 255
Adenosine diphosphate, anaerobic
glycolysis and, 94
inhibition of cell respiration and,

97-99
rate of oxygen consumption and,
96
Adenosine monophosphate, and

inhibition of cell respiration, 99
Adenosine triphosphatase activ-
ity, and oxidative phosphorylation,
86
Adenosine triphosphate, and amino
acid activation, 166
and anaerobic glycolysis, 94
and inhibition of cell respiration

99
and rate of nucleic acid synthesis,

105
and rate of oxygen consumption,

96
as source of energy, 101
in cell-free incorporation system,

164, 165
in enucleated cytoplasm, 4, 8-9,

11
specific activity of depression by
radiation, 85
ADP (see Adenosine diphosphate)
Adrenal steroid synthesis, radia-
tion on, 45-47
Adrenergic blocking agents, 102
Adrenoeorticotrophic hormone,

and steroid formation, 45
AET (see S, j3-aminoethyh'sothiouron-
ium-Br-HBr)

Amino acid incorporation, and

microsomes, 169
and nucleic acids, 174-182,

196-197,210,214
comparison of rate of, in pro-
toplasts and intact cells, 187
inhibition by antibiotics, 179,

180
inhibition by chelating agents,

180
inhibition by 8-hydroxyquino-

line, 180, 181
inhibition by nitrogen mus-
tards, 183
inhibition by ribonuclease, 186
inhibition by uranyl chloride,

187
inhibitors of, 179-182, 186, 187
in proteins, 7, 8, 9, 10, 18, 162,

163, 186
metals on, 182
nitrogen mustards on, 69
radiation on, in various tissues,

229
stimulation by deoxyribonu-

clease, 186
use of lysozyme in study of,
186
S, /3 - aminoethyh'sothiouronium •
BrHBr, on respiratory centres,
137
multiple equilibrium structures of,
. 123-124
protective ability of, 74, 121, 123-
124
Amoebae, enucleation on, 3-4, 8, 9,
10, 11, 12, 13, 14, 15, 18, 23
phosphorus metabolism in, 12
AMP (see Adenosine monophosphate)
Anaerobic glycolysis, and effects of
extraneous agents, 93-95
in spleen, 86
inhibition of, 47, 48, 49, 110-111

311

312

Subject Index

Antibiotics, inhibition of amino acid

incorporation by, 179-180
Antimycin A, inhibition of cell res-
piration by, 97
Ascites tumour cells, amino acid
incorporation in and con-
centration gradient, 223
and oxygen effect, 117
influence of radiation on meta-
bolism of, 212-222
rate of metabolism in, 222
Ascorbic acid in adrenal, decreased

by radiation, 46
ATP (see Adenosine triphosphate)
Auxin synthesis, from tryptophan,
52
inhibited by radiation, 51-53
Bacillus megaterium, and protoplast
formation, 185
cytological structure, 258
induced enzyme synthesis in,
187-188
Bacillus subtilis cytological structure,
257-258
induced enzyme synthesis in, 187
Bacitracin, inhibition of amino acid

incorporation by, 180
Bacteria, inactivation by organic

peroxides, 278-280
Bacteria - bacteriophage com-
plexes, inactivation by organic
peroxides, 283-284
Bacteriophage, inactivation by or-
ganic peroxides, 280-283
part-inactivation, 286-287
Batyl alcohol, therapeutic activity,

140
Blood transfusion, and recovery of

irradiated animals, 140
Bone marrow, cellular re-seeding of,
151
humoral factor in, 141-142
protection by, during irradiation,

132-133
regeneration of, 141, 155
therapeutic effect in irradiated
mice, 154-157
Cancer cell, destruction of repro-
ductive integrity, 266
Carbohydrate synthesis, radiation

on, 221
Carbon monoxide, therapeutic ef-
fect, 140
Carcinogenic property of maleic
hydrazide, 252

Carcinoma of the skin, therapeutic

action of radiation on, 71, 76
Cell death, after irradiation, 200-
201, 223
and growth rate of survivors,

213
site of action of nitrogen mus-
tards in, 264
site of action of X-radiation in,
264
Cell-free incorporation system,

components of, 164
Cell metabolism and effects of

extraneous agents, 92-103
Cellular oxidations, centre of, 3

rate of, 3, 4-5
Cell population, radiation-induced

changes in, 200-203
Cell respiration, and extraneous
agents, 95-101
inhibition of, 95-101, 108, 110
Centromere function, loss of, 259
Changing quotient, and protection,

30
Chemical effects of radiation, 59-

69
Chemical mutagens, induction of
chromosomal aberration by,
224
inhibition of oxidative phos-
phorylation by, 245, 248, 249-
250
Chemical protection against radi-
ation, 120-135
in mammals, 132
Chemical protectors, mechanism

of action, 138
Chemical treatments, influence on
radiosensitivity of bacteria,
120-135
significance for higher organisms,
120-135
Chimaeras, 152

Chloramphenicol, inhibition of
amino acid incorporation by, 179-
180
Cholesterol in adrenal, decreased by

radiation, 46
Cholinergic blocking agents, 102
Chromatin, effect of hypertonic salt

solution on, 207
Chromosomal aberrations, and
chemical treatment, 134
and oxygen effect, 242-243, 251
distribution of, 245-246

Subject Index

313

Chromosomal abberations

in Drosophila, 241

induced by chemical mutagens,

239-251
induced by radiation, 227-228,

239-251
in Tradescantia microspores, 241
Chromosome set, and amount of

deoxyribonucleic acid, 200
Chromosomes, translocation in, 148
Chymotrypsin, radiation on, 39
Citric acid cycle and oxidative

metabolism, 42
Citric acid metabolism, effect of
fluoracetate poisoning on,
221
effect of radiation on, 221
Coenzyme A, activity and irradia-
tion, 42-43
and inhibition of cell respiration,

97-99
role of nucleus in synthesis of, 5
Coenzyme level in X-ray cataract,

43
Coenzymes, activity in irradiated

tissues, 38-56
Colchicine, and mitosis in regenerat-
ing liver, 236-237
Colloids, radiation on, 35, 269
Corticoid output, decreased by

radiation, 45-46
Corticosterone synthesis, de-
creased by radiation, 45
Cysteamine, effect on respiratory
centres, 137
derivatives, protective ability of,

136
protection by, 121-123, 124
Cytochrome, a^ and inhibitors of cell
respiration, 108
h and radiation, 42
c and depression of oxidative

phosphorylation, 86
c and radiation, 42, 86-88, 111
c and rate of oxygen consumption,
96
Cytoplasmic damage by irradiation ,
260, 263, 265
network, structure, 161
structure, in relation to metabolic
activities, 3-20
Dead cells, effect on metabolism of

survivors, 203
Dehydrogenase systems, and ex-
traneous agents, 100

Deoxyribonuclease, and nucleo-
protein inactivation, 159
inhibitors of, 274
on enzyme synthesis, 189
on protoplasts, 190-191
stimulation of amino acid incor-
poration by, 186
Deoxyribonucleic acid, as genie
substance, 65-66
chemical effects of radicals on,

61-62
constancy per chromosome set,

209-210
constitution. Crick - Watson

model, 59, 65, 70
digestion on, 178-179
heat on, 60

in dying cells, as pool for re-
generating cells, 237
metaboHc stability of, 215-222
reaction with nitrogen mustards,

67
specificity, and effects of radia-
tion and organic peroxides,
275-297
synthesis, and delay in mitotic
cycle, 199-203
and enzyme activity, 39
cause of inhibition of, 203-205
failure of, 53
in absence of mitosis, 90
in ascites tumour, 202, 204,

213
in bean roots, 202, 204, 205
in mammalian tissues, 198-

199, 202, 204
in regenerating liver, 205, 225,

227-236
radiation on, 196-206, 307
recovery after irradiation, 131—
132, 203
transfer from dead to living cells,

214-215
urea on, 75
Dilution effect, 25-26, 27, 28, 33,

305
Dinitrophenol, as metabolic in-
hibitor, 253-254
Diphosphopyridine nucleotide,
and identification of pace-
maker steps, 104
and inhibition of cell respiration,

97-99
and rate of oxygen consumption ,
96

314

Subject Index

Diphosphopyridine nucleotide,

content in anucleated cyto-
plasm, 9, 11,20
content in fasted amoebae, 4
distribution of, in non-nucleated
cytoplasm, 20
Direct action of radiation, and
chromosome breaks, 248
on deoxyribonucleic acid, 74^76
on enzymes, 25, 27, 28, 34, 38
on haemoglobin synthesis, 45
protection against, 294
DNA (see Deoxyribonucleic acid)
Dose -reduction factor, and pro-
tection, 121, 133, 134
DPN {see Diphosphopyridine nucleo-
tide)
Drosophila sperm, chromosome

structural damage in, 117
Enzymes, activity of in irradiated
tissues, 38-56
and dilution effect, 25-28
and inactivation of amino acids,

170, 171
and oxygen effect, 28, 114, 155
and protection effect, 28
direct action of radiation on, 25, 27,

28
indirect action of radiation on, 25,

27, 28
ionizing radiations on in vitro,

25-34, 36-37
removal of nucleus on, 10-11, 18
respiratory effect of radiation on,

41
steroid synthesizing, 46
Enzyme synthesis, by reticulocytes,
14
deoxyribonuclease on, 189
in bacteria, 58
induction of, in Bacillus megater-

ium, 187-188
induction of, in Bacillus subtilis,

187
inhibition of, 188-189
in non-dividing mammalian cells,

54
in protoplasts, deoxyribonu-
clease on, 190-191
in protoplasts, ribonuclease on,

191-192
lipase on, 188
radiation on, 40
ribonuclease on, 189
trypsin on, 188

Energy transfer and deoxyribo-
nucleic acid aggregation, 293-294

Extraneous agents, and energy
supply, 101-102

Fatty acid synthesis, and coenzyme
A, 44
increased by radiation, 43, 45

Ferritin synthesis, 172

Fertilization and irradiation, 261-
262

Flavoprotein, role in hydrogen trans-
port, 100

Fluoracetate, toxic effects of, 102

Fluorocitrate, as enzyme inhibitor,
102

Formate incorporation in deoxy-
ribonucleic acid, 196-197, 210, 211
in ribonucleic acid, 211

Genetic damage, modification of,
133, 134

Globin synthesis, stimulation of, 45,
56-57

Glucose oxidase (notatin) and oxy-
gen effect, 114

Grasshopper eggs, negative growth
of, 108

Grasshopper neuroblasts, action
spectrum of, 259
effect of hypertonic salt solution

on mitosis in, 206-207, 303
mitotic delay in, 200

Habrobracon radiation damage in,
260-261

Haemin synthesis, stimulation of,
44^45, 56-57

Haemoglobin synthesis, stimula-
tion of, 44, 57

Hexokinase reaction, 93-94, 104-
105

Histone, influence on radiation
effects, 65-66
reaction with nitrogen mustards,
67

Hormones and cell structures, 103

Humoral hypothesis, 141-143

Hydrocortisone synthesis, reduced
by radiation, 45

Hydrogen, effect, 295, 298-299
peroxide and genetic damage, 241
transport,"99-100

8- Hydroxy quinoline, inhibition of
amino acid incorporation by, 180-
182

Hypertonic salt solution, influence
on effects of radiation, 206-207, 273

Subject Index

315

Incorporation of antigens by host,
151
of 32P in nucleic acids, 6, 9, 12, 196,
198, 204, 209-210, 228
Incubation medium, effect on cell

recovery, 126-128
Indirect action of radiation, and
chromosome breaks, 248
on enzymes, 25, 27, 28, 34, 38,

305
on nucleic acids, 76
protection against, 294
Indoleacetic acid formation, from

indoleacetaldehyde, 52
Initial effects of radiation, on

enzymes, 39
Ionic yield, for carboxypeptidase, 26
for catalase,26
for chymotrypsin, 26
for trypsin, 26
Interphase killing effects of radia-
tion, 201, 207
Jensen rat sarcoma, radiation on,

196, 204, 228
Ketogenesis, 100
Ketone bodies, induced formation

of, 100
a-Ketonic acids, and inhibition of

cell respiration, 97-99, 100
Leukaemia, experimental treatment
of, 158
transplantation of, 160
Lipase, on enzyme synthesis, 188-

189
Luminescent bacteria, radiation

on, 306-307
Lymphocytes, and cell-death due to

irradiation, 201
Lymphosarcoma, radiation on, 198
Lysogenicity in bacteria, induction

of, 260
Lysozyme, and protoplast formation,
185
use in incorporation studies, 186
Mammary sarcoma, radiation on,

198
Metabolic effects, enzymic explana-
tion of, 37
Metabolic processes, rates of, 92
Metabolism, and cytoplasmic struc-
ture, 3-20
and glucose-6-phosphate, 94
oxidative, and citric acid cycle, 42
Metabolites, transfer of, from dead
to living cells, 215

Metal effects, on amino acid in-
corporation, 182
Micro -beams, 258-260
Microsomes, and amino acid in-
corporation in proteins, 7, 8, 162,
164, 169
Mitochondria, adenosine triphos-
phatase activity in, 86
and specificity of radiation effect,

84
change in permeability after ir-
radiation, 44
in bird erythrocytes, 3
in liver homogenates, 3
morphological differences in, 80
oxidative phosphorylations, in, 3,
78, 86
Mitochondrial defect, nature of,

84-88
Mitosis, inhibition by colchicine, 208
inhibition by heparin, 208
inhibition by radiation, 77-78, 233-

234
in lymphocytes, 90-91
Mitotic activity, influence of de-
oxyribonucleic acid on, 214
cycle, timing changes, 199-200
index of tumours, radiation on,
198
Mutagenic effects of organic per-
oxides, 287-288
Mutations, dose-reduction factor for,
133-134
interference with, 133-134, 139
production of, 66-67, 70
Neurospora, radiosensitivity and
ploidy, 265-266, 268
induced mutagenic effects in, 287-
288
Nitrogen mustard mutations, and
primary site of radiation damage,
256
Nitrogen mustards, and amino acid
incorporation, 69, 183
and tumour inhibition, 69, 183
reaction with nucleic acids, 67-68
Notatin (glucose oxidase) and oxygen

effect, 114
Nuclear damage, 255-270

functions, disturbance of, 78-79
metabolism, relation to oxidative

phosphorylation, 82
structure, and metabolic activities,

3-20
transfers, 260-264

316

Subject Index

Nucleic acids, and amino acid in-
corporation, 174-182
and effects of X-rays and radio-
mimetic agents, 59-69
biological functions of, 174
digestion on, 178-179
permeability of cells to, 184
purity of preparations, 183-184
radiation on mixtures of, 304
structure, 59-60
Nucleic acid synthesis, and rate
limiting reactions, 105
and viability of cells, 132
metals on, 184
relation to protein, 172
Nucleolus, role in cell metabolism, 4,

6, 12, 17
Nucleoproteins, radiation on, 269
radiosensitivity of, 72-73
structure, 59

X-rays and radiomimetic agents
on, 59-69
Nucleus, role in cell metabolism,
3-20
transfer of, between two species, 23
Organic peroxides, effect on viral
and bacterial functions, 275-
297
inactivation of bacteria by, 278-
280
of bacteria-bacteriophage com-
plexes by, 283-284
of bacteriophage by, 280-283
of pepsin by, 288-289
of Pneufnococciis-tTansforming
principle by, 290-291
influence of light on effect of,

280
mutagenic effects of, 287-288
posteffect on bacteriophage,
284-286
Orotic acid incorporation, in de-
oxyribonucleic acid, 196
in ribonucleic acid, 171
Osmotic control in retinal tissue, 50
Oxidative phosphorylation, and
adenosine triphosphatase ac-
tivity, 86
and pacemakers, 96
and radiation dose, 81-82
and rate of oxygen consumption,

96
and the " Pasteur effect ",94
and tissue specificity of radiation
effect, 83-84

Oxidative phosphorylation

during radiation, 91

inhibition by chemical mutagens,

245, 248-250
in radiosensitive tissues, 77-89
relation to nuclear metabolism,

82
relation to cytochrome c, 86
site of, 3
Oxine, see 8 -hydroxy quinoline
Oxygen consumption, control of
rate of, 96
in isolated nuclei, 3
radiation on, 41
Oxygen effect, 106-115

and deoxyribonucleic acid in-
activation, 208
and metabolic activity of the

cell, 111-112
and mitochondria, 88
and radiation damage, 109
and respiratory poisons, 108
and sensitization, 28
in chromosomal aberrations,

242-243
in fresh yeast, 112
in low-nitrogen yeast, 112
in relation to protection, 113-

114
on enzyme activity, 114-115
on Esch. coll B, 116
on growth of Sarcina lutea, 108-

109
on metabolism (respiration and
glycolysis) of Esch. coli, 110-
111
on nucleoproteins, 73
on Pnetimococcus-transiorming

principle, 295
on rate of reproduction of
Sarcina lutea, 108
Oxygen uptake, effect of radiation
on, 43, 81, 82
in presence of glucose, 110
Pacemaker reactions, 92-93, 95,

96, 104
Parabiosis, and recovery after ir-
radiation, 140
Paramecia, induced mutagenic effects

in, 287-288
Particulate cell constituents and

protein synthesis, 162-168
Part- inactivation of bacteriophage,

286-287
" Pasteur effect", 94

Subject Index

317

Penicillin, inhibition of amino acid

incorporation by, 180
Pepsin, inactivation by organic

peroxide, 288-289
Peroxidases, prevention of radio-
lesions by, 275
Phosphate uptake, depression by

radiation, 80, 81, 82
Ploidy effect on radiosensitivity,
of bean roots, 272
of mammalian cells, 272
of multinucleate cells, 264-

266
of Neurospora, 272
of Tradescantia microspores,

271
of yeast, 272
Pneumococci, permeability to de-
oxyribonucleic acid and deoxyribo-
nuclease, 190
Pneumococcus- transforming

principle, inactivation by
organic peroxide, 290-291
inactivation by X-rays, 63, 76,
276, 291-295
Polyvinylpyrrolidone, therapeutic

effect of, 140
Primary sites of energy deposition,
associated with radiobiological
lesions, 255-270
Properdin, therapeutic activity of,

140
Protection against radiation, 28-30,

112-114, 133, 305
Proteins, metabolic stability of, 215-

222
Protein synthesis, and nucleic acids,
6, 7, 14, 15, 177
and particulate cell constituents,

162
and rate of labelling of ribo-

nucleoprotein, 162, 164
and recovery, 131-132
and ribonuclease, 7
and the nucleus, 6, 13
and the nucleolus, 7
effect of metals on, 184
in absence of deoxyribonucleic

acid, 194-195
in ascites tumour, 220-221
in disrupted cells, 176
in protoplasts, 185-195
inhibition by chemicals, 101,

179-180
mechanism of, 161-168

Protein synthesis

role of nucleus in, 17
use of lysozyme in study of, 186
Protoplasts, abihty to divide, 186
bacteriophage multiplication in, 185
deoxyribonuclease on, 190-191
formation, and lysozyme, 185
induced enzyme synthesis in, 187—

188
protein synthesis in, 185-193
removal of cell wall on, 185, 186
ribonuclease on, 191-192
Radiation syndrome, treatment of,
140-153
treatment of, by implantation of

spleen, 141, 143
treatment of, by injection of bone
marrow, 141
Radiobiological lesions, and prim-
ary sites of energy deposition, 255-
270
Radiomimetic agents, effect on
nucleic acids and nucleoproteins,
59-69
Radiosensitivity of cells, with

changing conditions, 36
Recovery, and cell-free extracts, 158
and parabiosis, 140
in bacterial cells, 124-125
in regenerating liver, 227-236
of deoxyribonucleic acid synthesis
after irradiation, 198, 203
Reducing agents and radiation

damage, 108
Regenerating liver, radiation on

metabolism of, 225-236
Repopulation of host from donor,

144, 150, 151
Reproductive integrity of the cell,

destruction of, 266-268
Reserve products, decrease in

utilization of , 4, 5. 9, 20
Ribonuclease, effect on enzyme
synthesis, 189
-effect on protoplasts, 191-192
inhibition of amino acid incorpora-
tion by, 7, 186,
Ribonucleic acid, and amino acid
incorporation, 171, 174, 178
and protein synthesis, 6, 7, 17,

166, 167
and stimulation of recovery,

158-159
cytoplasmic, decrease in enu-
cleated amoebae, 9

318

Subject Index

Ribonucleic acid

cytoplasmic specific activity of, 5
digestion on, 178-179
in chromatin, 21-22
in growing tissues, 161
in protein-secreting tissues, 161
metabolic stability of, 215-222
nuclear, as precursor of cyto-
plasmic ribonucleic acid, 5-6,
13, 20
nuclear, specific activity of, 5
nuclear, two types of, 20-21
specificity of, 183
synthesis, and recovery, 131-132
synthesis, during incorporation,

171, 172
synthesis, in absence of nucleus,

13
synthesis, in tumours, effect of
radiation on, 213
Ribonucleoprotein, role in protein

synthesis, 162, 166
RNA {see Ribonucleic acid)
Sarcoma I, 144, 152
Shearing stress, 72, 73
Skin grafts, 147, 153
Spleen treatment and protection,

132
Steroid excretion after irradiation,
47
synthesis, in perfused adrenals, 45,

46
-synthesizing enzymes, 46
Staphylococcus aureus, disrupted cell
preparation of, 175-182
effect of nitrogen mustards on,
68
Subcellular systems and protein
synthesis, 185-193

Therapeutic effect of radiation, 71

Transplantation immunity, 144r-
147

Tricarboxylic acid cycle, 93

Triosephosphate dehydrogenase
systems, 94

Trypsin, effect on enzyme synthesis,
188-189
radiation on, 38-39, 40

Tryptophan, destruction by radia-
tion, 35

Tumour inhibition, nitrogen mus-
tards on, 69

Tumours, radiosensitivity of, 71,
76

Uranyl chloride, inhibition of amino
acid incorporation by, 187

Viability of irradiated Bacillus sub-
tilis spores, 257

Vicia faba, root growth on radiation,
117

Viral and bacterial functions, radia-
tion and organic peroxides on, 275-
297

Virus synthesis, in infected proto-
plasts, 186

Vitamin K, and dicoumarol, 101
role in hydrogen transport, lOSO

Visual purple, effect of X-rays on,
49-50

Walker carcinoma, radiation on, 72
inhibition by nitrogen mustards,
183

Water, irradiation of, in presence of
oxygen, 251—252
irradiation of, in presence of oxygen
and hydrogen, 252

X-ray cataract, coenzyme A level in,
43

Printed by Spottiswoode , Ballantyne & Co. Ltd., London and Colchester

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