REPORT OF THE

UNITED-NAXI-ONS .

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EFFECTS OF !iit}ifffHiADIATION.. : . . .

·'GENERAL ASSEMBLY
OFFICIAL RECORDS : SEVENTEENTH SESSIONSUPPLEMENT No. 16 {A/5216}

New York, 1962

NOTE
Throughout the present report and the annexes thereto, references to theannexes are indicated by a letter followed by a number: the letter denotes the relevant
annex and the number the paragraph therein. Within each annex, references to its
scientific bibliography are indicated by numbers.
Symbols of United Nations documents are composed of capital letters combined with figures. Mention of such a symbol indicates a reference to a United
Nations document.

h we ut··m,.

ANNEX B

FUNDAMENTAL RADIO-BIOLOGYCONTENTSParagraphs

I. INTRODUCTION •••••••••• • ••••••• • •••••••••••••
II. INTERACTION BETWEEN IONIZING RADL�TION A�"'ll
LMNG MATTER • ,,,,,, , ,,, ,, •,,,,, , •, ,, , , , , ,,,Energy dissipation by X - and gamma-rays andby corpuscular radiations....................
Ionization density-LET ... .. .. ..•. .. ... ..• ••Transport of energy..........................
Ill. QUANTITATIVE ASPECTS OF RADIATION EFFECTS,.,,
Hit principle (target theory) and dose-effect relationships ........••••.............•..••...
The threshold problem•..••...•..........•....
Direct and indirect effects of radiation ...•..••.
Influence of dose-rate and dose fractionation.•..
Relative biological effectiveness.••..•...•.•...•
IV. RADIATION CHEMISTRY ••••••••••••••••••••••••
Water and aqueous solutions of simple compounds .....••••••....•••..••••.•.••••••,.•
Nucleic acids ................................ .
Proteins ...•.•...•••.•..............., ,,, ••,,
Polysac:cbarides •.•...•.••....•........••••..•
Macromolecular complexes •....•..... , ., ,....
Detection of free radicals in whole cells by electron spin resonance (ESR) ................ ..

Paragraphs

1-3

General considerations of radiation effects in cellular metabolism ...•...................•... 161-167
Effects of radiation on integrated functions..••. 168-171
Growth rate ......•......••................ 169
1Iitotic delay ..............•...••.......... 170-171
Inhibition of mitosis and cellular death: reproductive and interphase death................. 172-177

4-9
6
7-8
9

VIL BIOLOGICAL VARIABLES INFLUENCING RADIATION RESPONSE ••, ,• ,••,,,,• , ,•,,,,•,• ,••,,•••••,,••• 178-185
Concept of radio-sensitivity............. ..... 178
V ariations in radio-sensitivity with stage of division .. ,. ,.•..•••,.•...••••....•.......... 179-181
V ariation of radio-sensitivity with ploidy....... 182-184
Genetic control of radio-sensitivity in bacteria.. 185

10-35
12-18
19-22
23-26
27-30
31-35

VII!. PRIMARY GENETIC EFFECTS OF RADIATION......... 186-225
The genetic material.......................... 187-189
Point mutation .............................. 190-192
Radiation-induced mutagenic effects•.....•.•... 193-194
Oxygen effect ..•.....•......••.•.••.••....... 195-196
Chemically-induced mutagenesis . .•. ••...... ... 197
Uptake of abnormal p_recursors................ 198-199
Comparison between various mutagenic agents.. 200
Biochemical aspects of mutation processes....•. 201-204
Mutation expression •...•..... ....••••....... 205-206
Chromosome breaks .......................... 207-210
Factors influencing the production of chromosome breaks •••••......•..................• 211-213
Genetic effects of incorporated radio-active substances ,........ ,..............•........... 214-215
Possibility of transmutation effect with cu..... 216-217
Local consequences of transmutation••......... 218-222
Ionization dose per transmutation under uniform
contamination •.••...............•........•• 223-225

36-93
38-49
50-61
62-78
79-82
83-88
89-93

V. CHE:\UCAL FACTORS MODIFYING RADIATION RE·
SPONSE L.._ CELLS,•••• ,,,,••••,,.,,,.,,••,,,•• , 94-110
Oxygen effect ••••...•••..••••.,..•.•.•.....•• 94-96
Effect of gases other than oxygen.....••..••••• 97
Effects of gases under pressure.•..........••.• 98
Hydration .....•.•••••......•..•....•......•• 99-100
Peroxide after-effects •.•..•............••.••• 101-102
Chemical protection .......................... 103-110
VJ. EFFECT OF R..\DIATION ON CELLULAR STRUCTURESAND THEIR FUNCTION ......................... 111-177
DNA synthesis •..•••.•...•.........••.•...••. 116-132
fu'l'A and protein synthesis.................... 133-157
Effects of radiation on antibody synthesis...•... 158-160

IX. RECOVERY AT THE CELLULAR LEVEL••,•••••••,,••• 226-243
X. GE�·ERAL COXCLUSIONS ................. ,.... • • 244-250
REFERENCES

the impact of molecular biophysics on fundamental radio­
biology is greater than in the past. The molecular ap­
proach will eventually enable us to understand the effectsof radiation on the impairment of fundamental processes
in the cell. The effects of radiation on macromolecules
or subcellular structures are thus of great importance infundamental radio-biology.
3. This annex deals essentially with ionizing radia­
tion ; investigations with non-ionizing radiations are re­f erred to only in so far as they bear on our understandingof the effects of ionizing radiations.

I. Introduction

1. The effects of radiation on living matter must be
envisaged at different levels of organization, those ofindividual molecules and macromolecules, subcellular
structures, whole cells, tissues and organs, whole organ­
isms, and populations of organisms. To understand theaction of radiation, each system must be studied inde­
pendently and in its natural context. The actions become
more complicated as the organization level rises. At
each level and for each effect studied, it is sometimes
helpful to think in terms of the sensitive molecule or
structure, the sensitive cell, tissue, or organ.

II. Interaction between ionizing radiation andliving matter

2. The present annex deals chiefly with macromole­cules, subcellular structures, or isolated cells and cell
populations. Our knowledge of the molecular organiza­tion of various cell organelles is increasing rapidly and4. The absorption of ionizing radiation by matter isfollowed by a comple..x of events the nature of which41depei:ds on abso~b.ed dose ~d th~ chemical a;id physicochenucal composition of the irradiated matenal. Various
stages can be recognized in the development of radiation
effects. These are not sharply demarcated but blend into
each other. Distinctions have some value, however, because they permit a partial analysis of the temporalsequence of events.
5. (a) Elementary reactions. These occur in a very
short period of time,,...., 10-11 -10-15 seconds. They are pnmarily physical and result from the interaction between
photons or ionizing particles and atoms and molecules.
These interactions give rise to excitations and ionizations. Excited and ionized atoms and molecules are highly
unstable and chemically active; rearrangements in theelectron configuration of the excited structures lead tothe primary products of radiation action which may bestable or unstable molecules, or free radicals.
(b) Primary reactions. Radicals and excited molecules formed as the result of elementary processes reactchemically with neighbouring molecules and between
themselves. This stage, the chemical stage, may last froma fraction of a second to hours.
( c) Secondary reactions. Elementary and primary
reactions give rise to secondary reactions in which macromolecules of essential biological significance and major
metabolic pathways are affected. Secondary reactions
result, therefore, in alterations and impairment of cellular structures and functions, and may lead to biologicallyobservable radiation injury. This, the biological stage,
may last from a few hours up to years in long-lived
multicellular organisms.

existence in condensed systems since they are inherently
unstable. Although excitation can lead to dissociation of
the molecule, it is less likely to do so in the case ofn;or~ complex molecules where excess energy can be
distributed over many bonds. Energy degradation within
the same molecule is known as internal conversion.
Through internal conversion, the excited molecule isdegraded in energy from a higher to a lower excitedstate, or returns to the ground state; the excess energy isconverted into vibrational and rotational energy and may
be transferred to other molecules. Energy can also betransferred from one molecule to another through processes known as exciton interaction and resonancetransfer.1 The increasing emphasis on the mechanisms
by which energy migrates and on their role in radiation
effects is reflected in recent symposia and reviews. 2- 'm. Quantitative aspects of radiation effects
10. Known dose-effect relationships may be describedunder a limited number of headings. Their graphic presentation is often simple, linear in a few instances, andin general exponential or sigmoid. Thus, oxidation offerrous ions and reduction of eerie ions in aqueous·solution is, in certain circumstances, directly proportional to dose. These effects may be interpreted as due
to radicals induced in the aqueous medium. However,in somewhat more complicated situations, e.g. the inactivation of enzymes in solution or in the solid state,
there- may be an exponential relationship between remaining activity and radiation dose. This relationshipexpresses, in part, the fact that inactivated molecules arestill able to capture radicals and thus to decrease thenumber of radicals for inactivation of still intact
molecules.

ENERGY DISSIPATION BY X- AND GAMMA-RAYS A:ND
BY CORPUSCULAR RADIATIONS

6. The elementary characteristics of ionizing radiationand the way energy is absorbed by ionization have been
described in chapter II. Only part of the energy absorbed by an irradiated tissue gives rise to ionizations;the remainder, in a process called excitation, raises electrons of atoms or molecules to a higher energy level
without expelling them. In its chemical or biologicalaction, the energy absorbed in the excitation process is
not considered to be as important as that absorbed in
the ionization process. However information is incomplete on this point.

11. Even for complex systems like living cells, theexperimental relationship between dose and effect is?f~en a sirr:ple one. In the study of these relationships
it 1s essential to define the effect clearly. For isolated
cells, reproductive ability has been used most frequently
as the criterion of damage. Cells which have lost reproductive integrity may still divide a few times. However,ceIIs affected in this way can sometimes maintain theability to accomplish for a certain time some metabolicor ~hys_iological fu~ctions at .near normal rates, e.g.,
respi_rahon, 5 • ~ pr~tem synthesis,7 motility. 8 The doses
required for impamnent of such metabolic functions areusually much greater than those necessary to impair
reproduction.

!oNIZATION DENSITY-LET

7. I;i any interaction of ionizing radiation with matter,the ultimate transfer of energy is carried out by a charged
particle. pie rate o! loss of energy by a particle along~ts path is prop~rttonal to the square of charge andi?versely proportional to velocity. Hence, for any parttcl~, the rate ~f loss of energy is greatest near the endof its track. Lmear energy transfer (LET) is definedas t~e linear rate of los~ of energy (locally absorbed)
and is usually measured m keV/ µ..

HIT PRINCIPLE (TARGET THEORY) AND DOSE-EFFECTRELATIONSHIPS

12. According to the hit theory,11-11 the biologicaleffects of ionizing radiation on cells are due to hits in asensitive component of the cell; hits produced outside
this "target" are. ineffective. Although, as originallyformulated, the hit was considered to be an ionization
or excitation produced directly in the target, the theory
has been enlarged to include hits produced by diffusible
products involved in indirect action. 12

8 .. At a given dose the biological effect may varycons1.derably with LET; it may increase or decrease depending on the object irradiated and the effect measured.
There is as yet no complete theory on the influence of
LET (paras. 31-35).

~3. If a cell is inactivated by a single hit in a target
or m any of a number of targets, it can easily be shownthat the survival curve is exponential. The number ofcelis escaping biological modification (N) is then related
to dose according to the formula N = N 0 e-aD where N
is the number of cells originally present, D i~ dose,

TRANSPORT OF ENERGY

9. Free radicals, whose intrinsic lifetime is indefinite
us:13:11y disappear quickly_ because of their reactivity'.
Excited molecules have, m general, only a transitory

and

42

a is a constant expressing the sensitivity of the cells.

From this formula it follows that the number of survivors will be N/N o = e-1 ,....., 0.37 for the dose 1/a whichis the dose that brings about one hit per target on the
average. This 37 per cent dose is important in calculations of the volume of the target.
14. When two or more hits are necessary to destroy
one target or when two or more targets in one cell haveto be hit before the damage shows, the survival curves
are no longer e..xponential but are sigmoid and havean initial shoulder when the logarithm of the survival isplotted against dose. In the latter case (two or moretargets), the number of targets can be estimated fromthe survival curve by e..xtrapolating the linear part of thesemi-log-plot to zero dose. The value (greater than one)thus obtained on the survival axis is equal to the numberof targets.
15. As a rule, with high LET radiations and neutrons,
and in certain cases with X- or gamma-rays, exponential
survival curves are observed for the inactivation ofviruses and micro-organisms. 9 When the fraction of cells
or subcellular structures affected is small, the numberof responses is approximately proportional to dose. Thishas been found for the induction of mutations in bacteria,
Drosophila, and other organisms; the mechanism seems
to be one hit.
16. X-irradiated polyploid yeast cells13• 14 and isolated
mammalian cells15 have sigmoid dose-effect curves. The
type of curve often depends on the LET of the radiation.
Higher LET values may result in exponential survival
for cells having sigmoid type curves for low LET
radiations.18
17. Sigmoid survival curves are also e.xpected whena population of individuals is irradiated, the susceptibility of which obeys certain distribution patterns.
18. Both exponential and sigmoid survival curves may
have breaks (resistant tails). The interpretation usuallyoffered is that the population studied contains a subgroup which is more resistant to radiation. In general
there are two ways in which this could occur:(a) The heterogeneity may be genetic; the more resistant individuals are mutants of the more sensitive.
This situation can be recognized by isolating a clone
from cells surviving higher doses and by establishing a new survival curve with the population from thisresistant clone. The slope found corresponds to the slopeof the resistant tail in the original curve. However, insome cases, attempts to do this have failed. With thewidely used strain E. coli B, the rate of mutation toresistance is only about 10-5 per bacterium per generation and therefore probably too low to account for theappearance of the tail. 17( b) The heterogeneity may be physiological; in thiscase, if cells surviving at the higher doses are isolated,
the survival curve of the new population shows the same
resistant tail as the original one. This holds in haploidyeasts where budding cells appear to be more resistant. 18
There is similar phenomenon with Pneumococcus transforming principle. 19 A resistant tail may also be seen
with a bacterial population containing cells in both thelogarithmic and stationary phase of growth; the logarithmic phase is more radio-sensitive. 20 • 21
THE THRESHOLD PROBLEM

19. The observation of an exponential survival curve
may be interpreted as a one-hit process. The same applies
43

to the linear relationship for mutation induction when
the number of mutations is small compared to the number of loci at risk; any dose, however small has aprobability of producing the effect.
'
20. Sigmoidal survival curves may be interpreted as
an indication that inactivation results from multiple hits
in a single target or inactivation of multiple targets by
one or more hits in each. There is also a finite probability that any dose may produce an effect. Thus theexistence of biological responses with sigmoidal doseeffect curves do not necessarily prove the existence ofa threshold dose.
21. Even if recovery processes occur at the cellularlevel, these conclusions remain valid; such recovery
merely changes the slope of the dose-effect curve.
22. Without e.xtensive empirical data and detailed
knowledge of the various steps between initial absorption of radiation and expression of biological effects,
discussion of the threshold question is largely limited totheoretical considerations. In the only instance in which
it has been possible to obtain unequivocal experimental
data, the induction of phage growth in lysogenic bacteria,
no threshold was found; one ion pair per cell was effective. 22 It is therefore prudent to assume, as in the lastreport of the Committee, that "biological effects will
follow irradiation, however small its amount". 23
DIRECT AND INDIRECT EFFECTS OF RADIATION

23. Of the models proposed to explain observed doseeffect relationships, the simplest is the target theory basedon the assumption that inactivation is caused only byionizations inside the target-"direct action".
24. Although the concept of a "target" has been maintained in most theories, it has become increasingly apparent that at least part of the biological effect is due
to chemical events outside the target. In this event damage to the target is secondary-"indirect action".16• 24
25. As yet there is no general agreement on the relative importance of direct and indirect action in living
cells. The modification of damage by oxygen or chemical
protective agents has sometimes been interpreted as evidence that indirect action is predominant. It has howeverbeen shown that the effect of oxygen and some protectants is also consistent with direct action, if it is assumed
that the effect of radiation on the target is a two-stage
one. 2 ~· 26 The primary event might then be partly or
totally reversible.
26. The problem of direct versus indirect effects ofradiation has been comprehensively reviewed by Timofeev Ressovski and Rompe 2 with an analysis of mechanisms of energy migration and transfer in the heterologous system. Their theory allows for chance fluctuations in the occurrence of both direct and indirect effects,
and for the mechanisms of propagation of radiation
injury in time and space. Depending on the structure orfunction damaged, either direct or indirect effects may
be considered predominant.
INFLUENCE OF DOSE-RATE AND DOSE FRACTIONATION

27. Variation of the irradiation rate (fractionation ofdose or variation of dose-rate) may influence the biological effect in some instances. When radiation damage isirreparable, no modification of the response is e."Cpected;
if a modification is seen, it is generally assumed to repre-

sent a repair mechanism. Mice, Drosophila, plants, andseveral other species (C, table VII) have been extensively studied. Other examples are Arbacia eggs 27 andmammalian tissue culture cells. In Arbacia sperm, however, no repair has been observed. 28 • 29

which RBE and LET are directly related, inYersely related, in which RBE shows a maximum for a certain
value of LET, and in which RBE is constant. Other
factors make the picture even more complex ; RBE values
may depend on dose, dose-rate, presence of O>..")'gen, andphysiological conditions.
34. The LET concept itself is complex. The kinetic
energy loss of a particle is discontinuous and subject tostatistical fluctuations. 42 Furthermore, it varies along the
track. For these reasons an average value must be calculated. In principle, however, RBE not only depends on
this average value of LET, but also on LET distribution.
The following figure 43 attempts to summarize experimental data on bacterial, plant and animal cells.

28. If the phenomenon under study is single hit, e.g.
induction of point mutations, repair processes would
reduce the magnitude of the slope of the dose-effectcurve. Russell 30 discovered that low dose-rates were lessefficient than high dose-rates in inducing mutations inmouse gonial cells. This dose-rate effect was maximal at
0.82 r/min; further reduction of the dose-rate had nofurther effect on mutation rates. 31 Russell's finding,which stimulated similar studies by others, has been confirmed in several species. Low dose-rates also greatly
diminish the sterilizing effects of radiation in female
mice and increase survival of spermatogonia. 39

10.0

s.o

29. The effectiveness of fractionated doses to themouse testes has been demonstrated with doses in therange of 1600 rad.' 0 In experiments with Drosophila at
low doses and different stages of spermatogenesis, noeffect of dose fractionation has been observed. 32

3.0

30. The effect of dose-rate on multi-hit processes is
not difficult to explain. If the rate of delivery is reduced
so as to increase the time between two successive events(hits) significantly, and if the individual lesions due tohits can be repaired within a certain time, lowering thedose rate or fractionation of the dose will result in adiminished frequency of effects for a given total dose.
The role of chromosome aberrations may be of particularimportance in monkey or human embryonic tissue cultures. Some reports indicate that these tissues are twoor three times more sensitive than those of mice. 83 • 84
Investigations of the repair of pre-mutational damage
have been carried out with many species, including mammals35• 36 insects 37• 38 and plants. 264 • 1516 This subject is
discussed more fully in annex C.

0,50.3 .......~~~~~--~~~~~_._~~~~~-1000
100lLET, keV/µFigure 1. Variation in RBE with LET. for biological materials
irradiated in aerobic conditions43

A: plant cellssrr-5s2
B : animal ce1Is203, 577, ss3-ss5

C: two strains of bacteria1s3, 5SG-5S7

35. To assess the RBE of a certain radiation, doseresponse curves of the particular biological effect are
determined for both test and standard radiations. If bothcurves coincide when all dose values of the test radiation
are multiplied by a constant factor, the RBE is equal tothis factor. Sometimes the curves do not have identicalshapes; the RBE value then depends on dose. This comparison pertains to absorbed dose. If this dose is not
uniform throughout, the average value is used. This may
not be strictly correct ii the biological effect depends on
dose. There are many other complications that make experimental RBE values difficult to interpret. The values
are, however, useful in the practice of health physics,
where upper limiting values of RBE are used to transform dosages measured in rad to rem.

RELATIVE BIOLOGICAL EFFECTIVENESS

31. \\Tith radiations of different quality, the absorbed
doses required for a given effect are usually not the samefor different types of radiation. The extent to whichradiations of different quality differ from each other inthis respect is a measure of their relative biological effectiveness (RBE). The RBE of two radiations is definedas the inverse ratio of the respective doses that are necessary to bring about a given effect. The radiation
standard chosen by the ICRU is an X- or gamma-radiation having a LET in water of 3 keV / µ. delivered at a
rate of about 10 rad/min.

IV. Radiation chemistry

32. In the simplest cases, the mechanism underlying
the difference in efficiencies of radiations can easily be
e:cplained. For an event which is inhibited by the absorption of a minimal amount of energy, such as the inactivati?n of an enzyme or virus, the low ion density radiation
will be more effective than high ion density, because someof the latter ionizations will be wasted. On the other hand,radiation with a high density of ionization will be more
e~ective when larger amounts of energy are needed( ~m~t.dtaneously or within a relatively short time or
w1th.n~ a certain volume) to produce the effect in thesensitive structure.

36. Since water constitutes 70 per cent or more of cellmass, water molecules take up most of the energy imparted to cells by ionizing radiation and may be importantin the damage to vital cell components. Knowledge collected during the last decade about the chemical changes
induced by irradiation of water and aqueous solutions ofsimple compounds is therefore of great importance toradio-biology. Work has been done on the radiation
chemistry of solutions of nucleic acids and other macromolecules to gain some insight into the mechanism bywhich reactive intermediates generated in water attack
these molecules. The main results from those fields ofresearch will therefore be summarized in this chapter.
37. In interpreting these results, it is generally assumed that free radicals are important in the chemical. 33. Thus, RBE depends not only on the LET of agiven radiation but also on the effect studied, and thisdepen~ence may assume various forms. Thus, Zirkle 41
has pomted out that there are e:iqierimental situations in44

reactions resulting from ionization and perhaps fromexcitation of water molecules. At present, there isabundant evidence to support such a view. Recently,
development of the electron spin resonance technique
has provided a method for direct study of free radical
formation in certain irradiated materials.

cals formed in close proximity, and the damage to certain
molecules will be much less in cells than in dilute solutions. Secondly, the presence of great numbers of simple
and complex molecules in cells may give rise to secondary
and tertiary reactions which differ from those in simple
solutions.
45. Knowledge of the primary reactions in irradiatedwater has been derived largely from the study of aqueous
inorganic solutions. Much e.'Cperimental work has also
been done on aqueous solutions of organic compounds.
However, for many changes in solutions of simple molecules, the reaction mechanism has not been unambiguously established.

\VATER AND AQUEOUS SOLUTIONS OF SIMPLE COMPOUNDS

38. Most reactions in irradiated water can be e.xplained satisfactorily by assuming the formation of H 0
and OH 0 radicals. Recent reviews 4'-i 6 of the chemical
effects of ionizing radiation have shown the usefulness of
the radical hypothesis in interpreting the rapidly growing
body of experimental data, although some uncertainty
still exists with regard to the H 0 radicals and their distribution around the track of an ionizing particle. It
might be that what has been called an "H 0 radical" is inreality a hydrated electron, H 20-.
39. For each 100 eV of dissipated energy some 4
H 20 molecules are split into OH 0 and H 0 • OH 0 radicals
can combine to H 20 2 and H 0 radicals to H 2. A considerable fraction of the radicals react in this way to give
"molecular products" before there is any significant
diffusion or reaction with solute molecules. In chemicallypure water, however, only very small amounts of molecular products can be detected, because they are reverted
to water molecules through back reactions with freeH 0 and OH 0 radicals.

46. There is evidence for the formation of hydroperoxides in the presence of oxygen :
RH
OH 0
R0
H20
0
R
02
RO~

+
+

reduction

ROf
R02
R02 + H+ --~ R02 H

47. In some instances hydroperoxides are believed tobe labile intermediates, but stable peroxides have also
been found, e.g. after irradiation of solutions of various
amino acids and of pyrimidine bases 48 and their nucleosides and nucleotides.' 9 • 50 The formation of hydroperoxides may enhance oxydation, e.g. increase oxydationof ferrous ions in acid solution where there are organic
impurities. This can be prevented by addition of Qions; these react with OH0 radicals ( OH 0
Cl- ---+
OHCl 0 ) and thus modify the sensitizing action oforganic molecules. 51

40. When solutes capable of reacting with H 0 or OH 0
radicals, thereby preventing the back reaction, are present, the products H 20 2 and H 2 are produced in measurable amounts. Their yields depend on LET, a greater
LET giving rise to a larger amount of molecular products
through combination of free radicals. The molecularyield also depends on the efficiency with which free radicals are scavenged by solute molecules. Some very efficient scavenging solutes can depress the formation ofH2 and H202 considerably.

++

48. Reactions between radicals and oxygen, andbetween radicals and hydrogen-atom-donating compounds, have been shown to be important biologically.
In the bacterial spore, radicals formed that are biologically effective if they react with oxygen may be removedby hydrogen donators such as H 2S prior to 0 2 reaction. sz, 53 Such mechanisms have been proposed forother systems.'9 • 5 '· 55

41. A very common solute is 0 2. It reacts with H 0
radicals to give the radical 0 2 H 0 • This e.'Cplains why theyield of various radiation-induced chemical reactions isdependent on the presence of 0 2 • The 0 2 H 0 radical ismore stable than H 0 and OH 0 • When no solutes otherthan 0 2 are present, most 0 2 H 0 radicals will combine
according to the reaction 2 02H 0 ~ H 2 0 2
02.

49. Further chemical reactions which may bear onradio-biology are the oxydative deamination of aminoacids, 66 the decarboxylation of organic acids, 45 the oxydation of SR-compounds to the -S-S- dimer, 57 and thedecomposition of glucose by ionizing radiation.u+

42. The primary products in irradiated water may
have oxydizing or reducing properties depending on theredox potential of the solute concerned, on the qualitiesof other solutes (e.g. 0 2 • which converts reducing H 0
radicals to 0 2 H 0 radicals which may have oxydizing
action), or on pH.

NUCLEIC ACIDS

50. Irradiation of nucleic acids in aqueous solutions
leads to several different chemical changes which affectboth the purine and pyrimidine moieties and the sugarphosphate backbone. As yet, it is impossible to give a
consistent and quantitative description of these chemical
effects of irradiation. Because of the diversity and complexity of the chemical changes, only the main pathways
are considered to be established.

43. The influence of pH is explained by the following
ionic equilibria: H 0
H+ ±::; H 2•; OH 0 ±:::; H+ 0- and0
0 2 H ±::; H• + 0 2-. It should be noted that, in neutral
solutions, 0 2 H 0 radicals have far less oxydizing power
than at low pH's. The oxygen effect in living systems
can therefore probably not be interpreted as an enhancement of oxydation through the reaction H 0
0 2 ---+
02H 0 •+++

51. The chemical changes produced by irradiation ofdilute solutions of nucleic acids are, for the most part,
initiated by radicals formed in the aqueous media. In
agreement with results of e.xperiments with simple
nucleic acid components, there are two main reaction
pathways by which radicals attack nucleic acids inaqueous solutions : (a) destruction of the bases, the predominant site of chemical attack, 'and ( b) oxidation of
the sugar moiety. 5 11-oi The products of irradiationof the bases in the presence of oxygen differ from+

44. Application of these data to radio-biologicalsystems is by no means straightforward. In the firstplace, the diffusion range of free radicals in living cells
is very limited,47 because many molecules can react withfree radicals, thereby protecting more vital components.
Cell structures can be attacked, therefore, only by radi45

hydrogen-bonded structure has been shown by the increase in ultra-violet absorption near 260 mµ. after small
doses of irradiation,61 • 70 • 12 and also by titrimetricstudies.10, •s
56. Degradation of DNA proceeds for some timeafter irradiation, as judged by viscosimetric measurements.76 This "after-effect" is more pronounced if DNA
is irradiated in air-saturated solution.79-8 1 There areseveral hypotheses to e..x:plain this kind of instability;
the decay of some unstable pyrimidine hydroxyperoxides, 60• 61• 82 and hydrolysis of labile acyl-phosphatesn, 64 are the most plausible of them.

those formed in its absence. In o>..-ygen-free solutions,
pyrimidines are converted into products of undetermined
structure, without any specific ultra-violet absorption. 61
Some guanine residues are converted to 2 :4-diamino-5-formamido-6-hydroxy-pyrimidine which is attachedto the sugar by a labellized glycosidic linkage; fromthis they are gradually released as free bases. It isbelieved that the attack on adenine forms the corresponding fonnamido-pyrimidines, although this has not been
directly demonstrated in irradiated DNA.59 The yieldof chemically altered bases is highest for pyrimidine residues and lowest for purine residues, 59 • 61- 63 a circumstance which reflects their comparative radio-sensitivity.
52. In aerated solutions of nucleic acids, the hydroperoxides of pyrimidine bases are formed with thesaturation of 5, 6 double bonds, and under oxygen thisreaction becomes the dominant one. 61 In DNA, onlyhydro>..-yperoxides of thymine are stable and only theseremain attached to the sugar-phosphate backbone.61 In
the presence of oJ'..-ygen the sensitivity of all bases in
DNA solution is increased two to three times; underthese conditions a presumed 80 per cent of radicals
attacking DNA combine with the base components.
53. The attack of radicals on the sugar moiety leads
to formation of labile phosphate esters. Evidence for thisis seen in the large quantities of inorganic phosphate thatcan be liberated by the acidic hydrolysis of irradiatedsolutions.12 It is believed that this results from oxidative
formation of carbonyl groups in sugar moieties. 61 In
addition to the formation of labile phosphate esters, theattack on the sugar component breaks phosphodiester
bonds and liberates small amounts of inorganic phosphate.60• 6~ From experiments with simple phosphate·esters,65 it appears that inorganic phosphate must comefrom end groups present in the intact molecule, having
been formed during earlier stages of irradiation by main
chain scissions.
54. The direct measurement, with prostate phosphomonoesterase, of the number of breaks induced in thesugar-phosphate backbone has revealed that the yieldfrom this process is 20-25 per cent of the yield in termsof base destruction.66 The same percentage is found if
the release of free bases from irradiated DNA isused to measure the attack on the sugar-phosphatemoiety. 59, 62, e1

57. In dilute solutions, the indirect action of radiation
prevails. With increasing concentration of DNA the
relative importance of this effect decreases in favour ofthe direct action. This has been shown in experiments inwhich damage to DNA, as a function of concentration,
was studied in the presence of iodine ions which almost
entirely prevent the indirect effects of radiation. 8 3-8~Thus, Mekshenkov ascertained that 0.1 per cent solutionsof DNA are almost entirely protected against X-radiation by iodine ions (predominance of indirect effect).
With increasing concentration of DNA, however, theprotective ability of iodine ions decreases so that in a 20
per cent solution, 80 per cent of DNA molecules present
are damaged 86•87 (predominance of direct effect).
58. DNA molecules irradiated in the dry state or ina slightly moist condition are damaged mainly by the
direct action of ionizing radiation. With radiation dosesof ,_, 106 rad, in addition to the main chain scission, anintermolecular cross-linking takes place which leads tothe appearance of branched molecules as judged by
viscosity, sedimentation and light-scattering studies.
With increasing doses up to 101 rad (the threshold dose
depends on water content), this process renders DNA
insoluble in water and gives rise to gel formation. Bothprocesses proceed simultaneously, but their relative rolein the damage depends on moisture content, presence ofoxygen, and nature of ionizing particles. 68 •11 • 8 8-9 0
59. The rates of main chain scission and branching
induced by electrons are about the same at moisture contents up to 25 per cent, and are largely unaffected byoxygen. With swollen DNA gels having a water content
of 2? to 70 per cent, intermolecular cross-linking predominates over the scission of the main chain in theabsenc~ of oxygen. However, in the presence of oxygen,the ratio between the effectiveness of the two processes
is reversed. Above 75 per cent water content, and evenin the absence of oxygen, no gel is formed. 88 • 89 Alphaparticles are much less effective in the branching process
than electrons. With alpha-particles only a limited
amount of cross-linkage is found in the absence ofoxygen, and this is independent of the moisture content.
In the presence of oxygen one main chain break is produced by nearly every alpha-particle traversing a DNA
molecule. 90
60. It is believed that clusters of ionization are responsible for the main chain breaks; cross-links result fromthe combination of active points formed by ionization89 • 110
for which carbon radicals are likely candidates. Some
direct support for the formation of metastable species
is provided by the obsen'ation of strong gamma-induced
phosphorescence in frozen solutions of DNA and~NA. 91 With direct irradiation of dxy DNA preparat10ns by gamma-rays, the ESR method reveals thepresence of one radical per 105 DNA molecules for adose of 2 X 103 rad. 9 2

55. Studies of physicochemical changes in nucleic
acids after irradiation are, so far, chiefly confined todeoxyribonucleic acids. In the double-stranded helical
DNA molecule, both types of chemical lesions introducedby ionizing radiation, destruction of bases and breakageof phosphodiester bonds, must lead to an altered configuration in solution and consequently to changes inphysicochemical properties. The destruction of the baseresults in local dissociation of the double-stranded structure, and the break in one of the chains results in increased flexibility; two independent breaks at approximately opposite positions in each of two intertwined
chains lead to a scission of the whole molecule. There ismuch evidence supporting this general picture. Thus,
the critical temperature for the thermal denaturation of
i:r;radiated DNA is reduced. 68 Like,vise, the intrinsic
viscosity of irradiated DNA solutions shows marked
decreases that reflect coiling of the partially denatured
molecule and a fall in molecular weight. 6 11-11 Further
evidence for degradation is provided by light-scattering,71• 72 flow birefringence,72 •73 sedimentation and diffusion studies, 04 • 78 and chromatography on ecteola cellulose
column. 17 The breakdown of some of the secondary46

61. It is worthwhile to mention that ultra-violet radiation also causes aggregation of DNA 94 and, to a lesser
degree, of RN A in the dry state. 95 • 96 In water solution,
irradiation of DNA with ultra-violet light induces covalent crosslinks.96• 91 The native secondary structure isalmost preserved as shown by ultra-centrifugation in
caesium chloride. These cross-link processes are probably
connected with dimerization of thymine or uracil
residues. 98 ' 99

unpaired electrons in the protein molecules disappear.
This confirms the previous assumption that prolonged
retention of unpaired electron-excited energy is a causeof radiation after-effects. 114

69. Model experiments with irradiated myosin haverevealed "oxygen effect" at the molecular level. Inactivation of myosin's ATP function by irradiation has
two stages: first (without the involvement of oxygen)is the long-lived "excited" state of the protein molecule
capable of interaction with molecular oxygen; its enzymatic activity is still preserved at this time. Inactivation occurs in the second stage as a result of interaction
with oxygen. In an aqueous solution of myosin, "oxygen
after-effects" constitute most of the total "O:h."}'geneffect" ,113 • 1111-111 These results from a molecular systemcorrespond well with those from studies on a biologicalsystem and thus demonstrate the biological importanceof these events. In dry spores of B. megaterittm, oxygen
interaction with radiation-induced states can be almost"immediate" as well as post-irradiation.118 The radiationinduced species have proved to be free radicals in experiments involving post-irradiation heat, nitric oxide,and H 2 S treatments . 119-m coupled with physical experiments (paramagnetic spin resonance studies) of a similar kind. s:, 124 In these experiments, as in those describedabove with myosin, most of the oxygen effect can occur
for an appreciably long time after irradiation. Furthermore, an intermediate state (the metionic state), theconsequence of the reaction of oxygen with radiationinduced active species, has been postulated from studiesof another biological system. 125

PROTEINS

62. Changes in the structure of proteins irradiated indilute aqueous solution are mainly attributable to attackby free radicals and other active species from water. In
cells, free radicals account for ,_, y.1-~ of the effect; invery dilute pure solutions they account for almost theentire effect.100
63. Thiol groups, when present, appear to be the mostsensitive parts of proteins. These -SH groups become
oxydized, as shown by titration,1°1 thus creating new
disulphide bonds with a G* value of about 3. The sameprocess has been observed with enzymes,1° 2 although thehigh G value for the oxydation of those enzymes which
depend on -SH groups for activity does not always
correspond to the G value for inactivation.103 Conversely,
by other mechanisms, disulphide bonds can be reducedby irradiation, a process which leads to the formation of
new thiol groups. 104 ·1°5

64. Proteins, amino acids, and peptides, in solution,
can liberate ammonia on irradiation with large doses,
and can at the same time form carbonyl and amide compounds.106· 101 These products are formed in part fromamino-groups and in part from peptide bonds. This
reaction involves the formation of imino-groups as intermediates. The imino-groups are hydrolized, leading
to the rupture of polypeptide chains. 102

70. The damaging effects of heat and oxygen in theafter-effect response of irradiated myosin solution have
proved to be independent of one another. There are thus
two distinct forms of latent damage in the same irradiated protein molecule; this agrees with the data of Gordy
and his colleagues, who established, by ESR studies, thepresence in irradiated protein molecules of two typesof spectra-some modified by the action of oxygen andothers insensitive to it. 126•127

65. The effect on aromatic rings of amino acids inproteins resembles closely the effect on aromatic
amino acids themselves. Changes in optical density in
the UV absorbing region of some proteins, when irradiated, are similar to those produced in a tyrosine solution.108 Similarly, a decrease in optical density at 280 mµ
has been found for tryptophan itsel£1° 9 as well as forproteins rich in this amino acid.110

71. As a consequence of the chemical changes ofproteins under irradiation, one can e.xpect changes inphysical-chemical properties. Changes in chromatographic,1:9 absorptive,1 29 and electrophoretic130 properties have been seen.

66. Protein peroxides have been detected after irradiation of proteins in oxygen-containing solutions.111

72. In contrast to irradiation in the dry state, themolecular weight of proteins increases after irradiation
in solution. 131-133 From chemical evidence there may be
several reasons for this. Attack of the tyrosine moieties
may induce polymerization as with tyrosine solutions13•(melanin formation). In addition, disulphide linkages
may be formed among protein molecules. Finally, a reaggregation of broken molecules may take place, themolecules being held together by freshly formed hydrogen bonds.m

67. Model experiments with protein solutions haverevealed that the latent damage, caused by radiation in
myosin molecules responsible for the radiation aftereffects, can be eliminated by formation of comple.x compounds with actin molecules if these are introduced into
the solution immediately after irradiation. 112
68. Long-lived activated states persist for a few daysin protein molecules irradiated in aqueous solution.
Activation is associated with disruption of the protein
electron structure; this has been confirmed by the ESR
method. 113 • 114 The ESR method has revealed prolonged
retention, by protein molecules ( myosin, pepsin), of
unpaired electrons appearing after irradiation of protein
solutions. A close relationship has been established between these electrons and radiation after-effects in thesame system. vVhen irradiated solutions are slightly
warmed there is an accompanying "thermal effect", and

73. Irradiation of certain protein solutions (withdoses up to 6 X 106 rad) does not lead to perceptible
effects on physical, chemical and biological properties immediately after irradiation. However, exposure to heat, 136urea, 137 or UV, 110 alters X-irradiated protein solutions'(coagulation, denaturation) more than non-irradiatedsolutions.
74. In the case of catalase and trypsin inactivation,
an after-effect has also been shmvn.138•1311 The extent of
this depends very much upon the post-irradiation temperature to which the irradiated enzyme was exposed.13 9* "G" represents the number of molecules changed or produced for each 100 eV of energy absorbed.
47

The presence of oxygen after irradiation appears t? be,in general, unimportant; the after-effect may be attributable to the formation of protein peroxides, of therrnolabile molecules, or to other causes.uo, 1u

POLYSACCHARIDES

79. The most noticeable effect of radiation on polysaccharides is chain degradation. This holds for all
conditions of radiation152 as shown by decrease in viscosity, changes in light-scattering, electrophoretic andultra-centrifuge patterns. The most probable mechanismof degradation is one involving free radicals formed
from water, because Fenton's reagent, used as a sourceof free radicals, induces the same damage.153

75. According to present knowledge, enzyme i:iactivation is attributable to the action of hydroxyl radicals. m,m This hypothesis is supported by the obs7rva~on 0at
iodine ions serve as protectors for catalase mactivat10n;
it is to be expected that these ions react more readily
with hydro:x7l than with hydrogen radicals.14•

80. New acid and aldehyde-reducing groups areformed in polysaccharides after irradiation.107•154 Smallfragments have been found, e.g. gluconic and glucoronic
acids in the case of dex'tran. Mass spectrometry data
demonstrate the formation of H 2 , CO and C02 when dry
cellulose is irradiated.

76. Very little is known of the chemical changes inproteins brought about by irradiation in the dry state.
The involvement of disulphide linkages has been demonstrated by the close resemblance between electron spinresonance spectra of a number of proteins and that ofirradiated cystine, 127 and by the fact that irradiated ribonuclease, like ribonuclease with its S-S bonds reduced,
can be digested by trypsin whereas the native protein isresistant.145 A general increase in ultra-violet absorption,135•146•147 accompanied sometimes by a shift in theposition of the absorption maximum, indicates an attack
on aromatic amino acids. Changes in content of otheramino acids have also been demonstratedw,us and differences in sensitivity between particular amino acids
have been noted. 147 The formation of ammonia andamines with the development of carbonyl and carboxylic
end groups in the hydrolysates of irradiated proteins is
attributable to an attack on amino acids side chains andon peptide bonds.60 Susceptibility of peptide bonds tomain chain scission is apparently rather low because nosuch breaks have been detected in serum albumin irradiated with doses up to 2.5 X 108 rad.m The oxygen effectobserved upon irradiation of dry proteins seems to beconnected not only with the excitation of protein molecules but also with the e.xcitation of oxygen molecules
which in turn act on hydrogen bonds within protein
molecules.117 The most typical changes in physical,
chemical property are those changes which occur invivo: isoelectric point, decrease in sedimentation coefficient, or aggregation as a result of hydrogen bond formation between molecules with disorganized secondary andtertiary structure.133 • 135 •14 7

81. While the effects of irradiation on polysaccharides
in solution and in dry state are much the same, cellulose
and pectin, when irradiated in a dry state show an aftereffect, but only if stored dry in the presence of oxygen. 155
This is probably due to long-lived radicals formed withoxygen. In addition to degradation, branching has been
obsen-ed in the dry state. 156 The branches are random
in length and spacing. All branch points are probably
tetra-functional. Branching of polysaccharides in aqueous solutions has not been reported.
82. High molecular weight polysaccharides such as
hyaluronic acid in solution (synovial fluid) are depolymerizedm when irradiated with relatively low doses of
X-rays (9,000 r), and the process continues about
twenty-four hours after irradiation. Viscosity and lightscattering measurements have proved that, during theafter-effect, depolymerization continues. The most probable sites of depolymerization are the -0-C-phosphoester bonds. The addition of cysteamine158 protects thesynovial fluid, although in the absence of oxygen (presence of nitrogen) synovial fluid is more radiationsensitive. A detailed study of ESR of irradiated polysaccharide has not thrown any light on the observed
chemical changes. Internal crosslinking has been suggested159 although direct proof, using hyaluronic acid,does not exist.

77. The important aim of studies of the action ofionizing radiation on proteins is to understand themechanism of radiation-induced enzyme inactivation.
The catalytic capability of an enzyme is determined,
most probably, by an active site composed of only a verysmall number of amino acid residues maintained at thesurface of the enzyme molecule by secondary and tertiary
bonds. Thus, enzyme inactivation can be accomplishedeither by chemical alterations in the amino acid residues
within an active site or by disruption of essential configuration.

MACROMOLECULAR COMPLEXES

83. There is growing interest in relating the results
obtained by irradiating isolated compounds of macromolecules in aqueous solution, and even in the pure solidstate, to those from integrated macromolecular complexes (section VI below). N ucleoproteins are probably
the closest models of nucleic acids as they exist in thecell, although the status of nucleoproteins in vitro may
be very different from that in viva.
84. Protein has a protective effect because it traps
radicals that would otherwise reach the deoxyribonucleic
acid (DNA), but the extent of this trapping is unknown.160 However, some protective action of nucleic
acids on the denaturation of ovalbumin as measured by
the number of titrable sulfhydryl groups has been reported.1e1

78. The efficiency of inactivation through ionizationis very high, with G,..., 1. This implies that one ionization or cluster of ionizations anywhere within or near amolecule inactivates that molecule. This makes the
hypothesis of inactivation via an attack on the site ofspecific activity improbable. Consequently, inactivationof enzymes by radiation is discussed here in terms of
disruption of the secondary and tertiary structure following the production of an electric charge inside themacro-moleculem and migration of the ionizing energy
along the covalently bonded structure. Energy then becomes localized on weaker bonds, 150•151 particularly on
S-S disulphide bridges responsible for maintaining thevarious chains of the enzyme in the native structure.

85. Nucleoproteins from the same source but withdifferent protein contents show different radio-sensitivities. Dilute solutions of DNA nucleoprotein with N/Pratio smaller than 2 are more radio-sensitive than DNA
with N/P greater than 2. Radiation damage is established from a decrease in viscosity. These differences
can be attributed to the influence of protein content on48

-the configurations of DNA in the compk""<es rather thanto some protective action of protein. 162 • lsa

simpler systems it is known that the presence of evenslight amounts of impurities can modify the spectrum
appreciably. It is not yet possible to identify those free
radicals that give rise to the particular pattern of electron spin resonance absorption in irradiated biologicalmaterial. Therefore, attempts have been undertaken toshow a parallelism between radiation-induced ESR
phenomena ar!d biological effects on the same material.

86. If there is a radio-lesion, several possible sitesof disintegration and disruption of a nucleoprotein canbe envisaged. These include bonds between nucleic acids
and protein. Their response may explain why irradiatednucleoproteins do not swell in water as readily as unirradiated material, and why trypsin yields free DNA
more quickly from irradiated nucleoproteins.164
4

92. In seeds of the grass Agrostis stolonifera, theeffect of irradiation on growth inhibition decreases whenwater content increases. This has been related to theobservation that the fraction of free radicals persisting
for longer times after irradiation also decreases withincreasing water content. 174 In seeds of Vicia faba, boththe sensitivity and free radical concentration after irradiation decrease with increasing water content.175 In barleyseeds, studies have been made of the influence of waterand LET on radicals detected by ESR techniques. 176
Attempts to relate biological and ESR results on dry
pollen grains have been reported. 177 A parallel between
biological end points and ESR data has been establishedin bacterial spores in studies of the effects of oxygen,heat, and NO treatments on the biological and physicalresponses. 5 "· ss, us, 124 The ESR method, applied to theinvestigation of lyophilized tissues of whole-body
irradiated rats, also demonstrates the presence of stableradicals i,vhich vary with the different tissues. After
irradiation with 1,000 rad the amplitude of the spectra
does not change in any of the tissues with the exceptionof spleen where there is a sharp decrease immediately
after irradiation. 178 The ESR method has also been used
to study the effects of different gases 179 (air, N 2 , NO)
and of protective substances like cysteamine and AET
on the production of free radicals.186• 1 s1

6

87. On irradiation with electrons (2 X 10 -2 X 10rad), part of the DNA of sperm heads is cross-linked toform a loose gel-like network ;165 this does not appear tobe due to seconda1y valence forces. Such cross-linkage
has been postulated to be the cause of inactivation of bacteriopbages by ionizing radiation. 165 This seems lessplausible than the hypothesis that inactivation is due to
production of carbon radicals in phage DNA. Such radicals may combine with oxygen, react with a hydrogenatom donor, or become inactive by an unknown process
if neither oxygen nor hydrogen is present. 166• 167
88. It is not yet clear which chemical changes aremost important in the loss of biological activity of nucleic
acids. No data clearly relate radio-sensitivity of biologically active nucleic acids to chemical changes producedby ionizing radiation. From studies on the inactivationof transforming DNA by ultra-violet radiation, by heat
denaturation, 165• 169 and by radio-mimetic substances, 170
damage to the bases seems important. On the other hand,a break in one of the chains of double-stranded DNA, oreven scission of the whole molecule, does not necessarily
lead to loss of activity. The molecular weight of thetransforming DNA can be lowered approximately one
order of magnitude by ultrasonic disruption withoutcompletely inactivating DNA.171 The inactivation yields,from decay of P 32 incorporated into single- and doublestranded DNA phages indicate that, whereas all breaks
in single-stranded DNA inactivate the phage, bothstrands must be broken in double-stranded DNA phages,
a fact which accounts for the lower efficiency ( ca.10 percent) .172

93. The results obtained so far through the ESR
technique are summarized in the following propositions :11" (a) Ionizing radiation produces free radicals in
living material;" ( b) The concentration of free radicals producedby radiation increases with increasing doses ;" ( c) The measurable concentration of free radicals
depends on the surrounding gas and on the water content of the specimen;" ( d) The concentration of free radicals decreases
relatively slowly after irradiation and is still wellmeasurable for minutes or up to many hours according
to the material and environmental conditions (watercontent and gas) ;" ( e) The opinion, widely held up to the present,that absorption of radiation in biological material generally leads within micro-seconds to states stable in
the physical sense, must be abandoned;"(f) It has been proved in some cases that a molecular interchange exists between protective substances and the protected material, and that it plays
a fundamental part in protective action."

DETECTION OF FREE RADICALS IN WHOLE CELLS BY
ELECTRON SPIN RESONANCE (ESR)
89. Although the radiation chemistry of water andof macromolecules in vitro can provide useful information on models of primary reactions in vivo, complete
information depends on studies on the chemistry of thebiological constituents after irradiation of living organisms. Progress in this field has been obtained recently
with development of the electron spin resonance technique (ESR); this allows study of free radical formation in biological systems. 173
90. Through this method, unpaired electrons havebeen detected in a variety of materials. When applied
to detection of free radicals, the material irradiated must
be stabilized to prevent diffusion of the radicals, e.g.
measurement has to be carried out in solids, in frozen
solutions and suspensions, or in dry biological material.
In principle, quantitative estimates of the number of
unpaired electrons in a sample are possible. In practice
it is difficult to attain reasonable accuracy.
'

V. Chemical factors modifying radiation
response in cells

91. Dat~ d~rived from irradiated biological materials
are not easily mterpreted. They do not necessarily relate
to t?ose ~r~e radicals responsible for the biological effects
o! irr~diation be~use many unpaired electrons arise inbiologically less important molecules. From studies of

OXYGEN EFFECT
94. The influence of oxygen tension on the responseof biological systems to radiation is one of the fundamental phenomena of radio-biology. This influence, ex49

erted during irradiation, is generally called "o_xyge_neffect". Gray's recent review integrates the data m ~hisarea.1s2 The effect has been observed _in a _great vanecyof biological systems and can be descnbed m the following way:(a) In the absence of ~A.~gen, or C1;t ~e~uced oxygen
tension the effects of radiat10n are dimtmshed but notelimin~ted; oxygen acts as a dose m~lti~lying ag:ent.
Considerable clarification of the quantttat1ve relations
between radio-sensitivicy and oxygen tension has resulted
from work with the bacteriwn Shigella flexneri. 183
Since, for this organism, survival is exponentially related
to dose at all oxygen tensions, th~ slope. ~f. the curve
may be used as a measure of rad10-sens1tiv1ty. It has
been found that when a sufficiently dilute suspensionof bacteria is vigorously bubbled throughout the periodof irradiation with gases containing ?iffer~t_p~rcentagesof oxygen, the relation between radio:sens1tiv1ty, _S, ~dthe concentration of oxygen ( 02) .m 0e medium mwhich the organisms are suspended is fairly accurately
represented by the simple relation:s - SN)[02]
SN= (m - 1 [02] K

95. The nature of radio-chemical reactions in theo:i..·ygen effect including the possible role of H,O~ radicals and of other reactive products whose yields are
influenced by oxygen tension, have been widely discussed
in recent years. 208 Proof has been cited52 •53 • 11 8 ~ato:A.-ygen-free radical inter~ctio~ takes place in bacterial
spores to bring about b10logical damage by X-rays.
However, the spores are semi-dry, and the role of Wf!-terin these interactions has been studied as yet only m apreliminary fashion. 209 • 210 Consequently, a generalization involving the metabolizing cell cannot be made now.
The belief that the oxygen effect depends on cellul~r
aerobic metabolism is challenged by experiments m
microorganisms with normal and defective cytochrome
systems in which 9xyge? effect is tJie sam~. ~owever,oxygen effect vanes with the cell s phys10log1cal state.
For instance, freshly harvested yeast cells, before starvation, have a considerably higher oxygen enhancement
ratio ( m = 3.6) than cells which have been starved. The
ratio m decreases as the starvation period is prolonged,
reaching a minimum value of m = 2 after two days'
stanration. 212 The observation that oxygen alone causes
chromosome aberrations when in high concentrations213
complicates interpretation at this time.

where SN is the sensitivity under anaerobic conditions,obtained by bubbling oxygen-free nitrogen throug~ thesolution, and m and K are constants. In general, m is theratio between the effectiveness of a given dose whenoxygen is freely available and the effectiven~ss whenoxygen is absent. Thus, ( m - 1) may be cons1der~d asthe ratio of the OA.'J-gen-dependent to the oxygen-mdependent components of radio-sensitivity. The cons~t. K
is the concentration of oxygen at which the sens1t1v1ty
is exactly midway between anaerobic and fully aerobic
values. The ratio m varies around 3 for a wide range
of cell types and effects: inactivation of bacteria, 181i-isa
and yeast,163 growtl;, 187 chro!11'osome aberrati~ns18 ~· 200
and mitotic delay201 m plant tissues, as well as mact1vation of isolated mammalian cells. 202•208 The similarity
between values of K (in the range of 4.5-5.0 µ.M/l) for
irradiation of bacteria, yeast, 20~ ascites tumour cells, " ~and plant root cells, 189 may be fortuitous, since a somewhat higher value of K (10 ± 2.8 µ.Mjl) has been reported205 for Tradescantia pollen tube chromosomes.
( b) In wet metabolizing systems, the presence ofoxygen during irradiation appears to be essential sinceno effect has been seen in bacteria irradiated underanoxic conditions when oxygen is introduced only 20
milliseconds later. 206 Even stronger evidence is suppliedby studies of the inactivation of Serratia marcescens byvery short pulses of high intensity electron beams. 201
Cell suspensions were irradiated with 1.5 MeV electrons
delivered either in a single pulse of two microseconds
duration ( 10-20 krad total dose) or for five minutes at
a dose-rate of 1000 rad/min; both treatments were applied either in hydrogen or in a 1 per cent oxygen and99 per cent nitrogen mixture. \i\Then irradiation was very
short, the radio-sensitivity of the bacteria was the sameas under anoxic conditions, whereas with the longer
irradiation, oxygen enhanced the sensitivity by a. factorof 2.5. However. in dry bacterial spores two act10ns ofoxygen, one realized only if oxygen is present during
irradiation, the other at appreciable times after irradiation, have been shown. 52•53 • 11 8
( c) Oxygen effect is usually less marked when cells
are exposed to high LET radiation. An important aspectof the oxygen effect is that the enhancement ratio, m,varies with type of radiation, being highest with radiation of lowest LET.

96. This oxygen effect must not be coniused with theeffect of oxygen given in the post-irradiation period.
Since the development of radiation injury depends on
metabolism, it is likely that there are systems in which
the magnitude of radiation lesions can be altered bychanges in oxygen tension after irradiation. 21"-211 Several
papers have also dealt with the effect of anoxia; thesehave shown that anoxic conditions in metabolizing cells
after irradiation reduce damage in some cases, 218 inothers enhance it. 219

211+

EFFECT OF GASES OTHER THAN OXYGEN

97. If oxygen exerts its radio-biological effects by reacting with radicals induced by radiation, other oxygenlike substances may react similarly. 125 In Shiqne_lla
flexneri Y6R bacteria, 220 nitric oxide enhances radiation
damage in the absence of oxygen. Nitric oxide has beenfound to enhance the effects of ionizing radiation onplant roots 221 and on ascites tumour cells. 222 In Drosophila, nitric oxide present during irradiation enhances
the production of dominant lethals and sex-linked recessive lethals. 223 The system seems to differ from that inbacteria and ascites cells in that the same concentrationof oxygen does not show an equivalent effect Althoughthese studies have shown that nitric oxide may freque~tly
simulate oA--ygen, differences in the effects of the twogases have been shown in dry biological materials. Dry
grass seeds irradiated and stored in nitric oxide are lessaffected by radiation titan those irradiated in anoxia.
However, when the water content of the seeds exceeds 12
per cent, nitric oxide is as effective as oxygen.179 In
spores of Bacillus megaterium, two actions of nitricoxide are known: a small sensitizing action during
irradiation and a large protective action after irradiation.m The latter action is a consequence of removal of
free radicals. 52 •rn The degree of hydration may influence the size of the two actions. 182

0

EFFECTS OF GASES UI\"'DER PRESSl.JRE

98. The Q}q·gen effect on V icia f aba roo~s an~ ascit~stumour cells is prevented when cells are irradiated mliquids in equilibrium witlt different gases underpressure. 224 •225 The following gases have this effect:50

helium, hydrogen, nitrogen, argon, krypton, xenon, andcyclopropane; the same applies to nitrous oxide intumour cells. The mode of action has not yet been established; the structures normally injured by radio-chemical
reactions involving oxygen may be protected by anabsorptive layer of the other gas. Proof that these substances interfere with injuries directly or indirectly dependent on oxygen is provided by the fact that they neverreduce the effects of the oxygen outside the limits ofanaerobic conditions. This research may provide a mostvaluable clue to the mechanism of oxygen effect.

102. A possible clue to the action of peroxides has
been found through studies of radiolysis of purines andpyrimidines. The addition of hydrogen peroxide andpersulfate to irradiated solutions increases the G valueof pyrimidines but leaves the G value of purines unaltered. 241, 242
CHEMICAL PROTECTION

103. Certain substances of different composition anddistinct physical and chemical properties, when added tocell suspensions, can reduce the effects of subsequentirradiation. Study of the chemical protection of the cell
is potentially helpful for understanding the primary
events of radio-biological processes. Among "protective
agents", the sulphur-containing compounds ( cysteamine,cystamine, aminoethyl-isothiouronium, glutathione, etc.)
are the more important. A few inhibitors of enzyme
activity (sodium cyanide, sodium azide, etc.), somemetabolites (gluconate, pyruvate, ATP)2 4 s- 246 and alcohols,247-m have the same action. Chemical protection
requires the presence of the protector before or during
irradiation, and is more effective against X-rays thanagainst other ionizing radiations. However, some metabolites can also have positive effects after irradiation,
possibly by influencing repair processes. 245• 246

HYDRATION

99. The precise significance of water radiolysis in thereactions induced in cells by radiation has still to be
determined. New facts on this subject have been given
by experiments of Hutchinson et al. 226 They measured
inactivation of two enzymes (invertase, alcohol dehydrogenase) and of coenzyme A in wet and in dry yeast cells.
They found that the sensitivity of these enzyme molecules were two times and twenty times greater respectively in the wet state, than in the dry state. Wet versus
dry sensitivity for coenzyme A was estimated as 100 to 1.
It has been assumed that the difference between the wet
and the dry sensitivities is caused by the migration ofchemically active intermediates formed by irradiation ofwater in the wet cells. Hutchinson 47 estimates that the
migration distances of the water radicals are about thesame ( 30 Angstroms) in all three cases.
100. Although increased water concentration enhances
radio-sensitivity in Aspergilfas, 227 several investigations 22 B- 231 comparing radio-sensitivity of dried and wet
plant seeds show that it is higher in the dried. Experimental results on Artemia eggs 232 • 233 parallel results onplant seeds. It is difficult to draw a general conclusionfrom the few investigations made on the comparative
radio-sensitivities of wet and dry cells. The possibility
must be considered that, in some experimental conditions, radio-sensitivity is modified by an inadvertentchange in oxygen tension within cells which is very likely
to be different for different moisture contents. Also, it
may well be that effects of moisture observed in plantseeds and Artemia eggs are due mainly to alterations inphysiological state rather than to participation of water
radicals in primary radio-chemical reactions.~ 31 • ~ 33

104. Protection has for long been associated w1th theindirect action of radiation. It has even been used as acriterion for distinguishing indirect from direct action.
This view can no longer be justified. Experimental
evidence has been presented wherein no indirect action
can be envisaged. 141 ' 250- 252
105. One action of protective agents may be e.."\:plainedby a decrease of oxygen tension. 253 • 255 • 256 The anoxic
hypothesis implies utilization of oxygen by the protector,
e.g. in transformation of cysteamine into cystamine.
Support for an anoxic effect of protective agents stems
from experiments in which the dose reduction factor
with cysteamine is similar to that of simple oxygen
removal. 254 However, several investigators consider thatsulphydryl compounds are protective by other means
than production of anoxia. The most recent observations
supporting this have been obtained in Escherichia
coli, 2 "&- 255 in isolated rat thymocytes, 259 and in HeLa cells
in tissue culture. 260
106. Alternatively, protection may be achieved by
combination of the chemical protector with free radicals
produced by irradiation. By comparison with chemical
data 182 a competitive type of reaction may be envisaged.
This reaction involves free radicals, oxygen, and protector. The protecting molecule may act either by combining \vith free radicals, thus avoiding formation of an
unstable active peroxy-radical. or by attacking theperoxy-radical and making it stable, i.e., non-active. 261
No clear-cut evidence has been presented in favour ofeither hypothesis.

PEROXIDE AFTER-EFFECTS

101. If phage particles are irradiated in buffer andallowed to remain in the suspending medium after
irradiation, the number of damaged particles increases
with time. 23 4-236 Similar phenomena have been reported 237- 2311 in bacteria, in lysogenic systems, and inphage bacterium comple..xes. This after-effect may be
attributed to the presence of H 2 0 2 or of organic peroxides formed in the broth. However, doses exerting
profound effects on whole cells are often not high enoughto produce damaging concentrations of peroxides in thesuspending media. This holds particularly if cells contain catalase, but hydrogen peroxide and organic
peroxides in dilute suspensions which contain little
protective organic matter may also exert a marked effect.
In synthetic media, the concentration of peroxides responsible for the after-effect decreases with time during
twenty days after irradiation. During this period therate of decrease depends on dose, at least in the 1-5
kilorad range. 236 Artificially added inorganic peroxides,
e.g. persulfate and urea peroxide, 240 can also increase
sensitivity of phages and bacteria.

107. Another e..xplanation is that protecting molecules
attach themselves primarily to cell structures, thus masking sensitive sites. The complex so formed would guard
these sites from the attack of free radicals (indirect
action). This complex may also dissipate absorbed
energy less harmfully (direct action). With SR-containing compounds, Eldjarn and Pihl m have proposed achemical model embodying this concept. The maskingeffect hypothesis is supported by experimental results
showing that decrease of protective ability of cysteine
injected into animals parallels recovery of the metabolic
activity which that substance had initially lowered. 262 • 263

51

108. Other substances with lmown pharmacologicalactivities (hormones, amines, neurodrugs), protectors
after injection in animals, seem to have no action in cellsuspensions. Thus, little information about the primary
events of radio-biological action can be obtained fromin vivo experiments in which they are used except forthat concerning their possible interference with metabolic
processes.
109. The chemical protective agents are also effective
against chromosome aberrations 264 and induction of
mutations by X- and gamma rays. 265 • 266 However, thissubject deserves much more attention, the data being
scanty.
110. Accumulated evidence on chemical protection 243 • 244 does not now permit an unequivocal recognition of mechanism. New data are needed to clarify this.
The ESR technique may become useful in this area.

"genetic" or "non-genetic" factors necessary for recovery of the damaged structure are functional. The contribution of the cytoplasm in radiation injury has been
partially clarified by recent investigations. In particular,
the presence of toxic products 289-290 and the existence ofchanges in IEP ( isoelectric point) perhaps associatedwith changes in RNP ( ribonucleoproteins) localized incytoplasmic microstructures may imply disturbances innuclear cytoplasmic interaction. 291-296
115. Particular emphasis has been placed on themetabolism of deoxyribonucleic acid (DNA) and on its
interaction with ribonucleic acid (RNA) and protein
metabolism. These metabolic functions are so intimatelyintertwined in the way they influence cell division andreplication that it seems logical to treat them integrally
to assess how radiation may affect this complex.
DNA SYNTHESIS
116. Recently Kornberg and associates 29 1-2 99 havesynthesized DNA in vitro from deoxyribonucleoside
triphosphates using purified extracts from E. coli. The
system requires "primer" DNA which, during thereaction, replicates. The product has a base composition
identical with that of the native primer. Single stranded(denatured) DNA preparations also provide excellent
primers. soo

VI. Effect of radiation on cellular structures
and their function
111. Some of the more spectacular and most extensively studied effects, such as inhibition of cell division,
mitotic delay and mutation, are most readily associatedwith nuclear damage and are apparent after exposure to
relatively small doses of radiation. However, inhibitionof cytoplasmic functions should be carefully consideredin assessment of total damage. Since nuclear and cytoplasmic functions are so clearly intertwined, it is imperative to consider their possible interactions in weighing
the relative importance of nuclear and cytoplasmic
damage.
112. These interrelationships vary with different
systems and different functions. The early works of\Vintemberger,2 67 Zirkle,2 68 Hensbaw, 269 Hercik 270 andPetrova 271 showed that mitotic delay and cell death are
principally manifestations of radiation damage sustainedby the nucleus. Recent experiments dealing with partial
cell irradiation have shown clearly that irradiation ofgenetic material is far more effective than cytoplasmic
irradiation in producing cell lethality. For example 50
per cent inhibition of hatching of Habrobracon ~ggs
requires 107 alpha particles to the cytoplasm; only 1alpha particle to the nucleus suffices to inactivate theegg. 272 Comparable results have been obtained in similare?Cper~ments with newt heart cultures. 273 Conversely,
situations may be expected where cytoplasmic damage isrelatively more effective in impairment of specific cellfunctions. For e..xample, changes in isoelectric point of
mitochondrial nucleoproteins of the adult nerve cell occur
during or immediately after irradiation with small
doses. 21 s-211 This indicates alteration of metabolic
functions and, in particular, of oxydative phosphorylation. 21s, 2;s117. This mechanism is compatible with present concepts on DNA replication in vivo. These postulate that
double-stranded DNA may split wholly or partially into
single strands that serve as templates and receptors forcomplementary strands. Moreover, Komberg et al., 301
identifying all the dinucleotides in synthetic DNA, haveshown that the in vitro system produces double-stranded
DNA molecules with each single spiral running in the
opposite direction as compared with its mate; this result
provides excellent support for the Watson Crick model.
118. The presence of polymerase, first found in E. coli
extracts, has also been demonstrated in e>.."tracts of mam~ian cells from ascites tumours, thymus, regenerating
liver, etc. 302- 3o5
119. In the nuclei of tissue cells, DNA synthesis islimited to a definite period during interphase. In the firsthours a~ter mitosis there is usually no DNA synthesis( G1-penod). In the next period ( S-period), lasting
several hours, the DNA content of the cell doubles. Theinterphase is concluded by the G:rperiod. This sequence~f events i~ the i_nterphase may be subject to modifications; thus, m ascites tumour cells the G1-phase is absent.
Precursors of DNA are probably produced in theG1 -phase and activated (to nucleosidetriphospbates) at
the ex~ense of energy-generating processes (e.g. nuclear
oxydative phosphorylation). Nuclear synthesis of RNA
also occurs in this phase, associated with the productionof new enzymic proteins. In the synthetic period theas_sembly of activated precursors most probably o~curswith t!1e. help of the ne:vly synthesized enzymes and withthe ongmal DNA servmg as template and primer. In theG2-p~riod J?N~ is further.p_r~pared for its subsequentrole m the immment cell div1s10n. In cells of lower organisms this stratification into well separated division
stages does not occur. Probably, however, the sequenceof metabolic events is similar.

113. Non-nucleated cells (Acetabularia, amoebae.~ 79
Paramecia,zso• 281 tissue culture cells) 282 ultimately die,but they may survive for a considerable time andeven cont!nue _to differentiate (Acetabularia). 250,283,mLethal!~ irra~ated E. coli cells retain the ability tos:rnthesize active bacteriophage. 236• 25 s- 25 s Owing to thishigh degree o~ cytoplasmic autonomy, nuclear radiationda~age aff~ctrng cytoplasmic functions may escape detection dunng the observation period.

120. Since the discovery by Hahn and Hevesy 306 thatp~osphor.us_incorporation into DNA is inhibited by ion-11~. Conversely, cytoplasmic damage affecting thephysiology of the cell may not become permanent if theizing radiat10n, a fact confirmed by similar evidence on
incorporation of various labelled precursors such as

52

cells in vitro using doses of 750-3000 r. They foundinhibition of the incorporation of (2-C 14 ) uracil in DNA
thymine, but the possibility that this effect was due toinhibition of mitosis has not been excluded. Further
results have been reported by Budilova 320 on the incorporation of several precursors into DNA molecules ofisolated thymus cells nuclei; incorporation was greatly
reduced in nuclei irradiated in viva, whereas there were
no changes when nuclei were irradiated in vitro.

adenine, erotic acid, formate, phosphate and thymidine,it has been generally accepted that DNA synthesis is aparticularly radio-sensitive metabolic process. Recent
investigations have cast serious doubt on the correctnessof this opinion. They lead, rather, to the conclusion that
relatively low radiation doses do not affect the rate of
DNA synthesis in various types of cells. It is now
realized that a diminished incorporation of precursors
into DNA after irradiation may not necessarily represent
primary inhibition of DNA synthesis. It may be the consequence of other differences between the irradiated andthe control cell populations, 301- 311 namely:(a) Accumulation of cells in the G2 -phase as a resultof mitotic inhibition;( b) Changes in the distribution of the various cell
types of a mixed cell population;( c) Increase of the fraction of dead cells in the irradiated population. The same argument obviously
applies to the synthesis of RNA and protein.

126. In bone marrow cells in vitro, high doses ofradiation (>SOO rad) directly inhibit DNA synthesis.
Lower doses ( < 300 rad) cannot inhibit DNA synthesis
in cells already in the synthetic period. However, cells in
the G1 -phase at the time of irradiation enter the S-phase
only after an appreciable delay. More recent observations
by Uyeki 321 are in accord; the number of cells entering
DNA synthesis after 800 r is strongly depressed.
127. Low doses of X-radiation (50-140 r) prevent
division of root tip meristem cells of Vicia faba but do
not interfere directly with DNA synthesis. 822 • 323 However, cells not yet in synthesis at the time of irradiation
pass on to the synthetic phase only after a delay of 10hours or more. In contrast, Das and Alfert 32• have reported an immediate effect of irradiation on DNA synthesis; even a dose as low as 200 r enhances DNA synthesis, whereas 800 r increases the uptake of tritiatedthymidine to approximately five times the control value.
128. From studies in regenerating liver 325 •826 it has
been concluded that DNA synthesis itself is not primarily
affected after partial hepatectomy by relatively feeble
radiation doses. 325 •326 In resting liver there is no appreciable DNA synthesis, but when regeneration is inducedby partial hepatectomy, synthesis begins 15-18 hours
after the operation and reaches a maximum at 24-29
hours. In this first stage of regeneration there is reasonable synchronization of DNA synthesis. High radiation
doses (up to 2,000-3,000 r) are needed to inhibit synthesis once it has begun; a dose of SOO r is ineffective.
However, the latter dose is quite effective in postponing
synthesis when given before the beginning of the synthetic period.
129. Few e..xperimental data are available on the sensitivity of DNA synthesis in micro-organisms to Xirradiation. Billen 321 studied mutants of E. coli and, inparticular, the influence of "unbalanced growth" andradio-sensitivity. He concluded that X-irradiation inhibits the synthesis of protein required for DNA replication.
130. In dividing H. inflitenzae, E coli B and B/r,
irradiation with doses between 19 and 100 k rad is followed by breakdO\vn of cellular DNA; after a certain
time this process stops and is followed by an increase in
DNA.32s,3w

121. Recent developments in the use of microspectrophotometry and autoradiography for the study of single
cells often make.it possible to account for these complications and thus to arrive at a more correct evaluation of
the biochemical effects of irradiation. Another method,
although at present often more difficult, uses more or lesssynchronously dividing cells. The following survey considers investigations using these techniques.
122. Irradiation of HeLa-cells with 5SO r leads to aconsiderable increase in the fraction of cells synthesizing
DNA as compared with control cultures. 312 This increase amounts to 100 per cent six hours after irradiation(this represents a larger fraction than can be accounted
for by inhibition of mitosis). Apparently, cells irradiatedduring active DNA synthesis continue to synthesize forlonger periods than normal; this may be related to giant
cell formation. Moreover, Painter 313 found that whenpost-irradiation mitosis resumes, added tritiated thymidine results in a lower fraction of labelled cells inmitosis of these cells than in mitosis of unirradiated controls. This could be due to sluggishness of irradiatedcells in the G2 -phase and/or in mitosis of the next division stage.
123. In contrast, Harrington m did not see any direct
effect of exposure to SOO r on the fraction of U-12
fibroblasts in DNA synthesis. The percentage of cells
synthesizing DNA began to drop after an interval corresponding to the duration of the G1 -phase; this decline
must be wholly attributed to inhibition of mitosis.
124. A similar conclusion has been drawn fromstudies of L cells (mouse fibroblasts) 315•316 in which
DNA synthesis continued in the absence of mitosis until
the double premitotic content per cell was reached. Veryhigh doses ( 4000-5000 r) retarded DNA synthesis
instantaneously. After e..xposure to 2000 r the cells still
completed an average of three divisions, whereas after
5000 r, only 20 per cent of the cells were still capable of a
final division. Such DNA synthesis as was observed
thereafter was in giant cells and occurred at a considerably lower rate than in normal unirradiated cells.

131. In H. influenzae, the biological activity of DNA,as characterized by its transforming activity, has been
determined after irradiation. All remaining DNA andDNA formed after irradiation is functionally normal.
No relation has been found between killing and severityof DNA breakdown. From this it has been concluded
that observed DNA breakdown is not the immediateradiation-induced process leading directly to cell death.a:i9

125. X-irradiation (800-1250 r) of Ehrlich ascites
tumours has not been found to inhibit DNA synthesis. 311• 818 Mitotic activity is arrested instantaneously
but volume, dry weight and total nucleic acid per cell
continue to rise considerably. The DNA content per cellrises to the pre-mitotic level. Harbers and Heidelberger319 cultured and irradiated Ehrlich ascites tumour

132. DNA is in a highly polymerized state in bacteriophages330•331 and certain tissues. 332•333 After irradiation,
depolymerization is seen, 33s-335 and shifts in the purine/
pyrimidine ratio in DNA synthesized after X-irradiationof spleen cells in viva have been observed. 336 • 831 Changes
53

selective irradiation of the nucleolus by UV microbeams,
Perry et al. 355 have concluded that RNA transport into
the cytoplasm originates from both nuclear locations ofRNA. From recent autoradiographic studies of the incorporation of tritiated precursors into RNA of Hel.acells, in which several correction factors were applied
for the conversion of grain counts into actual incorporation, the same authors state that their data do not show
a transport of RNA from chromatin to nucleolus. 356
Moreover, a few instances are known where labelling of
the nucleolus precedes that of the chromatin.357

in the thymine/adenine ratio in DNA synthesized after
irradiation of plants have been reported by Kusin andTokarskaya. 338 • 33~ These changes seem to be closely
related to disturbances in nucleotide metabolism. 34 o-m
RN A AND PROTEIN SYNTHESIS
133. In contrast to DNA, most RNA is in the cytoplasm; only a small fraction resides in the nucleus.
134. Little is known about the secondary structureof RNA. It is probably single-stranded. Physico-chemical data suggest that it may fold locally into incomplete
double spirals stabilized by H-bonds ; these orderly
structures would be held apart by unarrayed segmentsof the RNA chain. 343

140. Little is kno\vn about the mechanism of RNA
synthesis. An enzyme, polynucleotide phosphorylase,that catalyzes the synthesis of RNA from ribonucleoside
diphosphates has been found in micro-organisms by
Ochoa and associates. 358 The purified enzyme requires aprimer, but any tri- or tetranucleotide may serve in thiscapacity, and it is not the primer but the available nudeotide diphosphates that determine the base compositionof the product.359-361
141. On the other hand, extracts, not only from microorganisms but also from animal cells, polymerize ribonucleoside triphosphates to RNA. 362• 363 \/\Then DNA ispresent, treatment with DNA-ase destroys its activity.
Enzymatic activity depends also on the simultaneouspresence of the triphosphates of all four nucleosides.
Furth et al. 364 and Weiss and Nakamoto365 have shownthat newly synthesized RNA is a copy of the base composition of the added "primer'' DNA. The enzyme produces polyadenylic acid or poly-uridylic-adenylic acidwhen primed with polythymidylic- or poly-adenylicthymidylic acid respectively. With M. lysodeikticus or
TrDNA as a primer, the newly synthesized RNA has
the same nearest-neighbour base frequency as theprimer. 366 The resemblence of tliis enzyme to the polymerase of DNA synthesis is striking.

135. Nuclear RNA is not homogenous; an important
fraction is probably in ribosomes, as observed in thymus
nuclei. Cytologically, RNA may be divided into chromosomal and nucleolar RNA. Biochemically, two fractionsof nuclear RNA may be distinguished, one extractableby low concentrations of saline (n-RNA1 ), another remaining undissolved (n-RNA2 ). Generally, n-RNA1
incorporates labelled precursors less readily than doesn-RNA2 •84 ' -346 According to Zbarskii and Georgiev347 • 848
n-RNA1 represents the chromosomal RNAandn-RNA2
forms part of nucleolar RNA.
136. In the cytoplasm, RNA occurs in the cell sap(S-RNA) and in the microsome (liver, pancreas) andribosome fractions. The molecular weight of S-RNA isrelatively small (20,000-40,000) ; that of microsomal
RN A is considerably larger (approximately 1.7 X 106 ).
The possibility cannot be excluded that the latter molecular weight represents aggregates of molecules of lm·ver
molecular weight as it has been shown that ribosomes
may disintegrate into smaller particles depending on the
Mg++ concentration of the solvent. The RN A in thesmallest ribosomes, the so-called 30 S particles, has amolecular weight of only 5.6 X 105. Small amounts of
rapidly turning over "messenger'' RNA of an intermediate size, between the latter RNA and S-RNA, arepresent in uninfected and phage-infected bacteria. 849 • 850
This RNA attaches itself to existing ribosomes and confers on them the code for protein synthesis.

142. From e..xperiments with labelled RNA precursors, it has been shown that synthesis of RNA occurs
during the entire interphase, although in some cells theprocess is slower during S-phase. During mitosis, no
RN A seems to be synthesized.
143. Within the nucleus, DNA transfers its genetic
information to RNA. 367 • 368 The presence of an RNA
polymerase requiring DNA for action, and copying its
base composition, supports this concept. RNA formedin the nucleus then passes into the cytoplasm, carrying
its information to protein synthesizing sites. Rich369 has
demonstrated that, in principle, a single-stranded RNA
molecule can unite with a complementary single-stranded
DNA molecule. Moreover, Hall and Spiegelman370 haveshown specific hybrid formation between single-stranded
T2-DNA and the RNA synthesized subsequent to infection of E. coli. Geiduschek et al. do not favour singlestranded DNA as a necessary intermediate in RNA
synthesis in vitro. 311
144. Apparently, the base sequence of the DNA istranscrib_ed into newly formed messenger RNA, triplets(or multiples of 3) of nucleotides carrying the information for various amino acids (para. 151). The most
~rect proof of the ability of RNA to carry genetic
mformation is provided by the information that purified!obacco n;iosaic virus RNA is apparently infectious. How
mformat1on transfer between DNA and RNA is effected
is not known. Leslie372 recently postulated, from studies
on human liver cells and from the literature, that coding
for micro-organisms and for somatic cells of higherorganisms may differ.

137. Recent studies provide evidence that RNA is
synthesiz.ed exclusively in the cell nucleus, and is transported from nucleus to cytoplasm after synthesis. Thus,
Goldstein and Plaut351 transplanted P 32 RNA labelled
nuclei from intact amoebae into enucleated amoebae ;after a while the cytoplasm of the host contained labelledRNA. As these amoebae were viable, it seems unlikelythat leakage from damaged nuclei was responsible forthe effect.
. 138. So far, the type of the nuclear RNA transported

mto the cytoplasm has not been established. Woods andTay~or35 ~ have suggested that RNA is primarily syn-thesized m chromosomes and subsequently stored in the
nucleolus; from there it would be transferred to cytoplasm. This hypothesis is supported by other investigators353•354 who have found that, with a labelled RNA
precursor, radio-activity is first detected in chromatin
and only later in the nucleolus; continued incubation in
the absence of labelled precursor leads to an earlier andfaster fading away of the radio-activity of the chromosomal than of the nucleolar RNA.
139. Whether this hypothesis has general validity for
all types of cells is not known. From experiments on
54

actu~lly demons.trated that the new RNA(which, according to Volkm and Astrachan,3 ' 8 has a base composition corresponding to that of the phage DNA) is
associated with pre-existing ribosomes and provides
them with the necessary information for specific protein
synthesis. Gro:; et al., 3 ' 9 in "pulse e.xperiments" withtracers, have shown that e."'<:actly the same situation prevails in uninfected bacteria where an RNA component
with rapid turnover and which is physically distinct fromribosomal RNA or S-RNA can be demonstrated. The
fraction behaves in the ultracentrifuge and towards preexisting ribosomes in high Mg<+ concentrations exactly
as the phage specific RNA induced by T 2 infection; it
becomes associated with the active 70 S ribosomes, thesite of protein synthesis. According to this concept, thetypical ribosomal RN A carries no genetic information.
The concept of messenger-Ri'\TA has been greatly elucidated and amplified by experiments of Matthaei and Nirenberg380 who demonstrated that, in cell-free extracts ofE. coli containing ribosomes, poly-urydylic acid can induce the synthesis of poly-phenylalanine. At present,
triplet code letters have been assigned by Speyer et al.
to 14 amino acids. 381

145. About twenty years ago, a relationship between
RN A and protein synthesis was independently advanced
by Caspersson367 and Brachet368 as a hypothesis; thishypothesis has now become a finnly established biologicalconcept.
"146. Protein synthesis has been most studied in microorganisms and in the microsomal fraction of the cytoplasm of higher cells. The first step is activation ofamino acids in a reaction with ATP resulting in anamino acid adenylate. The latter compound does notappear freely in solution but remains attached to the enzyme ; amino acid activation is therefore usually studied
from the exchange between labelled pyrophosphate (oneof the reaction products) and the phosphate groups of
A TP or by the chemical transformation of the aminoacid adenylate by hydroxylamine into hydroxyamic acid.
147. The activated amino acid then becomes attachedto the transfer or soluble RNA (S-RNA). It is boundin the manner common to all amino acids, via the tenninal
nucleotide sequence cytidylic-cytidylic-adenosine; theamino acid residue is bound in ester linkage to the C3 'atom of adenosine. Although the method of binding is
identical, each amino acid has a high specificity for the
S-RNA to which it becomes attached. There are different
S-RNA molecules for each type of amino acid. The
specificity of S-RNA resides in its base sequence.
148. The function of S-RNA is that of acting as acarrier which brings the amino acid to the template.
Investigations of Bosch et al. 373 have shown that S-RNA
can be firmly bound to the ribosomes. On the other hand,
it is possible that this "transfer"-Ri'\TA resides permanently in the ribosomes. Thermodynamically, this latter~ypothesis is more attractive; it may be significant thatm one of the very scanty examples of net synthesis of
enzymatically active protein in vitro this could be accomplished by a cell-free system in which S-RNA
formed part of the ribosome particles.m·
149. The last phase in protein synthesis is the assembly <;>f ac.tivated amino acids into polypeptide chainsby peptide linkages, and release of these chains fromribosomal particles. For this step GTP is required. The
process is greatly stimulated by SH-compounds.374 • 375
150. Protein synthesis has been studied in microsomes of cells of higher organisms. It is, however, by
no means confined to this system. Net synthesis of~yto:hrome-c has been demonstrated by Bates et a1.a 1am mitochondria. Moreover, it has been shown by Allfrey
and Mirsky3 11 that protein synthesis in the nucleus isvery similar to that in the cytoplasm. These investigators
suggest _that t:J:ie energy for protein synthesis in the
nucleus is proVIded by phosphorylation in mitochondria.
. 151. 1:he part played by RNA in carrying genetic
mformauon fo_r the production of proteins is clearlyshown by the discovery of Astrachan and Volkin 378 that
infect!on of E. coli by v~rious bacteriophages immediately induces the production of a new RNA which resembles, in base composition, the DNA of the phage.
Nomura et al. 319 found that, after T 2 infection there isno synthesis of typical ribosomal RN A and that phage
specific RNA sediments at a slower rate (8 S) than;ibosom~ (16 S and 23 S). Apparently, the genetic
mf?fill!1tlon for the s~thesis of phage protein does notreside m the usual nbosomal RN A but is induced in
pre-existing ribosomes by a phage specific RNA which
m::;y b: considered a messenger RNA. Brenner et al.,3sousmg isotope labelling techniques followed by carefulseparation of the various RNA-containing fractions,152. The influence of ionizing radiation on RNA andprotein synthesis has not been studied to the same e.~tentas that on DNA synthesis, and available data do not
permit a satisfactory analysis of the effects.
153. Painter, 313 using 1,500 r, did not find a significant
disturbance of the uptake of tritiated cytidine into the
RNA of HeLa cells. Neither did Harrington 314 see any
effect on the incorporation of tritiated cytidine into
nuclear RNA of U 12 fibroblasts after SOO r. Shabadash,o~ the other hand, showed that cellular ribonucleoprot~ms .an~ ext~emely responsive to penetrating radiati?ns.·11•·91 This _was rece~tly c_onfinned biochemically.m
Ribonucleoprotems localized m structures of different
organelles do not have identical physico-chemical properties, as indicated by differences in their iso-electric
points,3112 which are more acid in mitochondria than in
microsomes. The former is more sensitive to penetratingradiation. 203 •296
154. Klein and Forssberg321 irradiated Ehrlich ascites~our cells in vivo with 1,250 rand found no changes
m RN A synthesis. However, in vitro irradiation of thesecells inhibits incorporation of labelled uracil into RNAof the nucleus but not into that of the cytoplasm. 819 This
result is difficult to understand in view of the probablenuclear origin of most RNA.
155. From the studies of Logan and collaborators 393 •354 it has been concluded that irradiation of isolated liverand cal~ thymus _nuclei in vitro distinctly reduces therate of mcorporatton of labelled precursors into nuclear
RNA. A similar effect on the incorporation of P 32 into
!1ucl~r _RN.('>. CCJ? be obtained with regenerating liver, if
i;rad1ation. 1s given ~t the earliest stage of regeneration: au This ~bservation agrees with data on the synthesis of certam enzymes necessary for the synthesis of
DNA in regenerating liver. Thus, Bollum et al. 3 1>$ havefound th~ ~ynth~sis of the .e~Y!11es DNA polymerase
and thym1dme kmase to be mh1b1ted by radiation dosesof 375-1,500 r if irradiation is given 6 hours after partial
hepatectomy. The same doses, given si.'<teen hours after
the operation, are ineffective. Other authors have also
r_oun? that polymerase synthesis is inhibited. by irradiation m the first phase of the regeneration process. a8 7, as5156. Relatively low doses of radiation can postpone
the onset of DNA synthesis in various types of cells.

55

pears to be more resistant to radi.a~on. ~pparently, an~­bodies formed when the system is 1rrad1ated dunng thisperiod do not differ fundamentally from normal antibodies. Studies of the degree of radiation sensitivity ofthe secondary response to antigen injection have yielded
conflicting results ; there have been several explanations,each of which might be acceptable for the particularantigen studied. 398 • 894

It seems reasonable to assume that inhibition of enzyme
synthesis is at least one cause of this delay.
157. Ionizing radiation also reduces the synthesis of
enzymes in micro-organisms. Pauly3 89 has reported a
37 per cent dose. of 7 X 104 r for t?e inhibit~on of theinduction of lysme decarboxylase m Bactermm cadaveris. Radio-sensitivity was the same for the rate ofsynthesis. and the maximum lev:el of enzyme formed.
This finding leads to the conclusion that every cell possesses one or more "centres of synthesis", each producing a definite number of enzyme mole~ules. ~hese
synthetic centres would be destroyed according to smglehit kinetics. The induction of catalase by 02 in a diploidmutant of S. cerevisiae, however, is stimulated by aradiation dose of 105 r. This stimulation may be due tothe production of peroxides in the cell, as suggested by
Chantrenne and Devreux. 890 Using serological techniques
and also various tagged amino-acids in newly synthetizedproteins of individual organelles of cells, Ilina andPetrov391 • 392 showed that qualitatively altered proteinsare formed after irradiation.

GENERAL CONSIDERATIONS OF RADIATION EFFECTS
IN CELLULAR METABOLISM

EFFECTS OF RADIATION ON ANTIBODY SYNTHESIS

158. Inhibition of antibody formation is a special
case in the formation of specific proteins, and appears
to be highly radio-sensitive. It involves the formation ofa specific protein complementary in structure to the
inductor antigen. The normal processes of antibody
formation are only just beginning to be understood, anda generalized theory has still to emerge from several
contradictory hypotheses. Antibodies are formed in theplasma cells of lymphoid tissues which themselves originate from undifferentiated cells of the reticular system.
The mechanism of radiation inhibition of antibody formation, recently reviewed, 398• 394 thus must account for :(a) The effect of radiation on the multiplication anddifferentiation of these reticular cells and their descendants;(b) The process of antibody synthesis, which probably occurs in the microsomes of plasma cells.
159. One of the characteristics of radiation is i"5greater efficiency in inhibiting antibody production whenadministered prior to the antigen. The final titer ofantibody is lowered only if irradiation occurs some hours
before antigen injection. In this case, and also whenirradiation takes place immediately before or after antigen injection, the latent period before the titer begins
to rise is increased and the rate of synthesis decreased.
Taliaferro 395 has distinguished a highly radio-sensitive ·(effects become detectable on the final titer for doses of
100 r) pre-induction period but this is not well definedin cytological or biochemical terms. The cause of thisinhibition could be twofold :(a) Decreased production of plasma cells from their"reticular ancestors", or from other types of cells also
involved in the process;(b) Delay and inhibition of the synthesis of new
protein when antigen is injected.
160. Stevens 396 has shown a correlation between de-press_io!! of the number of plasma cells formed after
irradiation and inhibition of antibody synthesis. Furthermore, i;xperiments by Taliaferro suggest that antibody£o:mation ~epends on cell multiplication in irradiatedammals; this does not exclude the possibility that specificeffects o~ the induction of synthesis of new proteinsare also mvoh-ed. The antibody-producing period ap-

56161. The importance of radiation effects that areclosely linked with cell division and replication, andwhich include mitotic inhibition, loss of reproductive
power and mutations. has been stressed. It would be
attractive to describe these changes within the frame ofa unitarian mechanism, although such a treatment would
be arbitrary. At least two key effects indicate a disturbance in the genetic properties of the cell. 897 One ofthese is the production of mutations. The other is thatdelayed effect on cell division in which cells multiply
immediately after irradiation but nevertheless fail toform macroscopic colonies.
162. The failure of cells to divide even once when
given higher radiation doses is also probably due todamage of genetic material. The inhibition of mitosismight be explained similarly, although here the implication that genetic material may be directly involved is lessobvious. Much may be said for the concept that the mainradiation effects are at some stage mediated throughDNA; this e.~plains why emphasis is laid upon themetabolism of DNA. DNA synthesis has been used in a
restricted sense throughout this report to indicate thestage where precursors are assembled into polynucleotides. Subsequent stages may include many more biochemical reactions before the full-fledged DNA-protein
molecule is formed and incorporated into daughter
chromosomes. These late stages of DNA metabolism
presumably take place in late interphase and in prophase.
163. There is some evidence. at least with radiationinduced mitotic delay, that the. G2 stage and early prophase may be the most radio-sensitive stages in the mitotic cycle of many cells. 398 Painter's work, 313 mentioned
earlier, may also be interpreted in this way. The dependence of radio-sensitivity on division stage may not always
prevail in somatic cells of higher organisms ;3911 survival
curves of somatic mammalian cells usually show no evidence of resistant fractions. 400 Because of considerable
radio-sensitivity during the G2 period, metabolic processes during this period are important. Unfortunately,
biochemical knowledge of G2 and subsequent mitotic
stages is still extremely scanty. Therefore it is not yet
possible to describe the effect of radiation at a molecularlevel on these phases.
164. In cells of higher oragnisms two patterns ofsynthesis of DNA probably occur. In tissue cultures
and ascites tumour cells, DNA synthesis continues moreor less unhampered if irradiation occurs during any
period of the division cycle, at least when doses are notexcessive; in cells of bone marrow. plant root tips andregenerating liver, DNA synthesis may be delayed whenlower doses of radiation are delivered before svnthesis
has begun. This latter effect is probably due to irihibitionof the formation of necessary enzymes as a result ofinterference with RNA synthesis. No inhibition. andsometimes even acceleration occurs in either patternwhen all ingredients are available for synthesis. Mitotic
inhibition interferes eventually because a feed-back}vfitotic delay

homeostatic mechanism precludes, or at least inhibits,
DNA synthesis beyond the premitotic level.
165. This concept has been confirmed by Lajtha etal. 310 and by Berry et al ;401 they found that dose-effectcurves for inhibition of DNA synthesis in bone marrowcells differ from those in ascites tumour cells. For bonemarrow cells the curve has two exponentials, a "sensitive" one and an "unsensitive" one, characterized by 37
per cent doses of 500 and 1,300 r respectively. The
curve for ascites cells lacks the sensitive component.
Ord and Stocken40z have, from similar curves for thymus
tissue, suggested that the sensitive component may represent the inhibition of nuclear phosphorylation describedby Creasey and Stocken.403 This inhibition would leadto a shortage of DNA precursors. However, there is noevidence for such a shortage; Ord and Stocken404 reported an accumulation of deoxyriboside mono- andtriphosphates after irradiation of the thymus. The significance and reproducibility of the inhibition of nuclear
phosphorylation seems doubtful.
166. Both cell types also differ in ploidy; tissue-culture
and ascites tumour cells are usually aneuploid. The problem of the relationship between ploidy and radio-sensitivity is complex (para. 182) but it is not impossible thatthe high resistance of these cells may be a consequenceof the aneuploidy. This suggests that DNA itself is theprimary target. The work of Opara-Kubinska et al. 405
and many studies on bacteriophages indicate that thisis probably so, at least for transforming activity andsurvival in micro-organisms.
167. The "primer" function of DNA in RNA syuthesis by the RN A polymerase enzyme means that the
explanation given for the delay of DNA synthesis,
namely interference with RNA metabolism, is at least
not incompatible with a primary radiation lesion in DNA
itself (in this case, the primer) (para. 155). This does
not e."'\:clude the possibility that effects on DNA-RNA
protein metabolism, even when mediated through DNA,may not result secondarily from quite another primaryradiation lesion, e.g. lesions on larger subcellular structures, proteins, membranes, lipoids.

170. When a cell has been irradiated before prophase,division is delayed. This delay can be modified by dose
ratem and by o:i..ygen concentration; this may mean thatmetabolic processes are involved:m The most informative experiments have been those of Carlson and
Gaulden 414 with neuroblasts of grasshoppers' embryos.
During mitosis there is a critical stage coinciding withthe condensation of chromosomes into visible filaments
and with the disappearance of the nuclear membrane andnucleolus. If a dose as low as 1 r is given to a cell before
that critical stage,- development of mitosis is delayed.
However.. this delay does not occur when the same oran even slightly higher dose is given later. In this lattercase subsequent mitoses are delayed. More recent experiments have shown that the critical stage may besomewhat earlier in the mitotic cycle, i.e., in midprophase. Gaulden irradiated one of the two nucleoliof neuroblasts with a UV-microbeam and concluded that
all cells treated at stages from late telophase to the middleof mid-prophase immediately show a permanent cessation of mitotic progress. This picture of mitotic delay
looks slightly different when other types of cells arestudied. In particular. the critical sensitive period andthe duration of the various phases of mitosis may differ
in different types of cells. In consequence, precise comparisons are difficult.
171. The main characteristic of mitotic delay is its
temporary nature. Although the mechanism of mitotic
delay is still far from being understood, some attempts
have been made to explain it. Since DNA metabolism
is known to be affected by radiation, it is tempting toattribute mitotic delay to inhibition of DNA synthesis.·115
This explanation is speculative, and it may well be that
reduction in DNA synthesis, when observed, is theconsequence rather than the cause of mitotic delay. In
particular, the radio-sensitive period for producing
mitotic delay usually occurs when DNA synthesis isalready complete. In some instances, DNA metabolism
is apparently normal despite inhibition of cellular division, e.g. in irradiated mammalian cells in tissue culture.
This suggests that delay in division may be a consequenceof injury to an unknown mechanism controlling theonset of division, 416 and that there is no direct involvement of DNA synthesis. Yamada and Puck showed thata reversible mitotic lag is produced by a block in the G2
period after X-ray doses of 34-135 r in hyperploid S 3
HeLa cells. m They proposed that this reversible mitotic
lag, like irreversible reproductive death, is due to chromosomal damage, and that the reversible lag may reflect
interference with chromosomal condensation just before,and perhaps in, the early stages of mitosis. Other
hypotheses have also been advanced: interference ofradiation with oxydo-reduction of sulphydryl compounds
produced during cellular division, 417 • 418 and inhibition of
the division mechanism of the cytoplasm419 or of theformation of the spindle. 420 Production of antimetabolites may be responsible, as suggested by
Kuzin, 296 • 421424 who used plant material from which he
was able to demonstrate antimitotic quinones.

EFFECTS OF RADIATION ON INTEGRATED FUNCTIONS

168. When irradiated in comparable conditions, different cellular populations react in similar patterns. With
increasing doses, effects often become e.xperimentallymeasurable in the following order : modifications ofgrowth rate, mitotic delay, inhibition of mitosis, delayedor reproductive death and interphase death.
Growth rate169. Under chronic irradiation, the total mass of cell

cultures first increases and then decreases. 4 06-ios The
initial increase of the total cell mass of the culture accompanies the emergence of giant cells, the volume andusually the ploidy of which increase without division.
This phenomenon has been observed among bacteria,
yeasts and mammalian cells, and seems therefore to be
fairly general. As dose accumulates, the total weight ofthe culture diminishes and becomes lower than that ofcontrols. In general, radiation reduces growth rate and· creases generation time; however, under certain meta~~~ olic conditions, the gen~ratic;in ?rn~ c~ be s)iorter than:;~~j l1n control cultures once irradiation 1s discontinued. 409 • 410:~',' !Interference with growth rate has also been detected in
Lf~ ~solated cells. In Phycomyces blakesleeamts, Forss:'.~-:. iberg411 has shown a lowering of the growth rate of
f:j;: \sporangiophores with extremely low doses of ,..., 0.001 r.
;;.; i:::-'C_j

iij

INHIBITION OF MITOSIS AND CELLULAR. DEATH:
REPRODUCTIVE AND INTERPHASE DEATH*

172. With increased doses, cellular death usually occurs. Cells can be killed either immediately (interphase*Under doses higher than 100,000 rad, instantaneous death isobserved, due mainly to protein coagulation.

57

structures produced immediately after irradiation. Nor
can other biochemical processes affecting permeability,m,
436 the maintenance of ionic balance 437 • 438 or the disruption of nuclear and cytoplasmic membranes ,439 be ignored
as factors in the mechanism of cellular death.

death) or after a few divisions (delayed or reproductive
death). In general, the doses required to achieve interphase death are higher, although there are cells which
undergo interphase death even if irradiated by relatively
small doses, e.g. small lymphocytes, primary oocytes in
insects and mammals, mammalian neuroblasts, insect
ganglia cells. Reproductive death occurs in bone-marrow,425
intestinal crypt cells, lymphomas and spermatogonia.
It should be noted that the latter group consists of cellswith a high mitotic index; with these, interphase deathwould probably require a higher dose.

VII. Biological variables influencing
radiation response
CONCEPT OF RADIO-SENSITIVITY

173. The processes leading to reproductive or to interphase death are still unknown ; it is likely that mo:-ethan one mechanism is involved. In delayed death, chromosome breaks and mutations have been invoked as
possible mechanisms. The mechanisms resulting in cellular death may be better understood when the role ofrepair processes in irradiated cells have been studied,since the ultimate expression of a radiation effect depends
not only on initial injury but also on the ability of thecell to repair the injury.425 Most chromosome breaks
rejo~n; metabolic and synthetic processes take part inhealing,4 26 energy from ATP being required. 427 • 4 ~ 8
Recent experiments by Elkind and Sutton429 have madeit clear that repair operates in mammalian cells and influences the ultimate expression of late effects.

178. Various criteria, e.g. death of cells, inhibition ofmitos~s. impairment of biochemical and physiological

funct10ns, are currently used to determine radio-sensitivity. However, when radio-sensitivities of different
types of living organisms are compared, survival after
irradiation is usually chosen as the parameter. The selective acti?n of radiation on different parts of the cell andthe relations between differentiation mitotic activity andradio-sensitivity were described within a decade of the
discovery of X-rays. In 1906, Bergonie and Tribondeau440 formulated the principle that cells in active
proliferation are more sensitive to irradiation than nonproliferat~ng cells, and that radio-sensitivity varies inversely with degree of differentiation. Radio-sensitivity
depei:ds on various factors, physical (e.g. temperature),chemical. (e.g. oxygen tension, hydration), biological(e.g. ploidy, phase in the division cycle in ·which the cellis irradiated). Radio-sensitivity further depends on themetabolic state of the cell.

174. A clear distinction should be made between biochei:iical p_rocesses leading to delayed death and thoseleading to mterphase death. In the former, synthesis of
nucleic acids and proteins continues. 315 Radiationinduced interphase death is sudden and marked by ana:rest of n:etabolic p:ocesses. in cells with very wide
differences m metabolic behaviour, e.g. cells which areno~. dividing (lymphocytes), cells dividing infrequently( oo9'tes ), and cells continually dividing (B spermatogoma).

VARIATIONS IN RADIO-SENSITIVITY \\'ITH STAGE
OF DIVISION. 179. The <?fferent phases of mitotic and meiotic divisions have different. sensitivities to radiation. Attempts
hav_e been mad~ to link these variations in sensitivity tovarious phases m the formation of new chromosomes andto the synthesis of nucleic acids during division.

175. The biochemical causes of interphase death are
not understood, but it is possible that Creasey andStocken's work403 on nuclear phosphorylation provides
a ~st ~lue. Their data indicate that nuclear phospho1ylat1?n 1~ a~ .extremely radio-sensitive process and is:ap1dly in111b1ted. As yet, this process has been detectedm nuclei of so-called radio-sensitive tissues only; it has,
therefore, been suggested that cells dependent upon thissource of energy are those which undergo interphase
death at smal~ doses. Creasey and Stacken remark, howeve:, tha~ failure to show nuclear phosphorylation inradio-resist':llt cells may be due to an increased activityof degradative enzymes rather than to absence of thismetabolic process.
176. Nuclear phosphorylation could also be involved
in reproductive death if the energy necessary to heal
chromoso~es was provided by this phosphorylation. A:ole of m1tochrondrial oxydative phosphorylations in
mterphase and reproductive death cannot be excluded.
~-irradiation in viva, in fact, damages mitochondria inliver cells43 0-432 even at doses as low as 25 r. Mitochondrial oxydative phosphorylation in plants is immediately and gr~atly reduced after a single dose of 3,000 r,
the effec~ bemg more pronounced when cells are irradi~ted :n vi-z:o than in vitro. 433 Similar effects are also seen
m microbial cells. 4 3-1. 177. ~t is ~cult to draw a coherent picture of thebioc~emical ba~is of cellular death at this time. The
possibl.e role ot nucleic acids and protein synthesis has
been discussed, but much more extensive information isneeded on the cytological alterations of sub-cellular180. Cell survival, gene mutation frequency, and frequency of 0romosomal aberrations all respond differently accordmg to when the cell is irradiated. It is difficult t? define the most critical moment as it may vary
for different cell types and for different lesions. 413 • 414
Mos_t experimental efforts to clarify this issue have been
carried out on germ cells, in particular on both fertilized
and unfertili:ed eggs of several organisms. The ende!fects mo~t trequently used as criteria of damage are~ther survrval, ?r f~equency of chromosomal alterations
m 0ese c_el~s: It i~ widely held that variation in sensitivity~urmg d1v1s1on is a gen~ral _phenomenon and is present!n all. c~lls. 'vhatever lesion 1s taken as the end-point of
irradiation.
1~1_. ~eYertheless. some recent results suggest thatsens1t1vity of mammalian tissue culture cells to the lethal
7ffe~t. of radiation is independent o_f the division stage
m which the cells are e.xposed. Survival curves291,400,H1443 obtained with mammalian somatic cells both in viva
and in vitro have failed to show the e.'Cistence of a resistant fraction in cell populations despite the existenceof heter?geneity in stage of division. However. experiments with synchronized cultures of HeLa cells have rev~aie.d s~ me fluctuations in sensitivity during mitotic
5 Cellular morphology does not affect radiodivi~1?n:s~nsitmty of these ce.lls app~eciably since the LD 37 ofdifferent cellular ... strams ( epitheliod, fibroblastic, etc.)
ranges between 7;,-166 r only.

58

recombination tests.' 59 Adler and Copeland~ 60 have produced evidence which indicates that radio-sensitivity inE. coli K 12 is influenced by at least 4 genes. The approximate locations of the four genes have been determined in genetic recombination tests. In E. coli B,
Rousch et al.' 61 have recently found mutations at two
different loci which have a cumulative effect in increasing radio-sensi~v1ty. They too ha~e determin~d the approximate location of these genes m the ge1!-etic .map byrecombination tests. Furthermore, comparative biochemical studies of these two independent mutations showthat one leads to loss of the tendency to form filaments,
the other to a strong inhibition of growth and of nucleic
acid and protein synthesis after radiation or other treatment. Such comparative studies of mutant strains which
differ genetically in response, seem especially promisingin elucidating the physiological basis of radiation sensitivity and resistance.

VARIATION OF RADIO-SENSITIVITY WITH PLOIDY

182. Ploidy is one of the biological factors aff~cting
cellular radio-sensitivity at the level o~ the pnmary
radiation injury. The shape of yeast s1;1rvival curves ~i;pends on the ploi~y of the str~in. L<i:tarJet and Ephruss1
showed that survival of hapl01d strains expose~ to.X-rays
follows a one-hit curve whereas that of dipl01d cells
follows a two-hit curve. These authors, and subsequently
Tobias,m propounded the hypot.hesis that in.activation. ofa haploid cell is caused by a single recessive muta~ion
whereas to inactivate diploid cells two homologous sites
must be injured.
183. Extending such studies to higher polyploids,
Mortimer found that radio-resistance reaches a ma.""{imum for diploid strains and then diminishes with increasing ploidy. 14 Mortimer's res~lts have been. confir~edby Magni,m but these authors. interpret ~etr fi.ndings
differently. According to Mortimer, haploid strain~ aremainly inactivated through lethal recessive mutahons,
whereas with strains of higher ploidy dominant lethal
mutations are chiefly responsible for the inactivation.
Both types of mutations would be produced in haploidand polyploid strains, the problem being to ev~luate
quantitative relationships of the two types. Magni suggests that, in a~di~i~n to recessive and ~ominant mi.:tations, non-genetic lllJUry accounts for a sizeable fractionof radiation lethality.
184. In some other systems a positive correlation between increasing ploidy and radio-resistance has been
seen. Sparrow et al:" 6 • 447 found that, on the average,
doubling of chromosome number in plants increased
radio-resistance by a factor of 1.67. Analogous results
were obtained with polyploid cereal seeds448 and withhyperploid tissue culture cells. 44- 51 In contrast, Till452
found identical dose-effect curves for cell lines withdifferent chromosome numbers and Rhynas and Newcombe453 have described radiation-resistant cell lines of
the L strain with a lower number of chromosomes than
the radio-sensitive line. Of interest in a considerationof the influence of polyploidy is the inverse relation
between nuclear volume and radio-sensitivity in 23
diploid species of plants. 447 The role of ploidy in cellularradio-sensitivity becomes more complex when stage ofdevelopment is considered. Clark454 showed that, in
Habrobacon. diploid female embryos are more sensitive
to irradiation than haploid males during the cleavage
stage, whereas during larval and pupae stages haploidmales are more radio-sensitive. Tul'tseva455 and Astaurov
have found that, during certain stages of development,
radio-resistance increases with increasing ploidy in
Bomby:r mori but that tetraploids are more sensitive than
diploids at the end of the larval stage.

VITI. Primary genetic effects of radiation

186. The tremendous headway in the last decade in
the analysis of genetic function and genetic material has
led to a clearer view of the need for a more full understanding of the mechanisms of radiation mutagenesis.
Some problems are related to the already-mentioned
macromolecular chromosome structure, others are related more particularly to the function and structure of
the genes. Since Muller's discovery in 1927 that radiations are mutagenic, much work has been accomplished,
but no complete answer to the mechanisms of radiogenetics has been given. It has been clear from the beginning that genetic effects include visible chromosomal
aberrations. On the other hand, many mutations do not
involve any abnormalities at the level of the light microscope, and it has become practical to divide radiation
genetics into the studies of point mutation and of chromosome damage.
THE GENETIC MATERIAL

187. \Vhile one of the most important advances ingenetics came from the studies of Morgan, who discovered the linear arrangement of genes along thechromosomes from investigations on Drosophila, themost important hypothesis advanced in recent years,
derived from work on micro-organisms and viruses, isthat of the linear arrangement of genes along the DNA
double helix.* Recombination studies in bacteriophages,
bacteria, and moulds, in combination with the demonstration that the genetic information is effectively carriedin the DNA (or in some cases in the RNA), give convincing evidence."° 3 Furthermore, the existence ofviruses containing single-stranded DNA+s4 or of viruses,
whose infon11ation is coded in single-stranded RNA
molecules, indicates that only one of the two strands ofa DNA or RN A molecule may carry genetic information.
On the other hand, it has also become clearer in recent
years that DNA replication probably concerns doublestranded DNA. Even in the one-stranded cp X-174 virus,
there seems to be a double-stranded stage during replication,m although priming of DNA synthesis in vitro
is much more efficient if the double-stranded molecule
has previously been "melted" to single-stranded units. 300
188. Hypotheses concerning the structural integrationof DNA chains into chromosomes must take into accountthe existing basic proteins and ribonucleic acids which

GENETIC CONTROL OF RADIO-SENSITIVITY IN BACTERIA

185. A number of mutations causing differences inradio-sensitivity in E. coli are known. The increased
resistance of strain B/r results from a single mutational
step in its parental strain B.11 Later, Hill discovered andinvestigated a more radio-sensitive strain, B/s. This
strain also differs from. strain B by only a single mutational step.456 • 457 A stable strain containing about threetimes as much protein, RNA, and DNA per cell, isolatedby Ogg and Zelle458 after camphor treatments of strain
B/r, was about 2.5 times more radio-resistant to ionizing
radiations and in addition had a sigmoidal survival curve
rather than the exponential survival curve typical ofstrain B/r. This radiation resistance segregated in a
fashion similar to any unselected marker in genetic*For a review of the subject, see references 462 and 463.

59

s.equence of s~ecific bacteria! or bacteriophage proteins
like ,8-galactosidase and alkalme phosphatase; it is hopedthat correlations between alterations of DNA obtainedby mu~genic agents and protein sequences will throw
some light on the problems of genetic coding. The error
in DNA, then, would be replicated in a minutely altered"messenger"-RNA carrying specific genetic information to ribosomes assembling activated amino acids in asJi>ecific sequence. 350•m This very much oversimplified
picture of the mechanism of phenotypic expression enables one, however, to understand present concepts ofmutagenesis and abnormal phenotypic expression.

are beginning to be thought of as factors stabilizing,
regulating or repressing the genetic units. 872 • 468 These
more refined concepts, fairly well established for microorganisms, will have to be extended to more complex
metazoan cells.
189. A big bar to understanding genetic processes inhigher organisms is ignorance of chromosome organization at the molecular level. Although the chromosomesfrom thymus are 90 per cent nucleohistone, plus nonhistone protein, RNA and phospholipids,467 it is notknown how these are made up into the chromosome
structure seen under the microscope. Electron microscope studies have repeatedly shown strands of 200 A
diameter,468 but nucleohistone strands are ten times narrower. Urea and versene can dissociate chromosome
fibrils or nucleohistones; this indicates the importance ofhydrogen bonds and of metal ions (Ca++ and Mg++) in
holding structures together.4119 The fact that the U V
action spectrum for chromosome aberration 470 is similar
to that of nucleic acid indicates that nucleic acid may
well play a major role in forming the backbone of thechromosome. That this might well be DNA is supportedby the fact that lampbrush chromosomes can be broken
in vitro by deoxyribonuclease but not by ribonucleases
or proteases.m On the other hand, Ca++ and Mg++ deficiency is known to induce chromosome breaks and rearrangements in plants 472 and other organisms, whichindicates that these metal ions may play a role in chromosome integrity.

RADIATION-I:!'<!)UCED MUTAGENIC EFFECTS

193. Damage to DNA of cells by radiation cannot be
so controlled that mutations can be obtained independen.tly of lethal events. Although all lethal effects of radiation should not be attributed exclusively to effects on
DNA, any alteration of DNA is liable to cause deathor mutation of the particular cell. So far, the damage
caused in vivo by ionizing radiation is not precisely
known ; the absence of damage to purines and pyrimidine
in nucleohistones irradiated in vitro475 proves clearly
that effects found in nucleotides or pure DNA cannot
be e..~tended to the same material in vivo. There areindications that DNA from irradiated bacteria has aslightly lower "melting point", suggesting that H-bonds
have b7en "'.eakened. Different elution patterns of DNA
from irradiated thymus cells have been obtained ,..76
these. indi~te some change in DNA structure or mole~u­lar size. Fmally the sequence of a certain number ofsh_ort nucleotid~ chains may be changed. 477 UV irradiation of bactena appears to lead to the dimerization ofsome of the pyrimidines, but other reactions such as~ydration of pyrimidines, are also probable. More work
is need~d to f o~low the new leads. given by recent advances m rad1at1on and photochemistry.99, 47s, 479

POINT MUTATION

190. The definition of the mutagenic event deserves
special attention because of the analysis of the genetics
of bacteriophage by Benzer:162 The size of the genetic
material (DNA) depends on the test used to study themutations. According to the genetic test used, Benzer
distinguishes three units:(a) The cistron or unit of gene function is what isbeing studied when phenotypic changes are observed.
. ( b) T~e muto? or unit of mutation is the sequencem nucleotides which has to be altered for a mutation to
occur. Benzer has calculated that a muton could consistof no more than a sequence of 4-5 nucleotide pairs in
the r II region of phage T4. As the same phenotypicchange (loss of an active enzyme, for instance) may betJ:ie res~lt o~ the alteration of many loci, the size of tl:e
c1stron 1s difficult to determine precisely but it is muchlarger, probably of the order of several hundred nucleotide pairs.
( c) The recon-or unit of recombination-is what is
assayed when recombination tests are made. One alteredmuton can be made to recover through recombination,
as the result of the replacement of one or two nucleotide
pairs which constitute the recon.

194. DNA could also be altered as result of uptake
through,normc;l ~etabolic processes, of an X-ray-alteretl
prec1:1rsor; this is to be expected from work demonstra.n~g. the mutagenic activity of certain purine or
pynm1dme analogues. On the other hand, Doudney andHac;s ~~ve postulated that UV alteration of purine andpynm1d111e precursors RNA might lead to mutations
after having been incorporated into an abnormal RN A.'80. OXYGEN EFFECT

195. Mutation to streptomycin independence, investigated by ~derson481 is not influenced by changes inoxygen tension, whereas other mutations in the sameb:l;cterial strain depend on oxygen tension during irradiation by ionizing radiation.m-.i5s
1?6: Ai:other important point needs clarification. Does
rad.1ation mduce mutation by affecting DNA directlyor is the J?N~ altered as a result of secondary action?
When DNA m the form of transforming principle 45 'or bacteriophage,4 85 is irradiated in vitro under con ditiC?n~ where indir7ct effects are presumably reduced to a
mimmum. there is no oxygen effect. In bacteriophage,
D N ·i\ ~ppears. to be m'?re. se~sitive to reducing than tooxy~mg. radicals._ This mdica~es that X-rays do not
act pnmanly on DNA, but that m certain circumstancest?is molecule is altere~ as the result of secondary reaction. However, Hutchmson showed that inactivation of
DNA in solution becomes oxygen dependent in thepresence of cystein.4 u

191. At present there is no reason to believe that
mutation processes in complex organisms are very different from those in micro-organisms; it is becoming increasin&"ly evident that similar concepts will eventually
be applied. It has been demonstrated that the mutation
leading to sickle cell anaemia in humans results fromthe su?stitution of only. one amino acid by another in
one pair of the four peptide chains of the normal haemoglobm molecule; the 2A chains each have one of theirglu~ami.c acid residues substituted by a valine residue.m
This mmute error in the protein is likely to be the resultof a corresponding error in the DNA code.

1

192. Studies are being conducted on the amino acid60

can result from replacement of one base pair (A-T) by
another (G-C) (or vice-versa), then a mistake would
appear in the DNA chain.

CHEMICALLY-INDUCED MUTAGENESIS

197. Important progress has come from the study ofthe effect of several chemical mutagens on DNA or RNA
and their correlation with lethal and mutagenic activities
in viruses and micro-organisms. Both purine or pyrimidine are known to be chemically changed by a variety
of mutagens. Nitrous acid is able to remove the aminogroup of adenine, guanine, and cytosine ;487 formaldehyde can hydroxymethylate amino groups, but its mutagenic activity in Drosophila depends on the presence ofadenylic acid in the medium which, after alteration,
could become incorporated into DNA.458 Alkylating
agents appear 489 to react in many cases with the N-7 ofguanine ; this could become unstable and be removed
from the DNA chain. Glyoxal derivatives appear toaffect guanine. Hydroxylaminem appears to react chiefly
with cytosine; hydrazine, to remove pyrimidine; a low
pH treatment,491 to remove purine. Acridines, like proflavines, are mutagenic; their action is believed to result
from fixation of this reagent between two adjacent basepairs, thus increasing their separation. A comparison of
the mutagenic effects of these chemicals with that ofradiation could be of great value. The linear dose response curves found in several cases of chemical mutagenesis indicate that, as for most radiation-induced
mutations, the process involves a single event. In thiscase the alteration involves a single nitrogen base in one
DNA molecule.

202. In the mutagenic actionofbromodeoxyuridineon
T 4 phage, the analogue might take the place of 5-hydroxymethylcytosine and pair with guanine (error in pairing) ;this would lead to the replacement of a guanine-5hydroxymethylcytosine ( G-H) pair by an adeninethymine pair after three DNA replications. Alternatively,
the bromouracil moiety of the analogue might replace
thymine during the first replication (error in replication) and pair with guanine at the next. This would leadto the replacement of A-T by G-H after the third replication.502 Effectively, mutants appear in a culture after
the third DNA replication. 2-amino purine could also
lead to the replacement of G-C by A-T, and would, likebromodeoxyuridine, on the basis of this hypothesis, be agood agent for back mutating a mutation due to bromouracil incorporation ; examples of chemically-induced
mutation and back mutation, interpretable in these terms,
are now becoming known.
203. However, it is not at all certain that the reversion
of a mutation to wild type is necessarily the e..'Cact reversalof the forward mutation, and different base pairs might
conceivably be involved in the forward and reverse
process as postulated by Brenner, Barnett, Crick andOrgel. 563 It is very possible that the hypothesis of Freeseis an oversimplification of the facts. A mutation andback mutation with proflavine might result from addition
or deletion of a base-pair; this might lead to a much
more substantial alteration of the protein, such as abreak or an alteration of sequence in the polypeptide
chain. With radiation, it is difficult at present to makeany hypothesis, but the concepts of chemical mutagenesis
will certainly have to be considered in radio-biology whenradiation-induced chemical changes in DNA are better
known.

UPTAKE OF ABNORMAL PRECURSORS

198. A number of base analogues have also beenfound to be either lethal or mutagenic. Bromouracil (orbromodeoxyuridine) once incorporated into bacteriophage, .. 92494 bacteria, and mammalian cells 495 • 496 canproduce mutations and lead to increased sensitivity to
X or UV radiation. 465 •493 • 491
199. 2-amino purine, another mutagen, is believed to
be incorporated or to permit the uptake of another base(perhaps adenine) instead of guanine.m-5oo

204. It had been knovm for a few yearsm that thefrequency of mutants in bacteria increases with cell
division. More recently, Witkin has shown that if protein synthesis is inhibited by amino acid starvation or bychloramphenicol, a lower frequency of bacterial mutants
is obtained. 505 · 506 This suggests that irradiation produces
pre-mutational damage which can eventually be lost, or
which can become fixed as a result of protein synthesis.
In a study of lethal mutations in Paramecium, Kimball 5° 7
has shown that loss of premutational damage is probably
due to metabolic repair of localized chromosomal lesions.
Lieb has recently shown508 that when DNA synthesis isretarded by treating the cells with chloramphenicol, theincrease in mutants, observed when growth is continued
after the chloramphenicol "challenge", parallels the increase in DNA; this strongly suggests that the terminal
event in this mutational process is DNA synthesis. Much
has still to be learned about induced mutagenesis. The
role of RNA suggested by Douclney and Haas480 is not
yet clear. However, one important fact emerges: it ispossible to inhibit to some extent mutation fi.'Cation in
micro-organisms by delaying protein or DNA synthesis.

COMPARISON BETWEEN VARIOUS MUTAGENIC AGENTS

200. When the frequencies of spontaneous and chemically-induced mutations in bacteriophage T 4 are studied,it appears that some regions of the genome mutate much
more frequently than others; the same region does notnecessarily mutate with comparable frequency after
treatment with various mutagens.162 -001 Proflavine seems
to induce a pattern of mutations which differs from that
produced by base analogues; the patterns produced bybase analogues show some differences when comparedwith the pattern of spontaneous mutations. One must,
therefore, suspect the existence of several classes ofmutagens; of these, the base analogue class induces amutation pattern similar to those produced by five bromodeoxyuridine and the proflavine class. Close study of
specific chemical mutagens. and their comparison withspontaneous and radiation-induced mutations, will no
doubt bring much light on the molecular basis of mutagenesis.

MUTATION EXPRESSION
BIOCHEMICAL ASPECTS OF MUTATION PROCESSES

205. The biochemical processes underlying the synthesis of cell constituents are becoming better known
each year. One of the major problems of present-day
biochemistry is the way specific enzymes necessary forthese synthetic processes become synthesized themselves.
Nisman 509 has succeeded in synthesizing in vitro an

201. From work on mutagenesis of various analogues
and UV radiation, it appears very probable that. mutation becomes fi.~ed during DNA replication. Examplesof bromouracil-induced mutations are pertinent to thishypothesis. 500 If, as postulated by Freese, 502 mutation

61

enzyme of E. coli, ,8-galactosidase, in the presence ofribosomes of these bacteria, a mixture of the four ribonucleoside triphosphates, and the DNA of a strain of
E. coli possessing the enzyme. The ~ynth~sis does notoccur with DNA extracted from an mduc1ble but noninduced strain of the same bacteria. Furthermore, Novelli
has shown 510 that this synthesis can be inhibited by X- or
UV-irradiation, and that restoration can be obtained byadding the genetically competent DNA to the system.
These experiments are pertinent to an understandingof radiation-induced mutagenesis and, together withthose on chemical mutagenesis, are the first leads to an
analysis of mutation processes at the molecular level.
Treatment of the genetic material (RNA) of Tobacco
mosaic virus with nitrous acid leads, after infection of
the plant, to the synthesis of viral protein with only threeabnormal amino acids. 511 • 512

FACTORS INFLUENCING THE PRODUCTION OF
CHROMOSOME BREAKS

211. The effect of oxygen on the occurrence of chromosome breaks produced by radiation is comple..x. On
the one hand, anoxia during irradiation reduces the production of breaks ;219 on the other hand, since the rejoining of chromosome fragments is a phenomenon which
requires energy, the absence of oxygen diminishes thefrequency of rejoining. 518 Probably connected with theoxygen effect is the effect of temperature. 203 The numberof breaks increases with a decrease of temperature;
this is consistent with the fact that the tension, and therefore the availability of oxygen, is reduced at lower temperatures.
212. Strictly mechanical agents such as centrifugation
and ultrasonics, when applied at the moment of irradiation, increase the amount of chromosome breakage.
When cells are irradiated with ultra-violet519 or infra-red
rays either prior to or after exposure to ionizing radiation, the frequency of chromosome breaks is reduced in
the former case but is raised in the latter. Infra-red
irradiation seems to act through changes in metabolicalprocesses. 5:0, 521

206. The problem of mutation expression is therefore
one of information transfer from the DNA to the cellularsites of specific synthesis, many of which are cytoplasmic.
One major problem concerns the formation of ribosomes; the way in which they receive their information
for specific protein synthesis is at present being extensively studied (para. 140).

213. Biological factors also influence sensitivity tochromosomal damage. 522 Cells from different tissues
show different sensitivities.m,m On the other hand, thefrequency of breaks per unit of radiation depends on
the stage of division during which cells are irradiated. 525
The highest frequencies are observed when cells areirradiated during metaphase and anaphase. 52 s-m In the
meiotic process, the diplotene stage is most sensitive inanimals.529

CHROMOSOME BREAKS

207. Point mutations in higher organisms probably
result from processes similar to those described for
micro-organisms, but the complexity of the chromosomes
may complicate the process. On the other hand, chromosome aberrations have been thoroughly analysed in various organisms and described at length in many valuablereference papers. Ionizing radiations can induce breakage of chromosomes or chromatids followed by restitution or illegitimate reunions. This may lead to a variety
of aberrations 513 which are visible at the first division
after irradiation, or in some instances, only after very
many cell generations. However, these aberrations often
lead to unequal distribution of chromosomes between
daughter cells ; these usually lead to cell death. Restitution may be at the morphological level only, and a point
mutation, probably due to DNA damage may eventuallyappear.

GENETIC EFFECTS OF INCORPORATED RADIO-ACTIVE
SUBSTANCES

214. Radio-isotopes introduced into organisms may
be incorporated into critical molecules. Although mosteffects are due to ionization by the charged particle
emitted from the isotope, some may result from disturbance of the molecule by transmutation of the incorporated atom. The new atom not only has different and,in most instances, incompatible bonding characteristics,
but also, in transmutation, gives off recoil and excitational energy.

208. Similar chromosome damage may also occur
after UV irradiation,513 but is less frequent than after
ionizing radiation. It may also occur as an effect ofalkylating agents 512 or aiter incorporation ofor H 3thymidine514• m or of bromodeoxyuridine•01 in cellularDNA.

215. Ionization and excitation from the ionizing particle are so large compared with the energy from transmutation that they usually outweigh the importance oftransmutation in radiation injury. However, certain
isotopes incorporated preferentially in vitally significant
molecules could, by transmutation, cause unique effects
not accomplished by ionization or excitation from acharged particle. Accumulating evidence, along withtheoretical considerations, indicates that transmutation
should be considered as a factor in the toxicity of internal emitters. The atomic number of the radio-isotope,
its type of decay, the particle emitted, and the energy
released, are obviously important in gauging the significance of transmutation.

cu_209. Studies of agents influencing chromosome damage have led WolfP 18 to postulate the existence of two
types of chromosome breaks : some which rejoin rapidly
and which presumably involve linkages through metal
ions, and some which are influenced by post-irradiation
protein-synthesis and which are believed to involve
covalent links.
210. The relative role of direct and indirect mechanisms in chromosome breakage has been partially clarified by comparing the modifying effects of various
chemicals with damage due to chemically induced radicals and radiation. 63 • 65 The effect of radiation in producing breaks is mainly direct; it certainly is so for dry
DNA. Evidence in favour of direct effect on DNA
in vivo is provided by experiments carried out with bonemarrow cells in vitro. m

POSSIBILITY OF TRANSMUTATION EFFECT WITH C 14

216. The disintegration by which CH exerts its biological effect is1
C e4 - N 11•
f3 0.155 MeV(1}
The mean energy of the ,8-particles is 50 + 5 ke V; thus
the reaction gives rise to fast charged particles for which

+ +

62

the RBE of the energy they release is probably 1. Mostof the energy of the reaction ( 1) passes via the kinetic
energy of the emitted /3-particle into ionization and e.'<citation of the surrounding material; a lesser part appears
at the site of the transmutation reaction itself. 530 Because
carbon is a part of every organic molecule in living systems, transmutation may significantly affect key molecules, especially those of the genetic apparatus. Indeed,
Totter et al. 531 have suggested that the mutational consequences of 0• transmutations might be comparable inmagnitude to those from the associated /3-particles. However, according to Pauling, 532 they are unlikely to amountto more than about 10 per cent of the total.
217. Although it is certainly established that P 32 ,when incorporated into the genetic material of a varietyof or~nisms, produces biological effects by transmutation (E. coli,5°1 • 533 • 53'• 538 bacteriophage, 538 • 637 Paramecium, 038 Drosophila 539- 541 ), the data concerning cutransmutation effects are less plentiful and less consistent. Apelgot and Latarjet, in tests with H 3 , P 32 and 0 4
labelled DNA in E. coli B/r found that, whereas thelethal effect with H 3 was due largely to the emitted
beta-particle, transmutation was mainly responsible forthe effect with P3 2and cu.m Kuzin et al.m have reported that the efficiency of incorporated cu in producing chromosome breakage in Vicia faba is 10-20 times
greater than that of external Co80 gamma radiation. By

contrast, Williams and Scully54' failed to observe an
increased rate of somatic mutations in Antirrhinimi
majus grown in a cuo2 atmosphere as compared toexternal gamma radiation. The work of McQuade andFriedkin 5 H is especially interesting, for despite the factthat no comparisons were attempted with external radiation controls, the frequency of chromosome breakage inAlliimi cepa root tips was about twice as great when thechromosomes were labelled with 0 4 thymidine bearing
the 0' in the methyl group as was the frequency observed when the 0·1 was in the 2' position.
LOCAL CONSEQUENCES OF TRANSMUTATION

218. Three processes may cause disturbances at orvery near the site of a nuclear transformation in whicha /3-particle is emitted:(a) Chemical changes ; C ~ N;( b) Mechanical recoil of the nucleus which emits the/3-particle;( c) The production of residual electronic excitationenergy due to the non-correspondence of orbital electrons and nucleus following the transmution. m
219. These and other features of transmutation reactions of especial biological interests are summarized
below.

PROPERTIES OF CERTAIN ISOTOPES RELEVANT TO TRANSMUTATION PROBLEMS

cu
Half-life .••..•............ 5,760 yrs.
Max. P-energy (MeV) ......
0.155
Mean P-energy (MeV) •..•..
0.050
Max. recoil energy (eV) .•.•
6.9
Mean residual excitationenergy (eV) .•..••.•••...
44.5
Chemicalchange ..•....... C...--.N

P'"

P1I

SU

H•

14.3 d
1.701
0.71
77.3

25.4 d
0.27
0.093
6.0

87.1 d
0.167
0.055
3.0

12.5 yrs.
0.0176
0.006
3.2

60.3
P--+S

60.3
P--+S

61.7
S--+CI

24.5
H--+Hetum to vibrational and other kinetric energy of surrounding atoms may suffice to break significant numbersof important hydrogen bonds in these molecules.

220. Except for P 32, by far the largest part of theenergy locally released is the residual electronic excitation of the transmuted atom. This energy and its magnitude closely resemble the corresponding release in a
primary or secondary ionizing event by a fast charged
particle. The effects of this electronic disequilibrium are
therefore qualitatively indistinguishable, except for site,from those of the emitted ionizing particles.
221. In P 32 decay, the large recoil energy is clearlysufficient to remove the disintegrating atom from themolecule in which it was previously bound, and to carryit into a neighbouring molecule, together with its associated electronic energy. m The recoil energies of all ofother transmutation reactions summarized above aremuch lower and are comparable to the relevant covalentbinding energies. Moreover, experimentally determinedchemical-binding energies are presumably lower than
the activation energies for reactions, even if reactions
take place by optimal paths in phase space; the isotropically distributed but directional nature of recoil momentum is likely to make a substantial part of it useless inrespect of the optimal reaction path. Hence, even though
its chemical binding is simultaneously weakened by thecllange in its chemical nature, it is doubtful whether,in substances of biological interest, atoms undergoingtransmutation other than P 32, effectively leave the molecule in which they were bound. An interesting possibility, with a transmuted atom that does not detach froma macromolecule, is that conversion of the recoil momen-

222. The most interesting possibilities of C1' transmutation lie in the chemical change, C ~ N; this may
leave a molecule altered rather than destroyed in function, giving rise to a special class of subtle and viablechanges in the genetic system different from those induced by the more destructive ionization or excitation.
The significance of the possibility of such changes underconditions of uniform contamination is discussed below.
loNIZATION DOSE PER TRANSMUTATION UNDERUNIFORM CONTAMINATION

223. As will be shown below with uniform incorporation, the practical limitation upon the effect of transmutation itself is likely to be dosimetric. Under suchconditions, for every transmutation of a C14 atom within
an important molecule, ,_, 5 X 104 eV of ionization andexcitation energy will also be liberated; this proportionality will only break down when the molecule underconsideration is part of a unit of dimension significantly
less than the mean range of the 0 4 ,8-particle ( ,_, 30 µ)and isolated from other carbon-containing units by distances significantly greater than the range. If the efficiency of transmutation in causing a certain effect is
1JT, and that of the ionization-excitation energy of conventional ionization ( 34 eV) is 711, then the fraction63added to the ionization-excitation effect by transmutation is only 6.8 X lD-4 TJT/TJi. This relation suggests at
once that, even for high T, C14 transmutation can besignificant only when TJL is very small; unfortunately,
it is not of much quantitative worth, since appropriate
values are not available. The only estimates available
for 'TJT are from P 32 incorporated into DNA, where'TJT is probably 0.01 or lower, 501 • 546 although the efficiency
with which the DNA molecule is broken may be in theregion of 0.1 for a double helix547 and reach a value close
to unity for single-stranded DNA.~ 4 • 548 For the destruction of infectivity of bacteriophage by P 32 incorporation
in DNA, 'TJT and 'TJ' values are available, and the ratio'TJT'/ 'TJ' is about 10. 536 • 549

IX. Recovery at the cellular level
226. The concept of "recovery" at the cellular level
covers various phenomena with different mechanisms.
At least three should be distinguished:(a) Spontaneous recovery of damaged molecules andstructures of the cell ; this constitutes genuine recovery;( b) Recovery through action of physical or chemical
agents immediately or soon after irradiation ; this constitutes a kind of "treatment" of the damaged cells;( c) Replacement of damaged molecules or structures by corresponding molecules or structures from undamaged cells. Here there is no recovery but there is a
restoration of cell function.

224. Mutation does not necessarily consist only ofdamage of this kind in the DNA molecule. Changes inat least three types of material might cause mutation:

227. The interval between irradiation and the biological expression of the primary damage indicates a complex process and suggests the possibility of interferingwith it to promote the repair of injury. Much work deals
with phenomena in bacteria and their related bacteriopbages using ultra-violet light. Some results have been
exi:ended by the use of ionizing radiation. The inclusionof ultra-violet data in this chapter is justified by thesimilarities and differences found between the action ofultra-violet light and ionizing radiation. These can enlighten several aspects of molecular biology, in particular those associated with the structure, replication, andbiological activity of nucleic acids.

(a) The gene code itself, i.e., in the double-helicalDNA (in most organisms);( b) Associated stabilizing material such as histone;( c) The machinery (other than the original gene) by\vhich a gene-replica is made, whether or not this machine!)' at any stage embodies the gene-code itself in a
non-DNA physical form.
The P 32 data presented relates almost solely to the firstof these, and even there is limited to events in the backbone of the DNA molecule rather than the nitrogenbases whose sequence presumably determines the information. Four of the carbon atoms of each averagenucleotide of DNA are likewise in the backbone, butchemical transmutation of carbon into nitrogen at mostof the otbers-4 or 5 in the nitrogen-base, 1 in deox7ribose linking nitrogen-base to backbone-could conceivably give rise to subtle viable changes unlikely to be duplicated by gross ionization damage or by P 32 disintegration in the backbone. In bacteriophage, some protein
synthesis necessarily precedes DNA synthesis and gene
replication after infection. 550• 551 Experiments on inactivation by P 32 decay suggest the possibility of a stage
at which the genetic information itself is carried in a
non-P 32 containing form. 547

228. Restoration is sometimes obtained by· destruction of some intermediate compound before the damage
is irreversibly established, e.g. photorestoration of ultraviolet damage, 552 • 558 restoration by catalase of lysogenic
systems treated with ultra-violet, 238 , 239 • 554 and restoration by ultra-violet light of X-irradiated yeast and bacteria. sss, ssa
229. Photorestoration (restoration by radiations of
the range 3,100-5,500 angstroms) is very general andhas been verified in a great variety of biological systems.
The study of photorestoration of a transforming factor
in vitro has led to the discovery of an enzyme in yeast
and bacteria which is necessary for restoration. 551 Work
with this system will soon give valuable information on
the mechanisms of ultra-violet inactivation and photorestoration. Recently, Marmur and Grossman97 haveshown that the PR (photorestoration) enzyme is able
to reverse induced linking of DNA strands by UV light.

225. In conclusion:(a) From theoretical considerations based on thelarge ionization-excitation dose per transmutation, thecontribution of transmutation to the biological effectwould not be expected to be significant under conditionsof uniform incorporation of 0 4 unless the efficiencyof transmutation in producing the effect is very muchgreater than that of ionization. Although experimental
data are as yet meagre and inconsistent, certain data indicate that 0 4 transmutation may contribute significantly to chromosome breakage;230. Several radio-biologists have attempted to achieve
photorestoration after exposure to X-rays. Dulbecco 558
has shown that coliphage T 2 , inactivated by X-rays insynthetic medium (predominant indirect effect), cannot be restored by visible light, but that the same phage
inactivated in organic medium (predominant direct
effect) shows a slight photorestoration. Similar results
have been obtained by Watson, 55 P• 560 with coliphages
T2, T 4 , and Ta. In general, however, there is no photorestoration after irradiation with ionizing particles.

( b) Because the C14 recoil energy is low and theenergy of electronic rearrangement strongly resembles
the usual ionization-excitation energy, such a contribution is most likely to be mediated through the c~ N
chemical change ;231. Some of the lethal damage provoked by UVlight in the coliphage T 4 can be repaired by some cellular
reactivation mechanism linked to the presence in thisphage of the gene µ.. This gene determines the difference in ultra-violet sensitivity between coliphages T 2
and T 4. The primary UV lesions are identical in bothphage types, but the presence of the µ. allele in T ~ (asopposed to theµ. allele in T 2 ) results in reactivation of
about 50 per cent of the otherwise lethal damage. Lethal
UV damage reactivable by the µ. allele action is almost
identical to photoreactivable damage. 561( c) The area in which to seek such a contribution
would seem to lie in phenomena brought about with very
low efficiency by ionization: probably not in simple dam'.lge to the genie material but perhaps in abnormalities
1~ the components of replicative apparatus where ionizatlon-excitation "'ould, in contrast, be more likely tocause total inactivation.
64

phage incorporated by P 3 ~ decay, 537 a more recent study
has detected this phenomenon. 573
237. The fact that some of the phenomena of recovery
of genetic structures are only seen after UV irradiationis, in general, interpreted as being due to the different
primary effects which follow UV and X-ray absorption
in nucleic acid molecules. It appears that UV radiation
primarily damages bases whereas X-rays primarily produce breaks in the DNA backbone.
238. The damage produced by UV light in temperatebacteriophages can be repaired to a certain extent by thehost cell. 5 ss, 574- 5 ' 6 It seems that the normal host cells
possess a genetic component which is capable of repairing the UV damaged virus. This is explained by Garenand Zinder in terms of genetic homology between the
genome of the phage and the genome of the bacteria inlysogenic systems. The homologous part of the bacteria
could replace the injured part of the virus genome
through a process of genetic recombination. Similar phenomena have been reported with Rous sarcoma virus190
and with the measles virus 191 in host animal cells.
239. Another phenomenon of host reactivation has
been described by Weigle ;192 it applies to temperate andvirulent phages. Among the progeny of irradiatedphages grown in irradiated bacteria, a, certain fraction ofplaque-mutants is observed. These mutants are not seenamong progeny of the same phage grow"Il in non-irradiated bacteria. This suggests that the phenomena ofreactivation and production of mutants are connected.
240. A restoration phenomenon linked to diploidy has
been observed by Latarjet and Ephrussi13 in Saccharomyces cerevisiae; after X-irradiation, haploid and diploid cells can undergo a few abortive divisions before
dying (delayed death). In diploid cells, however, a restored cell with normal morphologoy may sometimes
arise after a few abortive divisions. Repair of radiation
damage may occur in diploid yeast cells if they are
starved after irradiation.193
241. The replacement of damaged macromolecules byintact ones inside cellular structures also offers a possibility of repair. For instance, survival of E. coli B/r toirradiation is higher on a synthetic medium enrichedwith yeast extract than on synthetic medium only.194
Similar experiments are those of Daniels et al. 19 5-197 withthe large multinucleate amceba Pelomyxa illinoisensis inwhich individuals lethally irradiated with ionizing radiation may be restored to reproductive viability by meansof fusion with fragments of unirradiated individuals.
When the contents of this amceba are stratified by centrifugation, the heavy third containing nuclei are mostactive in restoring irradiated cells. Some desoxyribonucleotides were reported to have favourable effect on
restoration of hematopoietic cells from radiation injury
in vitro as well as in vivo. 198
242. Restoration of cells can also be obtained by treatments that modify the post-irradiation metabolism of thecells such as temperature, presence of certain nutrients,
metabolic inhibitors. This subject, which is related to thevariations in the conditions of the cell populations after
irradiation, has been extensively reviewed recently by
Alper. 199 Characteristically, the results reported indicatethat most treatments which reduce the response to irradiation provide an environment which is sub-optimal for
growth.
243. Some physiological functions of cells impairedby radiation may also be repaired. At present, knowledgeof recovery mechanisms after ionizing radiation is in its

232. The restoration effect of ultra-violet light subsequent to X-irradiation has been observed by Elkind
et al. in yeast cells. 555 Ultra-violet light increases thefraction of cells surviving the exposure to X-rays by afactor of 3 or 4. _i\nalogous effects with spores ofStreptomyces aureofaciens have been reported by Goldat
et a/,, 556 In the latter instance, the restoring action of theultra-violet was observed for both lethal effects andmutation induction.
233. Restoration by catalase of ultra-violet-induced
damage 238 • z39 • m is more restricted, as it applies only tolysogenic systems and is linked to the destruction oforganic peroxydes formed in these systems during irradiation.
234. The supply of metabolites to micro-organisms
which have lost the capacity to synthetize them can beconsidered as one possible mechanism of recovery; inthis case, however, restoration is apparent only, since
the intrinsic damage has not been repaired. Restitution
would be achieved if there was a possibility of replacing the damaged molecules or sub-cellular units by nonirradiated ones.
235. The phenomenon of cross - reactivation or"marker rescue" was discovered by Luria with theT-even phages (T 2, T 4 , T 6 ). When a bacterium is infected with active and inactivated phages differing fromeach other in a few of their genetic loci, some genetic
markers of the inactivated parents may appear among
the progeny resulting from such a mixed infection.
These studies were subsequently carried out in great
detail by Doennann et al. 56 ~• 563 and were extended tothe coliphage ,\, 564 and to the Salmonella phage P 22 • Thisphenomenon may be explained by assuming that the
UV lesion, while preventing or delaying the reproduction of the whole phage, destroys only a small piece ofits genome. The cross-reactivated loci would be thoseof the undamaged parts of the irradiated phage which
would reproduce only after their "rescue'' from theinjured genome through genetic recombination with theunirradiated parent. 56 ~· m After X-irradiation and after
decay of incorporated P~, marker rescue has also been
observed in the T-even phages 559 • 566• 567 and in the Salmo1iella phage P 22 , 565
236. A bacterium infected with a single inactivatedphage does not yield active virus ; but if two or moreinactivated virus particles infect a bacterium, active
phage may be released. 508 The phenomenon of multiplicity reactivation has been interpreted by Luria as
being due to genetic exchange of uninjured parts of the
genome of the parental phages. Further studies5GD havenot supported some aspects of Luria's original theory ofmultiplicity reactivation, but recently Hann570 and Barricelli571 have amended Luria's theory to reconcile itwith the experimental data. Multiplicity reactivation
seems to be restricted to certain strains of phages and tocertain types of radiation damage. It occurs with the
T -even phages and T 5 with high efficiency; it is lesseffective with T 1 , ,\and P 22 , and not at all effective withT 3 , T 7 and the Pyocyanea phage P 8 •547 Multiplicity reactivation occurs with high efficiency only when the phagebacterium comple.x is exposed to irradiation. To explain
the different response to X-rays of intracellular andextracellular phage, Weigle and Bertani 572 assumed theoccurrence of an "early step" damage connected with
DNA injection which prevents the uninjured parts of
the irradiated genome from participating in the sequenceof events conducive to reactivation. Although it has beenreported that no multiplicity reactivation occurs in T,.

65

infancy. This subject is of such importance to radiobiology that research on all aspects of the problem shouldbe emphasized.
X. General conclusions

244. The main conclusions of radio-biology in the1958 report remain valid and will not, in general, be
repeated here. However, because of the importance ofthe threshold problem, it seems prudent to restate theearlier conclusion that "biological effects will follow
irradiation, however small its amount". This conclusion,
based largely on theoretical considerations and on the
exponential character of many dose-effect curves, issupported by new data on the effects in macromolecularsolutions, intracellular structures, viruses, bacteria, andother cellular systems.
245. The main development since the last report has
been spectacular progress in the study of biologicaleffects at the molecular level. This applies in particular
to the genetic material, DNA, and the way in which thissubstance replicates itself (DNA synthesis) and controls the synthesis of specific proteins transcribing its
information to RNA by a triplet code. In the wake ofmolecular biology, a molecular radio-biology is now developing and, although still in its initial stages, hasalready provided some important results. Thus, evidence
is now coming forward that the most significant radiation effects (inhibition of mitosis, reproductive andinterphase death, mutation), at least in a number of
instances, are due to primary damage of the genetic
material, namely the chromosomes and, in particular,
DNA. How these lesions interfere with DNA, RNA,and protein synthesis has already been much clarified;
it is expected that studies on cell-free systems i1i vitro

66

now in progress ·will provide many answers to still open
questions.
246. Understanding of radiation damage in nuclear
material has been increased by studies of the effects on
the physical and chemical properties of macromolecules,
especially nucleic acids and nucleoproteins in vitro andin vivo. The ESR method seems promising for detection
and determination of the fate of free radicals producedby radiation in biological materials.
247. New knowledge of the effects on cytoplasmic
functions has contributed to an understanding of the
problem of radiation damage to cells. Only by taking
into account the mutual interaction of damaged structures in the nucleus and cytoplasm can this complex
problem be understood.
248. The important role of recovery at the cellularlevel in determining final radiation effects has been more
appreciated, especially the partial reversibility of initial
mutational damage in cells of various origins. However,knowledge in this field is fragmentary; further researchis needed.
249. Biological effects after incorporation of P 32 , 0 4 ,and H 3 have been studied. It seems that under most conditions, biological effects are due to radiation rather thanto transmutation. However, it has been shown that undercertain conditions, particularly after P 32 and 0 4 areincorporated into essential molecules like DNA, transmutation may lead to chromosome breakage.
250. Radio-sensitivity studies have received new
stimulus from recent analysis of genetic factors determining radio-sensitivity in bacteria and from investigations of how these genetic factors are metabolically
expressed.

REFERENCES
1. Forster, Th., "Excitation transfer" in Comparative
Effects of Radiation. M. Burton, J. S. Kirby-Smithand]. L. Magee, eds., New York (1960).
2. Tmroq,eeB-Peco:Bc.ll'.Itit, H. B., 0 np~e nona,11.a-to radiation in Eschericliia coli. Proc. Nat. Acad.
Sci. 32 : 59-68 ( 1946).
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