IONIZING RADIATION:
SOURCES AND BIOLOGICALEFFECTS
United Nations Scientific Committee
on the Effects of Atomic Radiation
1982 Report to the General Assembly, with annexes

UNITED NATIONSNew York, 1982

NOTE
The report of the Committee without its annexes appears as Official Records of the General Assembly, Thirtyseventh Session, Supplement No. 45 (A/37/45).
The designations employed and the presentation of material in this publication do not imply the expression of
any opinion whatsoever on the part oft he Secretariat of the United Nations concerning the legal status of any country,
territory. city or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries.

UNITED NATIONS PUBLICATION
Sales No. E.82.IX.806300PANNEX L

Biological effects of radiation in combination with other physical, chemical or biologicalagentsCONTENTSParagraphs

INTRODUCTION ....................... .
I. MODES OF INTERACTION ......... .
A.
B.

1-20
21-72

General approach ................ .
Surface of response and isobolic diagrams .......................... .
Probabilistic assessment of the interaction .......................... .
Theory and practice .............. .

21-41

II. PHYSICAL AGENTS ................ .

73-113

C.

D.

A.
B.
C.

D.

E.
F.

~?mbina.tio_ns of various types of ion1zmg rad1at1on ................... .
UV and ionizing radiation ......... .
Electromagnetic and ionizing radiation ............................ .
1.
Experimental data ........... .
2.
Epidemiological evidence .... .
Su~op_timal temperature and ionizing
rad1at1on ....................... .
1.
High temperature ........... .
2.
Low temperature ............ .
Magnetic fields and ultrasound ..... .
Dusts and fibres ................. .

42-49
50-61
62-72

Paragraphs

111. CHEMICAL AGENTS ............... .
A.
B.

C.
D.

E.

Inorganic compounds ............. .
Organic radiosensitizing compounds
Carcinogenic chemicals .•..........
The special case of tobacco smoke ... .
1.
General .................... .
2.
Experimental data ........... .
3.
Epidemiological evidence .... .
Other drugs ..................... .

IV. BIOLOGICAL AGENTS ............. .
73-78
79-85

A.
B.

94-103
94-99
100-103104-107108-113

Introduction
1. In man's living and working environments situations are often encountered in which different ambient
factors of a physical. chemical or biological nature
could conceivably combine with ionizing radiation ingiving rise to undesirable effects. In this paper for the
first time the Committee considers the combined actionof radiation with potentially important environmental
conditions. Since this paper concentrates on radiationin environmental circumstances, three important areasof combined action between radiation and chemical
agents are not considered here. The first concerns the
combined action of chemical agents (both chemo-

114-120121-136
137-157
158-183
I 58-159
160-168
169-183
184-199200-217

General ........................ .
Hormones ...................... .
Infectious agents .....•............
I.
Viral infections ............. .
2.
Bacterial infections .......... .

200-201202-213214-217214-215
216-217

V. CONCLUSIONS .................... .

218-237

VI. RESEARCH NEEDS ................ .

238-244

C.
86-93
86-91
92-93

114-199

Page
References .•.••••.....•..•...........•.•.

765

therapeutic compounds and sensitizers of various kinds)
to enhance radiation effects in clinical radiotherapy (828,024. 025, H28. H29]. The second results from therestriction of this paper to radiation effects combinedwith agents which affect carcinogenesis, not therefore
including combined effects in mutagenesis. This area
may be considered by the Committee in the future. The
third area not treated in detail in this paper is theeffects of a combination of protective agents with acuteradiation exposure fAl2] because this subject is of only
minor importance in estimation or modification of risk.
2. There is a great scarcity of systematic data on
which an analysis of combined effects can be based, in727

spite of the large numher of reports where combined
actions were tested and interactions claimed. Thus, thisAnnex must be somewhat different from others inwhich a large body of literature data is reviewed andsystematically analysed. This Annex will be instead
more hypothetical and will attempt to suggest definitions, to identify suitable methods of analysis, to selectfrom a large amount of diffuse information the conditions and the data of importance for further consideration and to provide suggestions for future research.
3. The following review of experimental or epidemiological data should be simply taken as an illustration ofsome theoretical analyses using examples from the literature. These considerations point, on the one hand, tothe very preliminary character of this Annex and, on
the other hand. express a word of caution against hasty
conclusions in view of the present state of knowledge
and the large variety of situations encountered.
4. There are many instances of possible combined
actions in which different agents may interact withionizing radiation. Among the physical agents, forexample, temperature should be considered. It is wellknown that ambient temperature in different environments may vary within a range of about 70°C althoughcontrol mechanisms allow man to survive under themost extreme conditions. It is also known, however,that small changes in the temperature of cells may
result in striking changes of cell survival upon irradiation. These changes are presently being investigated
for their potential in cancer therapy [Cl, F2. 016].
Ultraviolet light, itself carcinogenic. sound, ultrasoundand vibrations are present in many living and working
environments and may give rise to combined actions.
The same can be said for static electromagnetic fields,
and high-frequency or very high frequency (microwaves) electromagnetic radiation.
5. Man-made(xenobiotic)
chemicals
intheenvironment are a major concern for toxicologists.
According to some estimates [M2] the number of identified molecules is now more than four million andevery year a few hundred thousand new items areadded to the list. There are some tens of thousandchemicals in common use in modern societies. notincluding pesticides, pharmaceuticals and foodadditives. The so-called "energy related pollutants" arealso to be considered in this category. They include theoxydes of carbon and nitrogen, sulfur compounds.
polycyclic hydrocarbons and some others. Among them3,4-benzo(a)pyrene (BP) is frequently used as an index
of polycyclic hydrocarbons withcancerogenic
properties [S2]. Its yearly production is estimated to beapproximately 5000 t [S31]. The time course of its
production may be followed, for example, by lake
sediment analysis (H11]. The concentration of BP in the
air of large industrial cities may reach values of 100 ng/
m3 [BIO]. BP is also one of the many chemical constituents of tobacco smoke and may be considered ofimportance for some sections of the population occupationally exposed to radiation. The circulation of BP andof other polycyclic aromatic compounds in theenvironment has been studied extensively [S2. S31].
6. The list of chemicals whose action might combinewith that of radiation in the environment is very
extensive. Special attention should be given to situations of practical interest where the chemical agentsthemselves have carcinogenic properties [H21]. For
example, many industrial effluents contain traceelements such as arsenic. nickel or chromium. These
728

substances may produce carcinogenic or mutagenic
effects [DJ. The same is true for dust and fibres. Dust isa very common and widespread industrial emission anda component of many occupational environments. It
has been reported that dust or fly ash from power
stations may have carcinogenic properties [K9] or may
serve as carriers of trace metals, radioactive nuclides or
polycyclic aromatic hydrocarbons [B22). In mines
mineral dust may combine with the organic products ofdiesel exhausts and with radioactive radon and thoron
daughters [C 16]. Asbestos fibres are also often a significant component of occupational and home environments which may include ionizing radiation.
7. High levels of mutagenic chemicals have beenreported in many types of food [S32]. Broiled meat andfish contain mutagcnic compounds arising from thepyrolysis of proteins and aminoacids. Mutagens andco-mutagens have also been reported in derivatives ofvegetable foods, such as caffeine. As mutagenicity often
correlates well with carcinogenicity, the above
substances may be considered potential carcinogens,both alone or in combination with radiation. According
to some estimates [H2] up to 20-50% of spontaneously
occurring human tumours can be attributed to diet.
Some pharmaceutical substances are also known for
their carcinogenic potential: depending on their use
and diffusion they could also be considered as candidates for combined actions.
8. Among biological agents, viruses may be regarded
as environmental factors likely to interact withradiation. It is well known that some viruses have an
important role in the aetiology of some radiationinduced animal tumours as specific agents. There is apossibility that specific agents of a similar nature may
be involved in the induction of tumours in the humanspecies and non-specific associations or combined
actions, even though on a purely speculative basis, may
be visualized. Natural hormones could also he viewed
as a special case of interaction in view of the wellknown dependence on the hormone level of someforms of radiation-induced tumours in experimental
animals.
9. There are two ways of carrying out an analysis ofcombined actions. The first is to search for any possible
effect. whatever its practical significance or quantitativevalue might be. The second is to concentrate on thoseeffects that may be of importance for the assessment ofrisk in man. The first approach is that to be followed in
the present preliminary analysis. The present practicein radiation protection is that of assuming sensitivity
values across the population which apply to all groups,
e.g .. to males and females of all ages. This practice does
not deny the existence of real changes in the susceptibility between various classes of people. but recognizes
the convenience that for practical purposes a single
average value of the risk is desirable and sufficient.
10. In acknowledging the merits of this approach, theCommittee wishes to emphasize that unless the effects
to be validated as synergistic or antagonistic are
extremely important (i.e., unless they might lead tochanges of at least an order of magnitude in the risk
estimates) and unless they also applied to substantial
fractions of the population at large, they presumably
may not be of relevance in assessing risk estimates inman. The above consideration applies to the estimationof risks for radiation protection purposes. It does notcontradict the fact that if some synergistic or antagonistic effects can be identified under specific exposureconditions of occupational or medical relevance.
appropriate actions should be taken to change suchconditions. Under such circumstances, however, theproblem would not be any longer one of radiation
protection philosophy, but rather one of practicaloccupational medicine. It would not involve basic
changes in the approach to such matters but specificremedial local actions.
11. Radiation effects with particular regard to carcinogenic and to genetic and developmental consequencesof irradiation were considered by the Committee in its
1977 report [Ul]. Non-stochastic effects of whole- or
partial-body irradiation (Annexes K and J, respectively) and genetic effects (Annex I) are also discussed
in this report. When reviewing such a broad field asthat of combined actions. no effects should be excluded
from consideration at whatever level (subcellular.
cellular, tissue, organ. whole-body) they may be
manifested. This is particularly true in view of the heterogeneity of the data available and of the fact that
understanding of combined effects will eventuallyrequire knowledge of the mechanisms involved. That is
why effects other than those mentioned above will be
discussed in this Annex. However. the main emphasis
will be on stochastic effects. Where possible, epidemiological data will be considered, even though studies ofthis sort are rare and often statistically inconclusive.

12. Each of the possible interacting agents may act
alone in producing biological effects or may only be
active in conjunction with other factors, particularly
radiation. Exposure to any of these agents may beacute, subacute or chronic, within a wide range of doses
and dosages. The pattern of exposure may also play arole. as the contemporaneous action of the various
agents or the order of their sequence and the intervals
between treatments may conceivably affect the qualityor the degree of the effect. Of all possible situations ofcombined actions the Committee chose to particularly
investigate conditions where long-term exposure to lowlevels of the agents on large human populations may
apply, because these conditions may possibly affectradiation risk estimates in man.
13. The combined action of several agents is not a
new problem in medicine. As early as 1928 Loewe [Ll]quantitatively reviewed the approaches to theassessment of the action of combined drugs. So-called"isobolic diagrams" were proposed in this regard. This
Annex will consider this approach in detail, as well asother approaches extensively used by toxicologists [MLT1 ]. Some of these ideas were adapted specifically forthe needs of the Annex and illustrative material has
also been derived and modified for the same purpose.
14. Nomenclature in the analysis of combined actions
was a problem that was recognized very early [Ll]. In
order to simplify the discussion to follow, it is appropriate to provide some clear definitions and terminology.
Two classes of combined effects will be considered. In
the first class, both ionizing radiation and the otheragent (or agents) produce the effect under discussion.
The second class includes the combinations where
ionizing radiation produces an effect whose nature or
amount may be modified by the other agent which byitself is inactive. This classification is only made as aconvenient approximation.
15. For the first class of interaction there are three
types of combined actions. When the end-effect of the
combined action equals the sum of effects of the two

agents acting independently, the resulting situation is
one of "additivity". If additivity does not apply, thenthere are two possibilities. When the effect of the
combined action exceeds the sum of the effects
produced separately by the agents. the situation is oneof "synergism". Finally, when the combined action
results in an effect which is less than expected from thesum of the action of the interacting agents, the situation
is termed "antagonism". The precise meaning of the"sum of effects'' will be expanded further in chapter I.
The notion of summation of effects pre-supposes theexistence of a quantity which may be meaningfully
added.
16. The concept of additivity cannot be extended tothe second class of combinations since radiation is here
the only agent capable of producing an effect. Under
these circumstances the comparison is usually between
doses of radiation producing the same amount of effectin the absence or in the presence of the modifying
agent. If, for a certain degree of effect, the dose ofradiation required is greater in the presence of themodifying agent, the resulting action is termed"protection". Conversely, when the dose of radiation isless for the same degree of effect in the presence of amodifying agent, "sensitization" occurs.
17. The above classification is not an absolute one.
For example, sensitizing substances which have been
assumed to be inactive, may be able to produce someeffect at high exposure levels. Also, if one considers
carcinogenesis as an effect. promoters may be viewed
as a special case of sensitizers and many promoters may
show initiating properties. The low environmental
levels of the interacting agents are mainly those ofinterest in this Annex. At these levels the threshold-type
dose-response curves of the sensitizers and promoters
may render their contribution negligible or zero. A
unified approach to both classes of interaction in termsof interaction coeflicient and more precise quantitative
definitions of the concepts introduced in the above
paragraphs will be developed in chapter I.
18. For exposure of the public the most significant
man-made source of irradiation is for diagnostic
medical purposes where the yearly dose equivalents
may be up to the order of a few millisievert (mSv) (seeAnnex G). Sources of occupational exposure are much
more varied and may range from exposure to radonin mines to x rays generated by electronic appliances.
The yearly occupational exposure according to ICRP
recommendations should not exceed 50 mSv (11].
Average yearly exposures to natural sources ofradiation are between 2 and 3 mSv (see Annex B). The
actual occupational exposure in industry has averagevalues of about 5 mSv (see Annex H). Thus the otherphysical. chemical or biological environmental agentswould combine with ionizing radiation at levels of thelatter of 1-10 mSv per year. These levels are usuallyreferred to as low doses.
19. It is sometimes held that in view of the ubiquitous
nature of background radiation all experimental or
epidemiological studies on the toxicity, carcinogenicity
or mutagenicity of chemicals or other agents areautomatically performed to account for the concomitant radiation risk. All the relevant risk assessments
would therefore be in essence assessments of combined
action [Sl]. This may be too broad a generalization for
the following reasons. Firstly, the actual levels of
exposure to ionizing radiation may be orders ofmagnitude higher than those cited in the preceding729

paragraph, and the levels of the other agents orders ofmagnitude lower than those at which experimental risk
assessments were performed. In view of the nonlinearity of the dose-response relationships for mostchemical agents, extrapolation of the risk assessments
between such widely different situations would be
unwarranted. On the other hand, some chemicals which
arc ineffective in producing detrimental changes whenacting alone, may instead provide a significant modification of the radiation action, as in the case of carcinogenic promoting substances. Animal experiments areusually carried out at levels of exposure to chemical orother agents which are much higher than those found in
the environment, which weakens the basis for extrapolation. Under such conditions of great uncertainty thebest course of action is to reserve any judgement and toinvestigate the facts.
20.
(a)

(b)

(c)

(d)

(c)

In summary, the scope of this Annex is:
To review possible quantitative approaches to theassessment of the combined action of radiationand other environmental conditions. based on theconcepts of additivity. synergism, antagonism,
sensitization and protection;
To explore whether and 10 what extent concepts inother fields of the biological sciences may be
applied to the special case of interaction withradiations, particularly at very low doses of the
combining agents;
To consider experimental results on the combined
action of radiation and other conditions, in order
to elucidate possible mechanisms of action that
may allow generalizations and extrapolations;
To review existing epidemiological data on
subgroups of populations living or working under
the action of radiation and other environmental
toxic agents;
To identify possible areas for useful research in
the field of combined effects.

1.

MODES OF INTERACTION
A.

l

X =

IC (t) dt(1)

0

Obviously, to give exposure some biological meaning,
the concentration of the agent. C(t), should be
expressed at the level of the target biological structure,
but this is often impossible. A useful type of exposure
characterization such as the pharmacological dose (thequantity of the chemical introduced per unit weight ofthe organism) does not provide such information. In
these cases special assumptions concerning the intake.
retention, metabolism and excretion of the agent underinvestigation must be made [L3. W4].
23. Even the relatively simple case of a chemical
acting on a culture of cells in vitro may require special
consideration of the kinetics of the substances involved
and of the different forms of their possible transformation [W4]. One may, for example, consider a scheme
whereby a chemical A is converted into intermediate B
which is in turn transformed into a cell-bound moiety C
leading to the observed effect:

Clearly the concentration of C is the quantity to be usedin equation (1) to express the exposure. lt often
happens. however. that the only information available
is on the chemical A, the most readily measurable
quantity, and this information may not be directly
proportional to the values for C. Thus. there might beapparent absence of effect. in spite of a high concentration of A. on account of absence of moiety C, at leastac the beginning of exposure.

GENERAL APPROACH

21. When examining the concept of combined action
it is useful to start with the definition of a quantity
referred to here as "exposure". X, which may apply to
any environmental agent [L3, L4]. Exposure is theindependent variable in exposure-effect relationships.
Without exposure to the agent there can be no effectover the spontaneous level and with increasing
exposure the effect appears to follow some kind offunctional "exposure-response" relationship. This
generalized concept of exposure is different from thenotion of exposure in radiation physics (see Annex A).
In the case of ionizing radiation the absorbed dose, D.
is used instead of the exposure and "dose-response"
relationships are established to functionally relate theenergy absorbed by the irradiated object with the
response observed. If radiation quality must be taken
into account, the quantity defined as dose equivalent,
H, may be used in place of the generalized concept of
exposure, X.
22. The definition of exposure (or dose), X, is moredifficult in the case of other agents [L3]. Often thisnotion includes the product of some intensive quantity(e.g., energy flux per unit area per unit time) multipliedby an extensive quantity (e.g., the time during which theagent acts on the biological system). It has been
proposed in the case of chemical compounds [El] to730

define exposure as the number of primary chemical
events leading to the final effect, but at present thenature of such events is only known in rare cases andtheir quantification exceptional. The concentration ofan agent may often meaningfully be taken as theintensive quantity, multiplied by time as the extensive
one. The notion of exposure is by definition extensive
and in the case of a chemical substance it could be
represented by the formula

24. Thus. the metabolic activation of chemicals into
active forms is of great interest [M19, S33]. Chemical
carcinogens are known to be subject to complex
processes of enzymatic reactions in vivo. The chemicalcompound introduced into the body may be considered
as a pre-carcinogen which. through various reaction
pathways. will eventually produce proximate andultimate carcinogenic derivatives. From a purely
chemical point of view, one of the important generalizations of the recent years is that the ultimate forms ofchemical carcinogens arc usually electrophilic (i.e ..
electron-deficient)
reactants.
Manyspecialized
examples of such processes are considered in the above
mentioned reviews [M19, S33].
25. In some cases the binding of chemicals with celt
constituents may be monitored by the use of radioactive
labels. Examples of such studies in vitro with two derivatives of nitrosourea were provided in (W4]. Experiments in vivo are also available [El, W9. P8] in whichcorrelations are established between the administereddoses of the compounds, the amount of bound moieties
and the biological effects. These studies help clarify theconcepts of administered versus active doses of thecompounds.
26. If the exposure, X. to a given interacting agent (orto several agents) may be satisfactorily defined, thedefinition of the effect, Y. should be considered. There
are different ways of expressing in quantitative terms
the response of a biological object. Y may be. forexample. the fraction of cells showing Joss of a specificfunction or the fraction of exposed animals affected bya given mutation or carrying a given type of tumour. In
such cases Y describes the probability of induction orthat given effect as a result of the exposure X. In othercases Y may describe the degree of a given effect: forexample, the weight Joss of an exposed animal, themean number of tumours per animal. changes invarious haematological indices. Graded effects may
sometimes be reduced to probabilistic quantities byappropriate analysis, but this is not always the case andit may represent a limitation.

B

0.5

'I
'II
I

27. The simplest functional relationship between
exposure and response, Y = F(X). is the linear one:
Y = Y 0 + kX

(2)

Here the term YO accounts for the effect produced in
the absence of exposure or of any other known cause inan apparently spontaneous fashion. The coefficient k
defines the sensitivity of the biological system to theagent. When the separate action of each agent is
described by equation (2), then the increment ofresponse of the system to each agent may be written as
l:l.Y = Y-Y0 = kX

(3)

If one assumes that the increments of response to one
agent are independent of the presence of the otherinteracting agent, the increment of response for thesimultaneous action will equal the sum of increments.6. Y1 . .6. Y2(4)
This is the situation of additivity.
28. However, the experimental value of .6. Y in case of
a combined action can be higher or lower than the .6. Y
expected from equation (4). If .6. Yobs > .6. Yexp thesituation is defined as synergism. If .6. Y abs < .6. Yexp thesituation is one of antagonism. As a measure of thedeviation of the experimental results from additivity
one may introduce an interaction factor(I)= .6. Yobs/ f:l. Y exp(5)

The value of ro = 1 will correspond to additivity, ro >to synergism and ro < 1 to antagonism.

29. The above concepts may be represented in agraphical form as in Figure I. Here a given level of
response Y* is chosen, which level may be obtained by
the action of each agent separately (X"i or X-2. respectively) or by the combined action of both agents at
variable exposures X 1 or X2. If additivity is operatingand equation (4) is applicable, all points (X1, X2)
producing the level of response Y* must lie on the
middle diagonal line of Figure I. This line is called theisobolic line and the diagram is called isobolic diagram[Ll]. The scale of Figure I is chosen in such a way thatthe co-ordinate value equals 1 for each agent acting
separately, that is. X1/X, = 1 and X2/X2 = 1.

0.1

'

:

'I

:

0.1

0.5

---~----------------: ----------

'
0.9

1.0

X, /Xlso~

Figure I. lsobollc diagram in case of linear additive responseto the action of two agents

30. The isobolic line in Figure I describes an ideal
case of additivity, but all experimental exposureresponse relationships are affected by errors. In realsituations therefore the line of additivity expands to anarea of additivity, such as that covered by the horizontal shading lines in the same figure. If the exposureresponse relationships for the agents acting separately
arc linear. the type of interaction may be defined bysimple graphical procedures. For a given level of effect.
Y*, several levels of exposure to both agents are tested:
if the experimental points (X1. X2) fall into the area ofadditivity. the interaction will be regarded as additive.
If the points fall to the left of the area of additivity, theinteraction will be one of synergism: and, conversely .
one will be dealing with an antagonistic interaction
when the experimental points are found on theright-hand side of the area. In Figure I the experimental
point A would be regarded as confirming synergism,
experimental point B as confirming an antagonistic
interaction.
31. As an example of the application of this analysis,
the experiments of l'vfurthy et al. [M3] on diploid yeast
BZ34 may be of interest. The cells were irradiated by
21opo alpha particles or by 60Co gamma rays separately
or in combination. The end-point studied was reversion
to arginine independence. Linear dose-response
relationships were found for both radiations given
separately with slopes of 25.5 ± 2.6 and 10.9 ± 0.4
reversions per 106 survivors per Gy applying to thealpha and to the gamma radiation, respectively. In thecase of combined simultaneous treatment with bothradiations (25% of the dose was by alpha radiation at
0.5 Gy/min and 75% by gamma radiation at 1.54
Gy/min) the slope of the regression line changed to17.7 ± 0.9 reversions per 106 survivors per Gy. The
results may be interpreted by an isobolic diagram. as in
Figure II. For the level of reversion Y* = 180 rev/106
survivors the dose of 60Co gamma would be 15 Gy andthat of 210Pa alpha 6.4 Gy. The dashed lines parallel tothe isobolic line in Figure ll establish the 95% confidence limits. If one plots the points corresponding to
the same level of reversions for the two agentscombined. one finds the point denoted A which lies
clearly to the left of the area of additivity. 1t is
concluded that synergistic interaction of the two agents
applies in this case. This is an example of isobolic
diagram analysis in its most simple form.
731

lthe response to Bo will be much greater than in the caseof heteroaddition. This is the reason why the isoboliclines of iso- and hctero-addition are so different in
Figure lllb.

)(

34. The area between two isobolic lines may be called
the envelope of' additivity [S3]. As a result or different
sequencing or the agents this envelope may reduce to aline [R7], as shown in Figure I I le. This occurs when oneof the two interacting agents produces an exponential
response. Other examples ( Figure l l I d. e, f. g, h, k)show how the f'orm of the response curves and theirrelative curvature define the form of the envelopes ofadditivity and the innuence of a different sequence of
the agents.

~--------,--------=
0.5

10

---·

x, • ??Gy l· RADIATION

Figure II. lsobollc diagram for reversion of yeast to
prototrophy (Y == 180 rev/10 4 survivors) under theaction of alpha radiation from polonlum-210 andgamma rays from cobalt-SO [M3)32. In the above example the mutation frequencies
could be meaningfully added because their increase
with dose was linear. The same procedure is not applicable when the effects change as exponential or sigmoid
functions of the dose, unless the dose-response relationships may be converted to linear or quasi-linear
functions.

35. The above considerations may be generalized to
any type of exposure-response relationship. Since any apriori judgement about the type of addition (iso- or
hetero-addition) is impossible, both possibilities shouldbe accounted for. The practical usefulness of theenvelope of additivity lies in the fact that if the experimental points fall within the envelope. additivity is to
be expected. When they fall to the left (point A in
Figure IV) synergism is operating; and. conversely.

C

1

*

33. The process of additon itself may be performed in
two ways. The first. takes the response to the dose A
from the survival curve A and adds it arithmetically tothe response to dose B from survival curve B. Bothdoses are counted from the origin of the co-ordinates.
Loewe [Ll] designates this type of addition as heteroaddition. A second process of addition. called isoaddition,is also possible. Let us assume that agent A is applied
before agent B (Figure Ill b, e. h).Figure Illa shows the
LI

b

,,~

(••-t

"ol

x,
....

DOSE

(b}

•••

(e}

DOSE OF B

Figure Ill. Examples of hetero- and lso-addl·
tlon for agents A and B In case of different
dose-effect curves and different order oftreatment by the agents [R7]case when the dose A 0 is given before B0 • In the case ofheteroaddition the dose B0 would be counted from theorigin of the co-ordinates. 1n the case of isoaddition. on
the contrary, the !alter dose would be counted frompoint O'. corresponding to the survival level on curve B
to which the biological system is brought by the actionof agent A. It is easily appreciated that for isoaddition
732

Figure IV. Envelope of additivity and areas ol
synergism and antagonism

antagonism will be operating if they fall to the right(point B in Figure IV). Enlargement of the envelopedue to experimental errors is also shown in the same
Figure IV. Attention should be drawn to the fact that
although in principle the area of antagonism extends
from the upper right-hand border of the additivity
envelope to infinity. the straight dashed lines in the
figure define the area beyond which the administrationof one agent requires application of the other at levels
greater than its single exposure level for the same effect.
Point C in Figure IV lies in such an area where
exposure X1 requires an exposure X2 greater than unity.
36. Discussion has so far been limited to the class ofinteraction where both agents may produce the effectunder study. A large number of agents are however
known in radiation biology which may modify theradiation response of the system without being
themselves active in determining the effect. These
modifying agents are called radioprotectors or radiosensitizers, without regard to their mechanism of action[MlO]. The same approach as that used in the preceding
paragraphs for the assessment of the interaction typemay also be generalized to the modifiers. However,since only radiation dose-response relationships are
considered here, a specific approach to sensitizationand protection may be developed.

OAB is the response in the absence of the modifier andthe line OCD the response in the presence of a sensitizer. In this particular case

37. Oxygen is one of the most important modifying
agents [D1]. Its action is extremely general at all levelsof biological organization in the sense that macromolecular, cellular and tissue systems irradiated underoxygen show an enhanced effect compared to that
resulting from the same dose delivered under anoxia.
This enhanced effect is often expressed as an oxygen
enhancement ratio (OER) defined as

co= Yc/Y /\OER = D(non-oxygenated/D(oxygcnalcd)(6)expressing the ratio of doses D under anoxia and underoxygen to obtain a given level of effect. Other similar
quantities may be used for the description of the effectof different modifiers. For example. the thermalenhancement ratio (TER) in the case of the combined
action of radiation and heat, is:
TER = D(standard tcmpcrature/D(enhanced temperature) (7)( I I)

and(12)
However. as the ratio Y c/Y /\ is equal to Xn/XA bothdefinitions coincide. In this special case of linearity thevalues of w and of DMF will be independent of thelevel of exposure, because the straight line is fully
defined by only one parameter (the slope at O exposureor the response at any specific exposure). In geometrical terms, the ratio Yo/Ye in Figure Vis the same as
Ye/YA.
39. The circumstances differ of course in cases of nonlinear exposure-response relationships that would mostprobably apply to the vast majority of the situations inpractice. Figure VI illustrates one such case where theyor the dose reduction factor (DRF) for radioprotectors
DRF = D(protcctor/D(no protector)(8)
Modified response

For radiosensitizers, the factor in common use 1s thedose modifying factor (DMF)
DMF = Deno sensitizer/D(sensiLizcr)(9)This quantity defined for a particular level of response
is often referred to as enhancement ratio (ER) or sensitizer enhancement ratio (SER) or dose modifying ratio(D~1R).
0

38. Also for modifying agents one may define theincrement of effect in the presence of radiation alone,/1 Y. and the increment in the presence of the modifier/1 Y M· The concept of an interaction factor may also be
introduced, as follows( I 0)
When linear relarionships apply both in the absence
and in the presence of the modifier, the value of theinteraction factor ro will coincide with the value of thedose modifying factor (DMF). In Figure V the line

x.,

x,,

X

FigureVI.
Non-linear
exposureresponse relationships In the absence(OAB} and in the presence (OCD} of amodifying agent

ratio Yc/Y /\ is not any longer equal to. but is actuallymuch smaller than the ratio Xe/XA. At exposure level
Xn the definition of an enhancement rario ismeaningless because the line through point D parallelto the abscissa will never cross the other response curve
OAB, but an interaction factor for a modified response
as defined in equation (10) may still be applied.
However, the rnlue of ro will depend on the level of
exposure or response.
40. The situation is further complicated when theapplication of a modifier significantly changes thegeneral form of the dose-response relationship. In suchcases the use of ro. that is the use of an enhancement
ratio in terms of increment of effects, may not be applicable. The solution requires specifically defining asuitable quantitative measure of the modifying effectunder the conditions applying to the experimental
situation.

Figure V. Linear exposure-response relationships
In the absence (OAB) and In the presence (OCD)of a sensitizing agent

41. The concepts and approaches outlined so far arequite sufficient for a discussion of the available scientific literature on the interaction of different agents withradiation. When possible, in the text to follow theconcepts of interaction factor and envelope of
additivity on isobolic diagrams will be applied.
However, further refinements and generalizations of
the concepts outlined may be of some value, as in the733

two following sections. These sections may however beomitted without significant detriment to the understanding of the experimental material reviewed in thechapters to follow.

horizonl.al planes at levels of response Y = 0.5 and Y
1.0 still transect this surface by straight lines. so that

B. SURFACE OF RESPONSE
AND ISOBOLIC DIAGRAMS

42. The methodology of assessment of effects incombined exposures outlined by Loewe [Ll, L5) allows
a much broader approach to the problem. If. for thesake of clarity. one assumes only two interacting agents,
the response to agent I is given by the function F1(X 1)
and that to agent 2 by the function F2(X2). The simultaneous action of the two agents will result in some newfunction F(X1.X2). The functions F1(X1) and F2(X2)describe the response on a plane; the new function
F(X 1,X2) describes the response in a three-dimensional
space. This new function is called the surface of
response. It may be used for any number of agents, andin these cases it will be described in multi-dimensional
space. The concept of a surface of response makes theapproach to the assessment of interaction geometrically clear. In this case the comparison is drawn
between the surface obtained as a result of addition of
responses to single agents (surface of additivity) and thesurface of response for the function F(X1,X2).

x,
Figure VIII. Surface of response In case of additivity
for curvlllnear functions

again the isobolic diagrams are of the same type as in
Figure I. According to this graphical representation,synergistic interaction is expressed by a deviation fromthe surface of additive response nearer to the OY axis.
The new synergistic surface of response is presented in
Figure IX and it resembles an inflated sail. The cross-

43. Let the functions F1{X1) and F2(X2) be linear witha simple law of addition operating for simultaneous
action. Then one obtains the surface of response (andthe surface of additivity at the same time) as theinclined plane in Figure YI I. Cross-sections of thisFigure IX. Synergistic surface of response

section of this surface by a horizontal plane at the levelof effect Y = 0.5, for example. produces a curve whichis the isobolic diagram of a synergistic interaction. The
case of antagonism is exemplified in Figure X, wherex,
Figure VII. Surface of response in case of
linearity and additivity for the combined
action of two agents

Figure X. Antagonistic surface ol response

plane at different levels of response (in Figure VJ I at Y
= 0.5 and at Y = 1.0) will always produce straightlines which are isobolic lines in the sense of Figure I. By
choosing the scales of the coordinate along the X1 andX2 axes it is possible to adjust the angle of the crosssection line with the coordinate plane so as to make itequal to 45°.

the antagonistic surface of response is further removed
from the OY axis. in the form of a sagging sail. Transection of this surface by an ordinate plane (Y = 0.5)
results in a curve with a concavity towards the origin of
the co-ordinates, i.e., a curvature in the opposite
direction than that of the synergistic action.

44. The linearity of the functions F1(X1) and F2(X2) is
however by no means a necessary condition for
obtaining linear isobolic diagrams. The case of Sshaped functions is considered in Figure VII I. The twofunctions are represented by the curves in the co-ordinate planes YOX1 and YOX2. The surface of additivity(i.e., the dotted surface in Figure VIII) has now also achanging curvature similar to that of a tense sail, but

45. The above interactions may be represented by theisobolic diagrams of Figure XI, where the isobolic lines
are the cross sections by a horizontal plane at the levelof effect Y = 0.5 of the three surfaces of response in
Figures VIII. IX and X. Such comparisons can be madeat any level of effect, but in the case of agents present in
the environment the levels would generally be low. It istherefore of interest to examine the shape of the734Synergism

x2

Additisity
y

y

l

i
Antagonism

x,
Figure XI. lsobollc diagrams obtained as the
cross sections of surfaces of response In
Figures VIII, IX, X at thelevel Y0.5

x,=surfaces of response around the origin of the co-ordinate
axes. It is not uncommon that the form of the surfaces
might go from a synergistic type to an additive type in
the region of low effects. At different levels of effect theinteraction might even change from the synergistic to
the antagonistic type, or vice versa.
46. Changing situations of this sort are illustrated in
Figure XII, where the form of the surface of response is

Figure XII. Cross sections of thesurface of response by 45° verticalplanesmade clearer through other types of cross sections. In
the case shown the planes transacting the surface are
diagonal, parallel to the Y axis and with an angle of 45°
with respect to the X1 and X2 co-ordinate axes. The areasof the planes transacted by the surface and covered by
the dashed lines show the extent of the difference
between the real surface and an ideal surface of regular
additivity. The plane nearest to the origin shows astrong antagonistic interaction, but the further thetransacting plane is removed from the origin, the lessimportant becomes the antagonism; until. at very highlevels of exposure, the interaction becomes synergistic.
Cross sections of this type can also help in assessing themode of interaction and are called "interaction
diagrams··.
47. In Figure XII the levels of exposure are limited by
the vertical planes chosen. but the levels of response
may change. Such changes of response which dependon the relative contribution ofX1 and X2 form the interaction diagram as shown in Figure XIII. The lower
curve in Figure XIII shows the line of interaction(antagonistic interaction) resulting from the transactionof the surface of response by the vertical plane nearest
to the origin of the co-ordinates in Figure XII. The casex,0

Figure XIII. Interaction diagrams In casesof additivity, synergism and antagonism

of additivity is represented by the line parallel to the
exposure axis, while the case of synergism is describedby the curve with upper convexity. Again, the line ofadditivity divides the space into two portions: an upper
one, where interaction is synergistic and a lower one
with an inhibitive type of effect. In essence, this methodof analysis relies on the comparison between two
surfaces: the actual surface of response and the surface
corresponding to the presumed additivity of the effectsof the two agents given separately. If the real surface of
response is higher than additive, there is synergism.
Antagonism would operate in the opposite case.
48. Construction of the surface of additivity is aprerequisite for all comparisons. It has however been
discussed already that the addition of responses incomplex biological systems represents a problem in
itself because the results of iso- or hetero-addition
depend on the sequence of the interacting agents.
Adding to these uncertainties the experimental errors,
turn the surface of additivity into a shell of additivity.
corresponding to the envelope of additivity of the bidimensional representations. Although the actual
comparisons should always be performed in relation toa shell of additivity, it is often more convenient to use
the two-dimensional representations under the form of
isobolic diagrams (or interaction diagrams). Elaborate
methods of analysis have been developed for the interaction of several agents [C21].
49. When all the above assumptions have been dealt
with. the comparison of the experimental data with theideal case of additivity is straightforward conceptuallyand technically simple. For a given combination of
exposures (X1. X2) the interaction factor ro may be
calculated as the ratio of the actual ordinate of responseto the ordinate of the additivity surface in the point (X 1 ,Xz). To this end interaction diagrams are particularly
convenient. In Figure XIII the interaction factor ro forthe combination X1, X2 as in point D will be equal to
the ratio AD/BD in the case of synergism and to theratio CD/BD in the case of antagonism. When iso- or
hetero-addition give different outcomes, they should be
used instead of the segment BD, and the upper andlower values of co will be obtained, rou and w1. respectively
C.

PROBABILISTIC ASSESSMENT OF THE
INTERACTION

50. As any biological end-point which is expressed ata sufficiently high level of complexity may be viewed as
the final outcome of a long chain of intercorrelated735

events, it appears quite natural and wholly justified toexpress any end-effect by the probability. P. that it may
occur. The dependence or this probability on theabsorbed radiation dose may sometimes be one of
direct proportionality but in most instances. and particularly for the most complex effects. the relationshipmay be more complex. Essentially the same can be said
about the effects of other physical and chemical agents.
51. In probabilistic terms, if two agents act simultaneously on a biological system. one possible assumption isthat the two agents act independently, which allows
comparison of the results of the joint actions with thepresumed outcome of the two agents acting independently. In some respects. this notion is similar to that ofhcteroaddition. with the difference that the probabilistic approach is conceptually much broader. The
discussion to follow will again be limited to the simple
case of two agents acting simultaneously but may inprinciple be extended to any number of agents.
52. The Committee agrees that the most important
effects of radiation in man are carcinogenesis andmutagenesis, effects that are described by the ICRP [11]as stochastic in the sense that their probability of occurrence increases linearly with dose and without
threshold; their severity is independent of dose; and no
causal relationship with radiation exposure can empirically be established for any given case. It is well known
that these effects do occur even in the absence ofartificial irradiation withfrequency which is muchhigher than would be expected if they were induced
only by natural background radiation. For the purposeof the present analysis. they may thus be viewed as
stochastic derangements of physiological processes towhich a probability of occurrence Po could be attributed.

a

53. If one assumes that the exposure X 1 to an agent
causes a probability P1 of a given effect. t, the overall
probability that this same effect can be observed. taking
into account the spontaneous level Po and assumingthat Po and P1 are independent is:(13)
Since in biological experiments control and test groups
arc run concurrently. the following formulae may also
be convenient:

Po1 = Pll - Po

Pet may again be calculated on the hypothesis ofindependent action:

Pc,= Pn + P1 + P2 -PoP1 - PoP2 - P1P2 - P11P1P2(19)

The absolute and relative increases in probability ofobserving the effect as a result of the joint action of thetwo agents will accordingly be:

(20)
and

(21)
55. When an experiment is performed on the
combined action of two agents, the observed total
probability of effect. P01 , will in general be different
from the expected probability Pet- One of the possible
definitions of the interaction factor ro might be simply
the ratio between the actual and the expected probabilities. Po1/Pe1- It is easy to see, however. that if this definition is adopted the value of the ratio will depend
critically on the absolute value of Po. When the effectunder study has a high spontaneous level of occurrence.
the interaction factor co may be about 1 despite theobserved absolute deviation between the experimental
values and the expected value based on the hypothesisof independence. On this ground. another definition of
the interaction factor. m. is preferred. as follows

(22)
where

LlPobs = P01 -

(23)

Po

The two definitions of the interaction factor will
naturally coincide if Po = 0. The probabilistic definition of w in equation (22) coincides in essence with thedefinition of interaction factor in equation (5).
56. The denominator of equation (22) is calculated on
the basis of the independence of action of the two
agents (equation (19)). Equation (20) may be rewritten
by using equations (14) and (17):

(14)
(15)

They show the absolute and relative increase of theprobability to observe the given end-effect following
exposure X1. A similar set of equations can also be
written for a hypothetical second agent:

Accordingly, the equation for the interaction factor willassume the following form(U

I ol + Po2 J(

2)

Pc,J P0
1 - Po---(25)

If Po1 and Po2 are small. the above equation reduces to

(16)

(17)

= ( P01 - Po) /(!)=(Po, -Po)/(Po1 + Po2)

(26)

or. changing the probabilities to the corresponding
ratios for Po
0, according to equations (15) and (18)

*

(18)

w = (Rohs 54. When the action of the two agents is combined.
the expected probability of observing the overall effect736

1)/(R, + R2 -

2)

(27)

where
(28)

57. To give an example of such a type of treatment,
the experiment on diploid yeast irradiated with alphaand gamma radiation lM3] and analysed by the methodof the isobolic diagram (see section I.A.), may now berecalculated to obtain the interaction factor. For a doseof 9 Gy of mixed irradiation (25% alpha and 75%
gamma) the level of reversion will be 180 per J06
survivors, corresponding to a P01 = 18 10-s. The
spontaneous level of reversion Po = 2 10-s. If one
knows the alpha and gamma doses and the slopes of
their regression lines, one may calculate Pai and Pa2 tobe 5.7 10-s and 7.4 10-s. respectively. The last term inequation (25) is negligible and one may use equation
(26)

O)

=---pol + Po2

18 10- 5 - 2 10-s

5.7 10- 5 + 7.410-5

1.22

(29)

The fact that ro is greater than unity suggests a synergistic interaction.

58. The question arises of establishing errors andlimits of confidence for the interaction factor. The
theory of error transfer may be applied to this end. The
value of ro as defined in equation (22) may beconsidered as the ratio of two stochastic quantities
flPobs and flPcxp• The mean value of this ratio is

60. To illustrate further. the confidence limits for theexperiment on yeast considered above [M3] inparagraphs 31 and 57 may now be calculated. In theexample b.Pobs = 16 J_Q_-5 and ~Pcxp = 13 10-1;
s(b.Pobs) = 0.8 !0- 5 : S(flPcxp) = 0.9 10-S. In case of asynergistic interaction there cannot be a negative correlation between b.Pabs and 6.Pcxp; equation (32) may
therefore be used without the third term within brackets
as an upper estimate of S2(w). Fitting the above values
to the equation, S2(w) = 0.01 and S(m) = ±0.1. The
estimated value is therefore= 1.22 ±0.10 and, for
95% confidence limits, W1 = 1.02 and rou = 1.42.

ro61. For complex biological systems a possible
situation of isoaddition should be kept in mind. In
probabilistic terms this means that if the action of agent
1 takes the system to probability level Pk then P02 will
depend not only on the level of exposure X2 but also on
the value of Pt,1. Therefore P0 2 becomes a conditional
probability P0 2(X2/Pt,1). The same is true for a reversedorder of application of the agents. In this situationdifferent conditional probabilities depending on thesequence of the agents and on the levels of exposure
should be used in equation (24). This will give finallyupper and lower limits for flPexp· Corresponding upperand lower limits for the interaction factor co may be
calculated as(34)-

6. Pobs(J) ==-Ll Pexp

(30)

and

The error matrix for KP0 b, and !:,, P0 ,p will be
2_ S (flPobs)
S(D..Pab;)S(ilPexp)q12][ S{b.Pobs)S(6.Pexp)q12
S (6.Pcxp)(35)( 3 l)

where S is a symbol representing the mean quadraticerror and q12 is the correlation coefficient. The mean
quadratic error of will be in this casero.!.f_the distribution of l:,,Pobs and b.Pexp is no~al and
flPexp/S(D..Pexp) > 5. then the distribution of ro should
also be approximately Gaussian.
59. The same problem of assessing an error to m wasconsidered by Rothman [R 1] for the case of epidemiological investigations and he used the same definitionof the interaction factor. If a log-Gaussian sampling
distribution is assumed, an estimator of ro referring to alarge sample interval may be written as

cD1 = exp [In ro Wu = exp [In w

ku S (In ro )]+ ka S (In w )](33)

where ka is the abscissa value for a given level of significance a. The methods for the calculation of S(ln w) for
cohort studies and case-control studies have also been
given in [Rl].

These limits will be further extended by the presence ofexperimental errors.

D.

THEORY AND PRACTICE

62. Before proceeding further to the analysis of someexperimental and epidemiological data. it is necessary
to discuss briefly the applicability of the concepts
reviewed in the preceding section to situations
involving complex biological effects. In doing so, it will
immediately be realized that even problems which may
appear of minor and mostly speculative importance inthe analysis of the action of a single agent, are likely to
become very difficult to disentangle when various
agents are combined, giving rise to much uncertainty inthe assessment of the type of interaction that might
apply.
63. The definition of an effect is hardly ever a
problem in radiation biology. The conditions underwhich the effect is manifested, its degree of expression
or its probability of occurrence may usually bedescribed with sufficient precision. What may be lesseasy to define is the dose-effect relationship at all levelsof exposure. particularly at the low ones. When two
agents arc combined. depending on the form of therespective exposure-response relationships. more or lesseffect might be obtained at a given exposure regime
than might be predicted on fragmentary knowledge ofthe relevant relationships. This points to the need toestablish with sufficient precision through appropriate
controls not only the response to be expected at the
exposure levels of interest for the particular experiment,
but to obtain a full dose-response curve for both agents
under study. The ultimate aim is to establish experimentally the surface of response corresponding co the full737

range of both agents. However. if the number of experimental points to establish a given exposure response
curve with one agent is N, to establish the whole surfaceof response with the same number of experimental
points in a given sequence of administration requires
N2 experimental points. Reversing the order of administration will in turn double the number of experimental
points to 2N2. Such an increase in the size of the experiments is often not feasible and complete series of thetype envisaged are almost never reported or conducted.
64. The definition of the level at which a supposedsynergistic or inhibiting action may take place isextremely important in the analysis of such actions.
Here the need for operational definitions of practicalsignificance and the need of resolving mechanisms inbiological experiments may often be at variance or evenincompatible. If. for example, one takes a very complex
biological short-term end-point. such as the death of ananimal (but the loss of reproductive integrity of a cell
may be sufficiently complex, depending on the level atwhich the mechanisms of action may be resolved).
exposure to any toxic agent in sufficient amounts could
produce such an effect. This of course will happen attimes and with mechanisms differing from one agent toanother and mostly specific to each agent. The
combined application of two agents may in principle
produce apparently antagonistic or synergistic effects
when some of the pathways of action of the two agents
happen to interfere with each other. But at this level ofcomplexity. even though the end-point might be ofpractical significance, the real existence of combined
actions may be difficult to assess. Only when themechanisms of action of the two agenrs are reasonably
well defined will there be any merit in making use of
the concepts of synergism or antagonism, in order toavoid misuse of the terms. Within this framework it
may also be discussed how the presence of one may
enhance the detectability of another interacting agent.
when both produce the same effect.
65. Confusion of iso- with hetero-addition could
result in the false identification of synergism. For
example. one could visualize two agents. both toxic tothe bone marrow and both inducing leukopaenia with avery curvilinear relationship to exposure. as is usually
the case. It is easy to imagine that the action of the
combined treatments might produce more effect than
expected by the same doses of the two agents separately, simply because of the curvilinearity of therelationships and of the isoadditive character of the
combined effect. It is also easy to understand that deathof the animals might ensue at levels of the combined
agents which are much below those of the two agentsacting separately. If leukopaenia and death were theend-points of reference. in the absence of any otherinformation one might be tempted to think of a synergistic action. Yet, to call such an effect synergistic
would be unjustified because isoaddition would be
operating in this case. Clearly. without knowledge of
the whole range of responses, it would be impossible toclarify the issue. It should be realized that too often thecases of synergism claimed in the literature have bee.n
insufficiently analysed in this respect and there 1s
ground to doubt that they might stand up to morerefined investigations.
66. As to long-term effects, it is usually thought thattumour induction is a sufficiently well-defined phenomenon to be taken as an end-point, as though all
tumours have the same aetiology and pathogenesis and738

there are not great variations in the incidence of various
tumours between species, strains and experiments. This
assumption is imprecise when different doses of thesame agenr are administered, because expressing the
response as overall tumour induction may mask
important effects on some tumour classes. The
assumption is however particularly dangerous instudies of combined actions because under these conditions changes in the tumour spectrum would certainly
be expected. It is essential therefore that the end-pointof the studies be specific and extremely well defined.
The same reasoning applies to the genetic and developmental effects.
67. Changes in the state of the biological system may
be brought about by sequential treatment. For example,
a large body of evidence on mammalian cells indicatesthat dose fractionation in radiobiology is a difficult
subject to investigate. Usually the first dose produces
partial synchrony of the irradiated population, so thatthe response of the surviving cells to the second dose
fraction is altered with respect to that of a non-exposed
undisturbed population. It would be very easy but
totally unjustified to think of antagonistic or synergistic
effects in the absence of information on the survival
curve of the overall population and of its constituent
sub-populations and in the absence of data on theamount and time sequence of synchrony induced by the
first treatment. There is every reason to believe thatsuch cases may occur also in respect co other chemicaltreatments and it should in fact be pointed out thattreatments with chemicals (BUdR, hydroxyurea, forexample) are orten used to obtain experimentallysynchronized cell populations. The amount of information available in respect to such effects by thevarious agents discussed in the following parts of thisAnnex is lacking or extremely limited. Efforts to clarify
the situations occurring in practice through experimental analysis might help to avoid misconceptions.
68. Another point calling for great caution concerns
the time parameters of the combined action. Two typesof treatment may be visualized. contemporaneous andsequential. Partial overlapping and fractionation of the
exposure to each of the agents could increase thecomplexity of the temporal patterns of exposure.
Contemporaneity of the treatment time does not necessarily imply a simultaneous action at the level of thetarget structures. For example, in the case of chemicalor pharmacological substances, variable time formetabolic processing of the agents might be required
and it would temporally displace the action on the
biological structures of interest. If hetero-addition isassumed to operate, administration of one agent before
another, or vice versa, should not in principle lead to achange in the end-result. But. on the other hand. if
reversing the order of administration does produce achange (qualitative or quantitative) of the response. theconclusion should not necessarily be drawn that someinteraction differing from additivity applies. This all
points to the relativity of the definitions and to the difficulties of translating into sensible biological terms theprecise statements of the theory.
69. There are biological effects for which the timing
and the sequence of the actions is all-important.
According to one hypothesis, for example, tumour
induction may be regarded as the result of two
independent phenomena, initiation and promotion.
Initiation is visualized as a fast irreversible processacting on normal cells and conferring upon them thecharacter of neoplastic ones. It precedes promotion but
without the latter could not result in a growing tumour.
Promotion, which on the other hand is ineffective if notpreceded by initiation, takes place during fairly longtimes and may be reversible. Many agents share theproperties of initiators and promoters in different
degrees at different doses. Thus, reversing the order oraltering the time pattern of administration of two
carcinogenic agents is bound to produce changes in thequalitative or quantitative expression of their final
action. This should be kept in mind when designing
experiments on combined action.
70. The issues discussed in the preceding paragraphare further complicated by the fact that the time fortumour appearance is important, as is the final tumour
yield. The rate of appearance of tumours in time (oncethis rate is referred to a given tumour type and iscorrected for competing risks) is an importantparameter since. in principle, it is related to thepromotive action of a treatment; while the final tumour
incidence is related to the initiation action. When
agents possessing both properties are administered in
combined experiments the precise nature of the interaction and its influence on the combined end-point
would not normally be resolved without detailed information of the mechanisms involved.
71. The decisive importance of the temporal patternof exposure to ionizing radiation vis-a-vis practically all
biological end-points is documented for a variety of
biological effects in the specialized sections of theprevious report (see Annexes H, I, J of [Ul]) and in
Annexes I, J and K of the present report. In general.
fractionation or protraction of the exposure lead to adecrease of the final effect, although in some cases
deviations from this general pattern are reported [H 19).
It is not unreasonable to expect that changes in theyield of effect may also occur by altering the pattern of
exposure to other agents interplaying with radiation. sothat the final effect of the combined treatment cannot
be predicted, particularly in the region of the low doses
which are of major concern in the present context.
Precise information about the temporal distribution of
the exposure is therefore required in evaluating the
combined effects.
72. In conclusion. the notions of synergism. additivity
and antagonism which may be defined in theory andevaluated by appropriate statistical analyses. are seen to
lose some of their clarity when confronted with thecomplexity of biological organization and the variability of experimental conditions. They may. on theother hand, acquire important practical connotations.
Normally the assessment of combined actions requires
clear understanding of the nature of the biologicaleffect under study; precise knowledge of the pattern of
its manifestation in time for the combining agents;
reasonable definition of the exposure-effect relationships for each of the interacting agents, particularly
when effects must be analysed over a range of
exposures; control experiments to check for the applicability of the effect to different conditions of exposure.
Without the detailed information described above, such
notions will probably remain confined to the realm of
theory and the subject of disbelief or overestimation, as
the case might be. Only studies of mechanisms might
eventually solve these uncertainties_II.
A.

PHYSICAL AGENTSCOMRINATIONS OF VARIOUS TYPESOF IONIZING RADIATION

73. The simplest type of interaction, where most of thereservations raised in the preceding sections do not
apply due to the similarity of the underlying
mechanisms, is that between two different types ofionizing radiation. Mixtures of high- and low-LET
radiations have repeatedly been tested for the presenceof synergistic or inhibiting effects due to the combination of two beams. since current understanding ofradiation action is not sufficiently advanced to allow
prediction of possible interactions. In other studies
external irradiation was combined with internal or theeffects of mixtures of radionuclides were tested.
74. Studies on the combined action of fast neutrons.
heavy ions and x rays were stimulated by possible
radiotherapeutical applications [N2, N4. N5, B16, 013,
F5]. Interaction of sublethal reparable lesions producedby neutrons and x rays was shown in experiments
where x-irradiation was delivered at different intervals
after neutrons [N4]. The actual survival curves of cells
in vitro Jay between those to be expected on the basis of
iso- and hetero-addition. Similar experiments withresults in the same direction were performed with neon
ions [N5]. Cells irradiated with ions, incubated for threehours and then exposed to x rays showed a partial
restoration of the shoulder of the survival curve.
However. the results of Durand and Olive [013) are in
disagreement with those reported above because they
showed no recovery after combinations of neutronneutron. x ray-neutron and neutron-x-rays. It should bepointed out that these experiments were not confirmed.
A theoretical description of the interaction of high- andlow-LET radiation based on the theory of dual
radiation action was provided by Zaider and Rossi [Z3].
Within the frame of definitions accepted in this reporttheir interaction would be confined to the envelope of
additivity.
75. Some insight into the nature of the underlying
processes may be provided by studies of repair. Whentested at the tissue level. the rate of recovery fromsublethal damage appeared to be independent of theradiation causing it [H 12]. It was suggested thatrecovery from sublethal damage does apply to the
low-LET component of the damage, whatever theradiation producing this damage [G3, Hl3]. Naturally.
in the case of neutrons which cause relatively morelethal than sublethal damage the final effect will not beclearly determined until one of the two components, thesublethal, has been repaired at sufficiently long
fractionation times [Hl5]. Further evidence shows [Hl4,F3] that tissues treated with neutrons or with x rays tosimilar levels of biological damage and then submitted
to an x ray course appear to be more radiosensitive
when neutrons had been delivered in the conditioning
treatment. Thus. the presence of different components
in the LET spectrum and the presence of different typesof damage to be repaired (sublethal. potentially lethal)
each with characteristic time parameters make thepicture rather complex.
76. New studies on combined radiation treatments
were reported on Chinese hamster cells in culture
irradiated first by neon ions (LET = 180 keV /µm) andsubsequently by 225 kVp x rays. Cell survival was theend-point analysed [N9. NlO. Nl 1]. The results for thethree levels of survival presented in Figure XIV show
739

I?
-

§

!0-

18:

20

Figure XIV. lsobollc diagrams at threelevels of survival for Chinese hamster
V79 cells after Irradiation with neon Ionsfollowed by x rays [N10]that the experimental points fall in the middle of anenvelope of additivity formed by application of heteroaddition (upper curve) and isoaddition (lower curve).
When the order of application is reversed (Figure XV)

hormonal state in the course of acute radiation sickness.
Other studies were also reported on the yield of
mammary tumours in rats following 131I and external
irradiation with x or gamma rays [M22]. For low iodine
exposure (0.04-0.08 kBq/g body weight) an increased
yield of tumours was seen in the combined treatmentgroup; for high iodine exposure the reverse was true.
Other experiments [V4, V5] tested tumorigenesis in rats
with 131J (3.7 kBq/g) and external thyroid irradiation(up to 3 Gy). No significant effects of the combination
were reported. The interaction in these cases is attributed to hormonal disturbances, about which moredata will be provided in chapter IV. Luz et al. [L14]reported on an enhanced osteosarcoma induction inmice by the joint action of two radionuclides, a shortlived alpha-emitter (227Th at 190 kBq/kg) and a beta
emitter (227 Ac al 1.9 kBq/kg). A higher than additive
osteosarcoma incidence was reported at 700 days postexposure, amounting to interaction factors of about 1.7
in terms of final tumour incidence and of about 1.3 interms of the time for 50% tumour appearance, ascompared to the effects of the two doses given individually. The authors attributed the interaction to thestimulation of osteogenic cell division due to the
protracted action of the low level 227Th formed from
221 Ac or to the continuing activation of a virus by thesame cause. It is clear however that without kinetic
studies and accurate physical dosimetry at the level ofthe sensitive bone cells it would be difficult to validate
the phenomenon as belonging to the class of synergistic
effects.
B.

Figure XV. lsobolic diagrams at three levels ofsurvival for Chinese hamster V79 cells after Irradiation with x rays followed byneon Ions [N10]the envelope of additivity is reduced to a line, asituation illustrated earlier in Figure Ill c and e. Under
these circumstances one would conclude for thepresence of synergism. if not for the fact that the actuallevels of survival do not change after the two sequencesof treatment. This apparent paradox is explained by thefact that radiation may be considered to be synergisticwith itself when survival is not exponential with dose.
77. The above examples suggest that in cases where
the sequence of treatments does not change the final
outcome, the isobolic diagrams should be constructedwith the order of treatment that gives the greatest
possible area of the envelope of additivity. As to theinteraction of ions and x rays, this does not appear ingeneral to exceed the isoaddition limits (interaction of xrays with itself) and only in few experiments true
synergism may be suspected. Further work on themechanisms at the cell kinetics and molecular level
could clarify the precise conditions of the interactions.
78. Moskalev et al. [M21], modelling the effects ofnuclear fallout, gave 1311 orally (0.3 kBq/g) to rats andirradiated them at the same time externally with gamma( - 6 Gy) or beta (surface dose - 24 Gy) radiation.
Other animals were only irradiated externally. Lethality
at 90 days was about five times lower in the combined
treatment group. which was attributed to changes in the740

UV AND IONIZING RADIATION

79. The combined action of UV and ionizing
radiation has been examined repeatedly in various
experimental systems of micro-organisms [HlO, Y4].
The experiments of Haynes in E. coli Bir [HlO] may be
examined as a good quantitative example. Pre-irradiation of the bacteria with different UV exposuresincreases the final slope of the x ray survival curve.
Changing the sequence of the agents leads to a disappearance of the synergistic interaction. but only if thecells are irradiated in rich medium. If cells areirradiated in buffer the order of irradiation is not soimportant. This suggests that post-irradiarion events
may affect the interaction mechanisms. Many experimental data point to these events in relation to repair
mechanisms [M24]. It appears that the repair of singlestrand DNA breaks induced by x rays may be inhibitedby prior UV irradiation. For some recent reviews of
DNA repair mechanisms and their genetic control see
references [D10, M23. S34].

SO. Experiments are also available on mammaliancells in culture [H 16]. Synchronized Chinese hamster
cells were irradiated in mid-S phase with fixed doses of
UV and then exposed to graded doses of x rays (case I):
alternatively, fixed doses of x rays were followed bygraded doses of UV (case II). In case I the resultant
survival curve may be obtained by isoaddition showing
that. despite the different nature of the molecularlesions, the damage by UV is fully additive with that ofx rays. The UV survival curves in case 11 are higher
than the theoretical curves obtained by isoaddition. but
lower than those obtained by hetero-addition. The sizeof the shoulder on the combined action curves in case
II is less than that of the pure UV survival curve. Thus.
the damage produced by x-ray pre-irradiation is only
partially additive with the subsequent UV damage. In
mammalian cells, according to these data, the situation

of survival additivity seems to prevail. These data havebeen analysed by others [L28] according to themolecular theory of cell survival.
81. Transformation of mammalian cells in vitro isalso a relevant end-point. Di Paolo and Donovan [Dl 7]
tested the morphological transformation of Syrian
hamster cells with UV and x rays. Irradiation by UV
alone (254 nm) gave a yield of transformanls linearly
increasing with dose. X-irradiation produced no transformation at all. X-irradiation (2.5 Gy) followed by UV( 1.5 J/m2) at 24, 48 or 72 hours resulted in a greatly
increased yield of transformants. An interaction factorof 3. 11 and 2.2 may be calculated at the above timeintervals. showing the interaction to be very timedependent. Increasing the UV dose to 3 J/m2 led to adecrease of the 48-hour interaction factor. thus showing
its dose dependence. The relevant biologicalmechanisms remain unclear owing to the lack of understanding of the phenomenon of transformation.
82. A synergistic interaction of UV and x rays wasfound by Holmberg and Jonasson [H22] for chromosomal aberrations in human lymphocytes. Go cells were
irradiated first by 254 nm UV (5 - 10 J/m2) and then byscalar doses of 260 kVp x rays (1.25-2.0 Gy). UV alone
gave a very small yield of dicentrics; UV followed by xrays doubled the yield of x rays alone. The interval
between the treatments was less than half of a minute.
Reversing the order of administration did not change
the interaction factor of about 2. When phytohaemoagglutinin-stimulated cells entering stage G1 were used in
the same experiments no synergism was observed [H23].
83. Experiments with chronic exposure to UV light ofd.ifferent spectral composition and parallel chronic oracute exposure to ionizing radiation were also reported.
They involved complex biological end-points such as
LDso or life span. Galanin et al. [G2] studied in miceand guinea-pigs the haemopoietic functions and the life
span under conditions of combined chronic irradiation
by UV and gamma rays (dose rate 0.5 Gy/day). The
experiments showed that animals receiving the
combined treatment lived longer and had haematological values closer to normal than controls receiving
only the gamma treatment. Acute damage was also
influenced in the same favourable way by the combination of chronic UV irradiation with lethal andsublethal doses of gamma radiation [L15]. In experiments by Yatzula [Y3) on rats the treatment by UV
preceded or was made concurrently with x-irradiation
or internal irradiation by 32P. Again. a decrease in the
LDso130 was seen after the joint treatment. Animals
under combined irradiation had a better recovery of the
body weight and showed less severe skin reactions.
Physiological adaptation mechanisms could be invoked
to explain such effects.
84. A comparison of the carcinogenic action on rat
skin of UV and ionizing radiation. was made by Burns
and Albert [B11]. The predominant tumor typeobserved following UV irradiation was a keratoacanthoma; after electron irradiation epidermal
tumours were mostly seen. The yield of keratoacanthoma in rats irradiated at four weeks of age bydifferent doses of ionizing radiation up to 30 Gy andthen exposed for different periods to high and low
fluences of UV was not influenced by the ionizing
radiation dose and depended primarily on UV
exposure. Absence of interaction was also seen in thecase of epithelial skin tumours. Only one UV treatment
schedule (high-fluence, 25.2 104 J/m2. from 5 to 16

weeks of age) enhanced the yield of epithelial tumours
for lower doses (5.5 and 11 Gy) but not for higher dosesof electrons (17 Gy). However, some of this increase
was also observed in the zero-dose group and neither ofthese increases was statistically significant at P = 0.05.
The absence of oncogenic interaction between the tworadiations is a particularly good illustration of the factthat there may be a difference in the targets specific to
the two radiations.
85. The examples reviewed in this section illustrate
several important points. They show that the type ofinteraction depends on the biological end-point
studied. on the level of exposure of the agents applied,on the order of their administration. on the stage of cell
cycle, state of growth of the cells, etc. Under thesecircumstances it is not surprising that no general
conclusion about the character of the UV and ionizing
radiation interaction may be drawn.

C.

ELECTROMAGNETIC AND IONIZINGRADIATION
1.

Experimental data

86. Many industrial,.scientific. military and domestic
appliances produce microwaves. electromagnetic
radiation having frequencies of from approximately 10
to 105 MHz. In some cases the same apparatus may
produce very soft x radiation. as well as microwaves; inother instances ionizing radiation from other sources
may be present in an occupational environment
together with microwaves. The assessment of a possiblecombined action of these two agents is very difficult
because exposure parameters for microwaves equivalent to the absorbed dose of ionizing radiation areabsent [823). Even such a simple characteristic as thedensity of energy flux (DEF) is lacking in some experimental work. The quantitative expression and theunderlying mechanisms of effects are far from clear.
Differing views have been expressed on the nature ofthese mechanisms. Some authors consider the effects of
microwaves tp result from the dielectric heating of thetissues [l\113]; others place the main importance on
specific actions of the microwaves. particularly on thecentral nervous system [P9, G4]. A combination of theseviews has also been considered [812).

87. In several early experiments [P18, M14, T3] thechanges induced in the lethal action of r.adiation by
microwaves were studied. The results consistently
showed an antagonistic type of interaction on thelethality induced by ionizing radiation following pretreatment of rats [P18]. dogs [M 14] and mice [T3] withmicrowaves. Later. interaction in the sense of additivity
was reported for the same biological end-point [B13]. In
more recent experiments Davydov et al. [Dl4] studied
the lethality to mice after high exposure rates of microwaves prior to acute gamma irradiation. Curves of theRashevsky type describing the relationship between
mean survival time and radiation dose were reported,and a shift to shorter survival times after combined
treatment was observed. The animals were irradiatedfor 10 consecutive days with microwaves at a frequencyof 2400 MHz with density of energy flux (DEF) 10, 20,
40 and 100 mW/cm2, and exposure times of 40, 20, 10
and 4 minutes, respectively. It is interesting to point out
that the degree of interaction was highest for DEF =100 mW/cm2 and depended rather on the intensive
factor (DEF) than on the extensive one (energy admin741

istered). An approximately linear decrease in theLOso130 with increasing OEF was observed. Extrapolation of this relationship to zero gamma dose would
give a OEF value of about 325 mW/cm2. The authors
argue that at this level of DEF death might be brought
about by microwave irradiation alone. If so. this would
imply additivity of the two agents for a very complex
end-point.
88. The slate of the haemopoietic system of theanimals in the course of the microwave pre-treatment
described above was studied by Tichontchuk [T4] after
31 day irradiation at 100 mW/cm2. Gamma radiation at
4 Gy was given after the last microwave treatment. The
haematological parameters that were followed in thecourse of these experiments included the weight ofspleen and thymus, and the number of cells in the circulating blood. An inhibitory action of the microwave
irradiation alone on the haemopoietic system wasnoted, with a pronounced leukopaenia. The subsequent
gamma treatment added further injury to the bloodforming organs, which could explain the additive typeof interaction observed in (014]. It is however fair topoint out that other data on an inhibitory interaction of
the same two agents have been reported in the literature[L19, F4. R8].
89. Rotkovska and Vacek [R8] exposed mice underconditions similar to those in (014]. Lethality following
low-LET radiation was again the experimental endpoint tested. A definite decrease of lethality wasobserved if the mice were exposed after the x-ray
treatment for five minutes to microwaves (2450 MHz.
1000 mW/cm2). Animals treated with microwaves
showed also an increased number· of haemopoietic
stem-cells surviving and increased values of erythroand myelo-poiesis. Differences of these results fromthose in [D14] could possibly be due to the reverse
order of application of the interacting agents. A more
recent paper by Rotkovska et al. [R14] provided further
details of the therapeutic effect of microwaves on
short-term mouse survival following whole-body xirradiation and attributed the antagonistic effect of thetwo agents to an increased survival of the stem cells inthe bone marrow.
90. For the purpose of the present document mostinteresting are the studies where clearly non-thermal,
down to environmental, levels of microwaves were
tested. Sakovskaya et al. [S25, S26] modelled in the
animal a situation of chronic irradiation by microwaves
and low-energy x rays. Female mice were irradiated in
31 or in 82 sessions. each delivered every second day
and including 20 minutes microwave irradiation at
OEF 2.5 and 5.0 mW/cm2. and x irradiation (effectiveenergy 10 keV) up to doses of 0.15 or 0.3 Gy. The endpoints studied comprised body weight and weight ofseveral organs (i); number of mice producing litters (ii):fertility (iii); weight of the litters at one month of age(iv); fraction of bone-marrow cells carrying chromosomal aberrations (v): lysozyme content of the blood
serum (vi). Control groups and groups irradiated with
only microwaves or x rays were also included in theexperiments.
91. Statistically significant (P < 0.05) changes of thecontrol values were obtained for the end-points (ii),(iii), (v) and (vi). A decrease of the lysozyme content ofblood serum was observed, approximately to the same
degree, for both the experimental groups irradiated742with microwaves alone or with x rays alone. In thegroup receiving the combined irradiation the decreaseof the enzyme was slightly greater, but the corresponding point on an isobolic diagram would fall into
an envelope of additivity. For the chromosomal aberrations again an increased yield was seen in the groups
receiving the separate treatment and again to approximately the same extent. The group under combined
treatment showed a slightly higher incidence of aberrations. but the corresponding experimental points were
not outside the envelope of additivity. Microwaves
alone seemed to show a slight stimulating action on thefertility of the mice and on the fraction of mice
producing litters; but in the combined treatment groupa decrease of both end-points was observed. An
additive type of interaction would appear more likely
to apply in general to these experiments.

2.

Epidemiological evidence

92. The combination of ionizing radiation and highor low-frequency electromagnetic radiation is characteristic for a number of occupational environments inelectronic and radiotechnical plants. Increased ambient
temperature, constant electric or magnetic fields, soundpollution and vibration may also be part of theseenvironments [02]. Wolfovskaya et al. [W3] studied thehealth of female workers employed in assembling,
testing and vacuum pumping of high-voltage electronicequipment. They were exposed to electromagnetic
fields of different frequencies (electric field strength
600-2500 V/m and magnetic field strength 50-320 a/m)
and to x irradiation at dose rates of up to 25 µGy/hour.
The effects studied included frequency of functional
disturbances of the nervous system. blood pressure,dysmenorrhoea, changes in the sedimentation rate oferythrocytes. thrombo- and leuko-cytopenia. It wasclaimed that the important determinant of thesymptoms was x irradiation. However, a highpercentage of disturbances of the nervous system foundamong these workers was attributed to microwave
exposure. The nature of the end-points, their variability
between groups and the lack of any precise dosimetry
and statistical analysis make it difficult to validate suchconclusions.
93. Similar comments may be made with respect toother epidemiological studies on the clinical effects ofcombined exposure to microwaves and ionizing
radiation. A survey was reported of workers testing
microwave generators [B9]. Four groups of people (200subjects in total) were included in the survey: twogroups worked under combined exposure to microwaves and ionizing radiation; one was exposed tomicrowaves only and the last one was the non-exposed
control. A group exposed to gamma radiation alone
was not included in the study. Asthenia and migraine
were characteristic complaints in all exposed groups.
For the first two of them the symptoms were 20-50%
more frequent than for the third group and between 2
and 2.5 times more frequent than in the fourth.
Dysfunctions or· the autonomic nervous system were 2
times more common in the groups with combined
exposure than in the third group and 3-4 times higher
than in controls. The combined action of ionizing
radiation and microwaves was also investigated inworkers by Lysina [Ll 7] but loosely defined conditionsof exposure render any judgement of the type of interaction impossible.

D.

SUBOPTIMAL TEMPERATURE AND
IONIZING RADIATION
1.

High temperature

94. Broad quantitative studies on the effect of heat on
cells and the interaction between heat and ionizing
radiation started in the 1960s, stimulated by thepossible application in the treatment of cancer. Several
conferences and symposia have hy now taken place on
this subject [C17, C18, P12. D20) and good reviews areavailable (Dl 8, F2]. A full discussion of this subject isbeyond the scope of this Annex. which will only briefly
cover the most basic aspects. Heat alone may damage
mamma\ian cells and tissues at temperatures of 42°C
given for a sufficiently long time [F8]. Thermal inactivation curves as a function of the temperature or of thetreatment time at a given temperature may be
produced, having characteristics similar to those of theradiation inactivation curves. There are reasons to
consider that the target for cell killing by heat may beplasma membranes [018] but other targets such aslysosomal membranes or macromolecules cannot be
excluded.
95. Treatment of cell cultures with heat increases their
sensitivity to radiation in the sense that the final slopeof the x ray survival curves becomes steeper after preheating [F8]. Thermal enhancement ratios as defined byequation (7) may be used to quantify the effect and for
different cell lines the values of this ratio seem tocorrelate well with the sensitivity of the cells to heat[R9]. After pre-heating for 1 hour at 42.5°C the TER
may reach values higher than 2 [R9]. These values seemlo increase for irradiation at lov.· dose rates [B24]because recovery from sublethal damage is sharply
reduced by treatment with heat (LI 8]. A delay ofrejoining of strand breaks in DNA [C19] and inhibition
of DNA synthesis, including repair synthesis (S37],
were observed after heat treatment. The targets forenhancement of radiation sensitivity by heat are
different from the targets for simple heat inactivationand include all the repair systems and the chromatin[WlO, D18. 026. S49].
96. The temporal pattern of treatment is very
important for the synergistic interaction of radiationand heal [S35, D19. S36. 03]. Maximum interaction isusually observed with the simultaneous presence of thetwo agents and it declines as the interval between treatments increases. Figure XVI [F2] illustrates the time

Figure XVI. The time course of the decay of heatpotentletlon of x-rey damage In normal tissues for
hyperthermla given at different times before or alter Irradiation. The tissue responses ere normalized to the percentage of the maximum response
which occurs, for each curve, et the shortest time
Intervals. Date from [F2]course of the decay of heat potentiation of x-ray
damage in a variety of normal mammalian tissues for
hyperthermia given either before or after irradiation.

97. Data on the life span of animals irradiated for theduration of their life under conditions of high environmental temperature are reviewed in Annex K. A study
with pre-implantation mouse embryos exposed in vitro
to 39°C immediately after x irradiation (V8] showed agreat increase in the number of micronuclei in thecultured cells, indicating an enhanced chromosomal
damage after combined treatment.
98. For animals, and for mammals in particular, it isdifficult to foresee under what conditions a direct sensitizing action of high temperature in the environment
might come about. since normally heallhy mammals
maintain a fine regulation of their body temperature.
However. Dobrovolsky (011] reported experiments onrats where the effects of chronic irradiation due to thedaily intake of 35S (0.55 MBq/kg), 45Ca (1.3 and 2.8
M Bq/kg) and 32p (0.37 MBq/kg) in the course of a year
were studied in combination with daily exposure fortwo hours to a temperature of 40°C. Survival, bodyweight. fertility (mean number of litters per female),haematological parameters and histology of the ovaries
were the end-points studied. During the first period oftreatment the changes characteristic of a chronic
radiation injury appeared to be aggravated by the
combined treatments. During the second half of thetreatment. however. the combination of irradiation andhigh temperature appeared to increase and accelerate
repair processes. Fertility of the female animals
depended on the mating time. but in general the
combined action group had enhanced fertilitv incomparison with the control groups [DJ 2].
·
99. Epidemiological investigations of combined
actions were made on workers at metallurgical plants
who were exposed to ionizing radiation and also
received periodically high temperature exposure [M12].
Primary functional disturbances of the nervous systemwere seen with higher frequency in this group ascompared to other groups of workers. People having ashorter occupational history were reported to show
vascular dysfunctions attributable to dystonicity of theautonomic nervous system. For longer occupationaltimes asthenia also accompanied the above symptoms
in a higher percentage of workers. Owing to the obvious
difficulties in the quantification of such subjective
symptoms any judgement about this type of interaction
should be reserved.
2.

Low temperature

100. Cold-blooded animals arc good experimental
material for studies of the influence of low temperatureon radiation sensitivity. Much relevant information was
published in a specialized symposium [RlO]. In very
general terms. regeneration of tissues [H24], lethality
after fractionated irradiation [£7] and recovery fromradiation injury in self-renewing tissues [E8. E9] in fish
are considerably inhibited when the animals are kept at
suboptimal temperatures. A detailed discussion of thesedata is beyond the scope of this report which is essentially centred on mammalian systems and on endpoints of practical importance for man.
JOI. Trujillo et al. [T5] reported that RF/Un female
mice showed a linear decrease of their ability towithstand a standard cold stress (6°C - 7°C for 14days), as a function of increasing age. Mice exposed to
protracted 60Co gamma exposures at 0.5 Gy/day andthen allowed to recover for 90 days showed a similarlinear decrease with increasing radiation exposure in743their ability to survive the same stress. This radiationinduced effect was considered similar to lire shortening
through natural aging and was estimated to be equivalent to 9.3 days/Gy. Other data on the combined effector duration of life irradiation in animals under conditions of low ambient temperature are discussed in
Annex K.
102. In experiments by Gambino et al. [GS] rats were
irradiated whole-body or on the adrenals only with astandard exposure of 5 Gy and then were exposed for 3
hours daily to 0°C. Reduced longevity, growth retardation, cataract, greying of the fur. and induction oftumours were the long-term effects seen in animals that
had been whole-body irradiated, while animals
irradiated only on the adrenals did not show suchphenomena. The treatment at low temperature did notmodify the incidence of these effects. with the exceptionof a slight reduction of the accelerated onset of tumours
seen in whole-body irradiated animals. Since thetreatment with low temperature as such gave rise to a
reduction of the life span and to differential effects in
the incidence of inflammatory and neoplastic conditions, the experiments are not easily interpreted [Hl 7].
103. In many of the experiments described thetemperature could not act as such. but as a condition
producing physiological adaptive changes. Some information on the influence of miscellaneous physical treatments (permanent or transitory high altitude, high or
low ambient temperature. mechanical damage, severe
metabolic or physical stress) in respect to tumour
induction in animals were already reviewed by theCommittee in its 1977 report (Annex I) [Ul]. The
findings were on the whole negative. When interaction
effects were reported they were not very large andexplanations in terms of physiological adaptation
mechanisms to the exposure conditions could readily
be produced. In respect to life shortening, which at the
low doses and dose rates of interest in radiation
protection is mostly associated with tumour induction.
some data are reported in Annex K. They concern low
and high environmental temperature and specific andnon-specific stress. Here again, the effects reported
were marginal and orten of antagonistic character, i.e.,leading to an increased life span by the joint treatments.
These effects could be explained on the ground that
suboptimal living conditions frequently act by
decreasing, rather than by enhancing. the susceptibilityof the animals to the effects of radiation. However,even though the impression is in favour of the lack ofpositive synergistic evidence. the data arc few, theeffects unspecific and the underlying mechanisms
obscure so that no definitive statement can be made.
E.

MAGNETIC FIELDS AND ULTRASOUND

104. A fairly extensive body of literature exists on theeffects of magnetic fields in biological systems [PI4] but
studies of their combined action with radiation are
relatively few. A review is to be found in [N6]. This
problem may conceivably be of practical significancefor workers in thermonuclear fusion devices. It should
also be recalled that the use of transversal magnetic
fields to improve the dose distribution of high-energy
electrons in radiation therapy has recently beenenvisaged.
105. Sikov [S38] tested various combinations of highintensity magnetic fields with gamma-irradiation in
mice. Of the various end-points considered (lethality,744

developmental changes, biochemical effects) only two
appeared to be susceptible to the action of magnetic
fields applied alone (2 to 4 10s Tesla. T): audiogenic
seizure and the level of lryptophan pyrrolase in liver. In
both cases radiation alone had little effect and theresults of the combined treatments could be attributed
to the action of the magnetic field. A decrease in theslope of the probit line of mortality (gamma rays, 5.8.
7.5, 8.6 and IO Gy) without change of the LDso value
following the contemporaneous exposure to the 4 1QST
field indicated a decrease of spectrum of radiosensitivity values induced by the joint treatment. Fields of
2 JOST were inactive to this end.
106. Although some indications of synergism werereported for biochemical indices following localized
liver irradiation [W11 J. experiments on the survival ofcell cultures in vitro were negative in this respect [R1 I,
N6]. Uniform magnetic fields of 1.4 107 T in combination with radiation produced no changes in the formof the survival curves of cells in vitro or in the patternof recovery from sublethal damage, as compared withradiation alone [R 11]. Higher intensities of themagnetic field (2 108 T) or non-uniform fields were also
without effect for similar end-points in other experiments [N6]. There is therefore on the basis of presently
available evidence little ground to expect anenhancement of the effects of radiation by the joint
application with magnetic fields.
107. Ultrasound is widely used for diagnostic andtherapeutic purposes. as well as in many industrial
appliances. Some experiments considered its possible
interaction with ionizing radiation. Harkanyi et al.
[H18) irradiated mice by ultrasound (800 kHz,exposures of 0.1. 0.5 and 1.0 W/cm2) followed twohours later by 0.5 Gy of x rays. Single-treatment groups
were also set up at the same time. The yield of chromosomal aberrations in the bone marrow of the animals
was taken as the end-point. None of the ultrasoundexposures produced any significant increase over the
spontaneous level, while the effect of the ionizing
radiation dose was easily assessed. No change in thislevel of effect was found in the group of mice undergoing the combined treatment.
F.

DUSTS AND FIBRES

108. For many industrial environments the combination of radiation exposure and exposure to dusts isquite usual. as, for example. in mining, metallurgicalindustries, power plants and construction works. Many
dusts and fibres have been shown to be carcinogenic or
pathogenic by themselves. Direct experiments on
mammals about the action of dusts arc available [C3,C16. K9, P2. PIO. P11]; concerning fibres, asbestos andother minerals have been given particular attention[W12. 825). Since dusts may be soluble or insoluble,
according to the different types of materials. studies of
their combined action with radiation could be covered
under the chemical or under the physical section,
respectively. In the first instance the chemicalcompounds dissolved from dust particles would be theactual agents taking place in any combined action; inthe latter the size and the distribution of the dust
particles would be the parameters of relevance.
109. Panov et al. [PIO] studied the respiratory andrenal systems of rats after intra-tracheal instillation of aneutral 210Po solution (37 kBq/rat) and quartz dust (50mg in saline suspension). Lung fibrosis was found to bemore pronounced in the combined treatment group.
Malignant tumours of the respiratory tract were also
said to be observed more frequently in this group,
although no precise description of all the histologicaland statistical aspects of these tumours was presented in
the work. Similarly. in kidneys glomcrulo-tubular
lesions were found more often in the group under the
combined action of the two agents.
110. Ponomareva et al. [Pl 1, P2] used different typesof mineral dust with admixture of highly active thoriumoxide. Rats were made to inhale or were instilled intratracheally for periods of time up to one year. The
chronic action of these agents gave rise to inflammatory
lung processes and to fibrosis. Tumours of the lungwere also observed after 1.5 to 2 years. When an
additional chronic whole-body irradiation course wasgiven to the animals (gamma rays. 20 mGy/day. total
dose 2.5 Gy) the lung tumour yield was increased by afactor of two, in comparison with the group under
combined treatment and a group receiving external
irradiation only. There were no experiments performed
to define the specific role of dust in combination withinternal or external radiation treatment. The dataobtained from the group combining internal irradiationand dust were used to standardize conditions ofoccupational exposure including a combination ofthese agents [B15).
111. Experiments on the combined action of internalalpha irradiation (239PuQ 2) and chrysotile asbestos
fibres (mean fibre length 1-10 µm) were performed by
Sanders [Sl2, S24, S13]. Insoluble particles were administered to rats by intra-tracheal instillation. Animals
receiving only the Pu02 had a more homogeneous
distribution of plutonium particles in the lung, while
the combination of treatments led to a concentration of
the radioactive particles within the asbestos-induced
scars in the peribronchiolar regions of the lung. In
groups receiving plutonium alone the pulmonary
retention half-time of the nuclide was about 200 days;in the combined-treatment group it was 450 days.
Correspondingly. the cumulative absorbed doses to thelung two years after instillation were 4 and 12 Gy. The
incidence of pulmonary carcinoma was 4.5% in rats
given the asbestos, 32% in rats receiving plutonium
alone and 21% in the combined-treatment group. Thus,
per Gy of absorbed dose, the incidence was about fourtimes greater in the plutonium group than in the
combined-treatment group. An explanation for thefinding could be that by a reduction of the number ofepithelial cells receiving alpha dose a reduction of theresulting yield of tumours could come about. In
another series [S12] the two agents were injected intraabdominally. The agents both tended to concentrate in
the fibrous adhesions of the peritoneum and theomentum, inducing sarcomas and mesotheliomas to a
final incidence which was not appreciably different
from an expected sum of effects.
112. Lafuma et al. [L16) reported preliminary resultsof experiments with rats where internal or external
irradiation were combined with intrapleural injectionof crysotile asbestos. In a first series 8 rats were exposed
to 3000 WLM (see definition of WLM in Annex D) of
radon-222 over 1 month and they received about 70
days after the beginning of exposure, 2 mg of crysotile
in suspension intra-pleurally. As in the case of previous
experiments with radon inhalation [L8] a very small
proportion of animals developed lung tumours afterradiation exposure and no mesotheliomas wereobserved at all. Exposure to crysotile only resulted in avery low incidence of mesotheliomas. However, thecombined treatment led to the appearance of lungcancer in all rats, 7 of them being mesotheliomas. A
clear synergism is here obtained. The same type of
results was obtained in a second experimental series
where whole-body mixed gamma-neutron reactor
irradiation was given (2.3 Gy of 0.5 McV neutrons witha gamma component of 0.75 Gy). The animals were
injected with the same amount of crysotile 125 days
after the radiation exposure. The results on lung tumor
induction arc given in Table 1 and show that, in
addition to an increase in total tumours. mesothcliomas
only appear in the irradiated group given crysotile
intrapleurally. These preliminary data should be
confirmed in larger experiments.

113. Sanders et al. [S22) studied the effects ofberyllium oxide aerosol inhalation in combination withplutonium oxide aerosol on more than 600 rats. Aerosol
particles were of micron and submicron sizes.
Exposures up to initial alveolar depositions of I to 91µg beryllium and 0.15 to 6.7 kBq of 239Pu were
performed. The results obtained by the two agents
given separately and by their combination (beryllium
aerosol being introduced prior to plutonium aerosol) as
total incidence of pulmonary tumours show that thechanges in lung tumour incidence due to the combination of the agents were insignificant. This in spite of
the fact that the alveolar clearance of plutonium wasdecreased by exposure to beryllium and the translocation of plutonium to the thoracic nodes wasincreased.

lll.
A.

CHEMICAL AGENTS

INORGANIC COMPOUNDS

114. Changes in the physical and chemical character1st1cs of the water matrix of biological systems may
bring about changes in radiosensitivity. Chinese
hamster cells were exposed to media containing
deuterium oxide (D20) following 60Co gamma irradiation and cell survival was scored as the end-point[Bl7]. Under these conditions the cell response toradiation was greatly enhanced. Depending on theconcentration and the treatment time of D20. dose
modification factors of up to 4.5 could be found. Preirradiation incubation had. on the contrary, a veryslight effect on the radiation response. The sensitizing
effect of D20 depended clearly on the conditions of cell
metabolism, since it was influenced by the type of
media and by the temperature. It was found that theradiation damage capable of interacting with thedeuterium oxide was repaired by the cells when they
were kept for three hours at 37°C in the growth medium
and split-dose experiments suggested that the sublethal
damage repair capacity was reduced in the presence ofD20. The heat sensitivity of the cells was unaffected by
D20 and the enhancement of radiation response
induced by heat was also independent of the presenceof D20.
115. Some natural mineral components of the dietmay change the radiation response of the animals[KIO]. Rats were kept on diets with low (50 mg/d Ca
and 0.2 mg/d F) or high (150 mg/d Ca and 3 mg/d F)
content of calcium and fluorine and after 5 weeks ofsuch diet were given radioactive 90Sr. As a result of the
combined treatments the haemopoietic system of thefirst group of animals was more severely damaged andtheir mean life span shortened by 50-70 days, as745

compared with the group with high Ca and F in thediet. The protective action of the high Ca and F diet isachieved at intakes of the two minerals not higher thanthe upper limits of physiological intake for humans.
Similar results were obtained if external gamma irradiation was added to the internal 90Sr irradiation. In
other experiments rats were subjected only to gamma
irradiation and to diet changes. In all cases the survival
at short term and the life span proved to be higher ingroups with high calcium and fluorine intakes.
116. The different trace metals found in the air, foodand water of some parts of the industrialized world [T7]
may alone induce adverse health effects, including
malignancies and teratological effects, at sufficiently
high concentrations. They may also conceivably
combine with the action of ionizing radiation at thebackground level or under special conditions of
exposure. The universal spread of these metallic
contaminants make studies of their possible combined
action particularly important. Data on the combined
action of silver ions and radiation in bacterial systems(spore or vegetative stage) have been provided by
Richmond and Powers [R12) and Held and Powers[H25].
117. Lead chloride (PbCli) in concentrations of 0.1
and 1 µg/cm3 was studied in combination withradiation (doses of approximately 1 Gy) for its ability
to induce various effects in vitro on embryonic systems[S15]. The number of nucleated cells per mouse embryo.
the labelling and mitotic indices and the number ofmicronuclei per cell were among the effects scored. At
both concentrations a synergistic increase of the
micronuclei was found. accompanied by an inhibitionof embryonic development. For cadmium, thecombined effects with radiation were found to be
additive in the same system [M26]. Lead was studied by
Kudrizkaya [K3] for its capacity to damage spermatogenesis in the mouse. Exposure was given chronicallyover a period of about six months up to cumulated
concentrations of 0.3 mg/g of lead chloride and 81
kBq/g of 90Sr, administered in drinking water. Testis
weight or the number of spermatocytes were unaffectedby lead alone, while 90Sr significantly decreased thecontrol values of both end-points. Combination of thetreatments produced a final effect which was lower
than that caused by radiation alone. an antagonistic
type of interaction. Lappenbush [L25] injected adultmale rats with cadmium chloride (125 to 250 mg)
intraperitoneally for 30 days twice per week and subsequently irradiated them with x rays. The 60-day
survival was unaffected by doses of the contaminant
lower than 125 µg. The radiation LDso130 was found to
decrease linearly with increasing exposure to cadmium.
The numbers of red and white cells in the peripheral
blood were affected by the combined treatment in acomplex way.
118. Platinum (cis-dichloro-bis platinum, DBCP) andradiation affected the survival of ovarian-derived
Chinese hamster cells in culture according to a synergistic type of interation [C8]. Chromatide aberrations
were also induced in higher percentages. In order toobserve synergism the chemical had to be administered
between four hours before and two hours after irradiation. Two or three days elapsing between the chemical
and the radiation treatment abolished the interaction.
The synergistic effect was considered to result fromradiation-induced single-strand breaks in the DNA
which occurred in linear proportion to dose, opposite
to a single platinum complex intra-strand cross-link
746

which occurred linearly with respect to platinum
concentration. The combination of the two lesions led
to lethality. A simple mathematical model to describe
the experimental data was developed.
119. The nitrocompounds, especially the oxides, arerather common pollutants of the air. Sensitization ofanoxic bacterial spores was reported when they were
irradiated in N02-saturated water [P15]. A study isavailableinmammals[K4]
whereinhaledplutonium-239 under the form of plutonium pentacarbonate ammonium (69 kBq/kg of lung tissue) was
administered to rats. after which the animals were also
made to inhale nitrogen oxide (0.09 mg/I) or chlorine(0.05 mg/I) for 15 minutes. After the combined treatments the incidence of lung cancer was almost doubled
as compared with the irradiation treatment alone.
Tumours were multifocal and different types oftumours were seen in the combined than in the single
treatment. Pneumosclerosis was also enhanced in the
combined treatment group.
120. Occupational situations where exposure toionizing radiation may be accompanied by exposure toother detrimental chemicals should not be uncommon
in industrial practice, but epidemiological data in rhis
field are very rare. In one case observations werecarried out on workers exposed to gamma rays forindustrial radiography and also to vapours of hydrofluoric acid (HF) [S21]. The changes investigated (levelsof T-lymphocytes. C-reactive protein and auto-antibodies) were mainly immunological. The group under
the combined influence of radiation and the toxic
chemical was reported to have lower levels of Tlymphocytes and higher levels of C-reactive protein
and auto-antibodies than the groups exposed to only
one of the agents.
B.

ORGANIC RADIOSENSITIZINGCOMPOUNDS

121. The present section includes what is essentially areview of substances which may enhance the radiation
response of biological systems, and are called radiosensitizers. Compounds inhibiting the radiation response
are called radioprotectors. In many cases thesesubstances were specifically developed for theirprotective or sensitizing properties. The study ofradioprotective chemicals has been strongly pursued[R2, M7. M29. 1329]. More recently, the application ofsuch compounds in clinical tumour therapy has been
discussed [Y7]. Also. a new field has grown and is still
rapidly expanding, that of the radiosensitizing
compounds [M10. R6], whose potential in clinicalradiotherapy is being tested.
122. The relevant data will be reviewed briefly
because it seems unlikely that situations will arise inwhich these substances may pose significant problemsof public or occupational health. For a review ofbiological effects. mostly lethal, of the combined actionof acute irradiation with other common industrial
poisons (at high toxic levels) the reader is referred to
Tiunov et al. [T2]. Annexes J and I of the 1977 report[U 1J reviewed the action of radioprotective and radiosensitizing chemicals in respect to the production ofembryonic and foetal damage by radiation and oftumour induction, respectively. The available information on the action of chemical radioprotective drugs
for life-shortening effects in animals is reviewed in
Annex K of this report.

123. Several
classificationsofradiosensitizing
substances have been proposed (M10. P16, S39], basedon their mechanisms of action. Keeping in mind that insome cases the molecular mechanisms are still
unknown and that some agents may act through morethan one mechanism. one classification may be as
follows: I. Agents modifying the primary radiation
chemical processes, including (a) electroaffinic agentsand (b) iodine compounds; 2. Agents interacting with
DNA metabolism (DNA-base analogues); 3. Antibiotics and other agents interfering with repair processes(see section Ill C); 4. Agents reacting with nucleophilicgroups (SH groups); 5. Other radiosensitizing agents.
124. The best known example of the first class ofagents is oxygen whose level in biological systems at thetime of irradiation greatly influences the yield ofradiation effects. A massive body of literature exists on
the action of oxygen and the interested reader isreferred to [Al 1, PlJ. A large number of electroaffinic
compounds or hypoxic cell sensitizers is also known
but their detailed discussion is beyond the scope of thisAnnex. Such compounds may contain one of thefollowing chemical groups: the carbonyl (CO). thealdehyde (CHO). the nitro (N02), the cyano (CN)groups, homo- and hetero-cyclic rings. Stable free
radicals are also electro-affinic agents. The radiosensitive prope1ties of such compounds are manifest when
they are present in biological systems at the time ofirradiation or if they are irradiated separately and then
immediately added to the biological system.
125. The same is true of the iodine compounds which
may also change the concentration of radiationinduced free radicals. If cells arc exposed to irradiatediodoacetamidc within milliseconds after irradiation cellkilling takes place, which is not observed if irradiatedcells are exposed to non-irradiated iodoacetamide, thus
showing the role in sensitization of short-lived transientcompounds [D9]. Radiosensitization takes place alsowith other iodine compounds: iodide. iodoacetic acid.
iodopropionic acid, methyliodide, p-iodophenol.
iodobenzoic acid and others. Reactions with -SHgroups may account for part of the sensitizing effect ofsome of these compounds [M7].
126. Attention has recently been given to the radioscnsitizing properties of iodine contrast media used inradiodiagnostics [S43, N7, A2, M25]. Sensitizing effects
on bacterial killing were first reported [S43] and then an
increased yield of chromosomal aberrations inperipheral lymphoctyes of children undergoing x-ray
angiocardiography with contrast media [A2, N7]. It has
also been reported that sensitization of mammalian cellkilling by iodine compounds would occur for x but not
for gamma rays [M25]. These data are explained by thedifference in doses due to photoelectric effect in thecase of x rays. An accurate physical dosimetry should
clarify this issue.
127. Quinones are unsaturated carbonyl compoundswith conjugated structures and electron affinic
properties. Several quinones and their derivatives havebeen found to sensitize bacterial and yeast cells underoxygenated and anoxic conditions [A4, Ml 1. S16, S17].
It has been postulated that the sensitization of E.coli
B/r by vitamin K5 is mediated by radiolytically
produced hydroxyl radicals [SJ 6]. Diphenylquinone
was found to enhance the action of radiation in mice[A4}. ln some bacterial systems under anoxia the valueof DMF could be about 3 (10-3 M indanetrion monohydrate [B7}) or even 4 (100 ppm vitamin K5, [S16]).

Newly synthesized isoindole quinoncs showedpromising characteristics when tested in vivo on soft
tissue sarcomas transplanted into mice [C13].
128. Electroaffinic compounds containing nitro
groups can specifically increase the radiosensitivity ofanoxic cells, leaving that of oxygenated cells unchanged
or even decreased. These properties would be advantageous for tumour radiotherapy [A5, D8, H4. H8, Pl 7.
Y5]. The radiosensitiziation by mizonidazolc wasproved to occur for hypoxic mammalian cells in vitro
and in vivo [A12]. Under aerobic conditions no sensitizing effect of the compound at any stage of the cell
cycle was observed [P17] and under anoxia thestrongest effect occurred in middle-S. Toxicity of theagent under anoxia requires low exposures to the agent.
129. Yuhas and Li (Y5] studied the effects of thecompound at a concentration of 6 mM in combination
with the ra<lioprotective compound cysteine (8 mM) on
mammalian cells in culture, showing protection underconditions of oxygenated irradiation and sensitization
under anoxia. Hall et al. [H8] tested eight different nitro
compounds: for all of them the DMF was an increasing
function of the concentration and for some it reached avalue of about 3.5. equalling the average value of the
OER in the cells tested. In generaL 2-nitroimidazoles
were more effective sensitizers than 5-nitroimidazoles.
Other nitrocompounds. the nitrofurans, may be evenmore effective, specifically under anoxia [R3, R6].
Sensitization by nitrocompounds was greater when they
were administered prior to irradiation [D8]. Radiosensitizing properties were also described for nitrogencontaining stable free radicals such as triacetoneamide-N-oxyl (TAN) [E5. 821] and 2,2,6,6,-tetramethyl4-piperidinol-N-oxyl (TMPN) [P6].
130. DNA base analogues belong to the second classof radiosensitizers. Extensive studies were madeespecially on halogenated DNA base analogues such as
5-fluorouracyl (5-FU). 5-bromouracyl (5-BU) or 5bromo-2-deoxyuridine (5-BUdR) [K13). Significant
enhancement of killing was shown for viruses, bacterial
and mammalian cells [S18]. Some attempts for a clinicalapplication of these substances have also been reported.
For a review of the relevant studies see [M7, Ml OJ.
131. The third class of radiosensitizers will beconsidered in section lll.C. Here various substances
capable of modifying the biochemical cellular processes
should be mentioned, belonging to classes 4 and 5.
Several organic chemicals capable of enhancing
radiation damage share the property of being -SHreactive. Since -SH compounds are known to be radioprotectors. the correlation has been investigated between
the ability to bind -SH groups and the capacity tosensitize the cells to the action of radiation. Bruce et al.
[B8] found that the capacity to sensitize was well correlated to the amount of p-hydroxymercuribenzoate
bound to cells.
132. Sensitization of anoxic cells is an important goal
for tumour radiotherapy (A13. R6]. Radiosensitizationof bacterial cells under anoxia by N-cthyl-maleimide(NEM) was shown as early as 1960 by Bridges [B6].
Other data on bacterial and mammalian cells are alsoavailable [L12, Ml8, Kl I]. A DMF of 1.5 with humancells in vitro irradiated with x rays was reported by
Klimek [Kll]. The known property of NEM to bind-SH groups led to the hypothesis [L12] that NEM could
bind the free non-protein -SH groups, thus preventing
DNA repair through donation of hydrogen from these747

groups. Other experiments by Klimek and Zemanova[K 12] showed that under concentrations of NEM too
low to inhibit DNA synthesis a high proportion of theoriginal free thiol groups was still present, thus
implying other mechanisms for NEM sensitization.
However, the role of intracellular thiol groups would
also be supported by experiments of Sinclair on oxygenated [S40J and anoxic [K17] Chinese hamster cells
exposed to NEM and radiation. Repair of lethal
damage is inhibited by the presence of NEM but themechanisms of such an inhibition are still unknown.

20

~

16

w

12

:-

8

"'
8

""'',.,1'

0

2

4

6

a

BCNU COIICE!HR,",TIQI; (ug/ml)

133. Another organic compound that may produce
cytological changes is carbon tetrachloride (CC1 4). Its
administration to animals induces, for example. liver
cell proliferation [A6J similar to that induced by partial
hepatectomy. Cole and Nowell [C20] examined theeffect of CCl 4 on the induction of hepatomas in fastneutron irradiated mice with doses of 1.7 to 3.1 Gy. At
various times after irradiation some animals received
the compound subcutaneously. Sixty-one percent ofanimals receiving the combined treatment developed
hepatomas. as compared to 19% of the mice irradiatedonly. Since CCl4 alone produced no hepatomas, theinteraction factor is approximately 3. Histologically thetumours were similar in both groups but tumours oflarger size were more frequent in the combined modalities group. The authors concluded for a promoting
effect of CCl4 in liver cancerogenesis. Procainehydrochloride. a local anaesthetic acting on cell
membranes. has been shown to sensitize bacterial andmammalian cells to the action of radiation [S19, S20].
134. Alkylating agents may react with DNA bases andthus directly influence the radiosensitivity of cells. The
alkylating agent spirohydantoin mustard (SHM) wastested in combination with x-irradiation on brain
tumour cells in vitro [D 15]. The enhancement of cellkilling was greatest when the cells were irradiated fourhours before the drug treatment. The doses ranged fromzero to 20 Gy and the subsequent chemical treatment
with SHM lasted one hour at concentrations of 0. 2. 3. 4
and 5 µg/ml. The results were normalized and thecorresponding isobolic diagrams were built. At levels ofcell killing down to 10% a synergistic interaction wasapparent, although for lower levels of survival down to
0.1% the interaction turned into an additive one. Thisand another paper [D2] by the same authors are some of
the rare examples where the analysis of the interaction
type was carried out according to the approach
outlined in chapter I of this Annex. involving the use of
isobolic diagrams.
135. The same brain tumor cells cultured in vitro were
exposed for one hour to 1, 3, 5, 7.5 µg/ml of 1,3bis(chloroethyl)-1-nitrosourea (BCNU), followed 15
hours later by a series of x-ray doses of up to 20 Gy[D2]. Survival curves for the x rays alone, the BCNU
alone and for the combination of both agents wereobtained and on their basis isobolic diagrams forsurvival levels of l, 2 and 3 log cell kill wereconstructed as in Figure XVll. The figure shows theexperimental points for the combined treatment
connected with a dashed line: all the points except one
fall into the envelope of additivity applying at eachsurvival level. The point at the lowest level of survival(7.5 µg/ml of BCNU. 4 Gy of x rays) falls outside therespective envelope. although the displacement is not sogreat that it might not be explained by experimental
uncertainties.
748

Figure XVII. lsobolic diagram
for the combined action of
BCNU and x rays on 9L rat
brain tumour cells [D2]136. The scope of this brief overview of chemicals
capable of modifying radiation sensitivity is simply thatof illustrating the very wide range of processes whosealteration may in turn lead to a synergistic or antagonistic interaction in irradiated biological systems. The
data reviewed are as such of little relevance for themain scope of this Annex, because the doses ofradiation used are usually very high (up to several Gy,
depending on the susceptibility of the systems tested)
and the concentrations of the chemicals often toxic. It
is however appropriate that they should be mentioned
because the processes governing different aspects of cellradiosensitivity might also be relevant at lower levels of
exposure. No practical situation where the above
mechanisms would be significant can currently beenvisaged at the low doses of interest for the purpose of
the present Annex.
C.

CARCINOGENIC CHEMICALS

137. Organic substances which are known to havecarcinogenic properties should be discussed separately.
Some, such as the alkylating agents, have already been
mentioned in section Ill. B. Carcinogenic agents areusually divided roughly into initiators and promoters,
following the two-stage theory of carcinogenesis [B 18].
It is known however that such a subdivision is not rigid
because many agents share the properties of bothclasses. In combination experiments it may be expectedthat the final tumour yield may depend on theproperties of the interacting agents, as well as on theorder and time pattern of their administration. A potent
initiator followed by an active promoter might be
expected to give the highest carcinogenic response andreversal of their order of administration a drastic
reduction of this response. Another important trait is
the spectrum of the tumours induced, as some agents
may be extremely specific in this respect and their interaction with radiation might change this selectivity.
138. Precise quantitative data were provided by
Di Paolo et al. [021. 017, D22] and Kennedy et al.
[K14] on the morphological transformation of
mammalian cells in culture in regard to the interaction
between ionizing radiation and the carcinogen
benzo(a)pyrene or the promoting agent phorbol ester.
The experiments elucidated the dose-time relationships
for an effect of special significance for practical
purposes, showing enhancement factors of up to 9 fold.
depending on the conditions of exposure and on thedoses of the agents interacting. This series of experimems also attempted to elucidate the mechanisms ofinteraction. For the same biological end-point, thepromoter l 2-0-tetradecanoylphorbol-13-acetate (TPA)
administered after x-ray or neutron irradiation to
C3H/10 T 1/2 cells in culture was shown to act synergistically. with complex relationships as a function of
the radiation type and dose [H27].
139. A study showing an increased yield of leukaemia
in mice pre-irradiated with x rays and subsequently
treated with methylcholanthrene was published by
Furth and Boom (F9]. An increased yield of leukaemia
in mice induced by x rays, methylcholanthrene oroestrogens was also shown by Kawamoto et al. [K6]
when the animals were simultaneously treated byurethan. After Berenblum investigated the interactionof x rays and urethan in mouse leukaemogenesis ingreat detail and showed that the order of administrationof the combined agents was of decisive importance [B2.
B26] many other authors reported enhancement ofleukaemia under the same agents [D4. L22, V6. Gl]
particularly in young animals [Bl, L23] whereenhancement is especially pronounced. This may be on
account of differences in the drug distribution or catabolism as a function of age [C2]. Data have also beenreported for croton oil [I2], myleran [US] and novoembicyn [A8] in conjunction with radiation.
140. Combined treatments of pre-implantation mouseembryos in vitro with x rays or phenols (which arepromoting and mutagenic chemicals in some test
systems) showed that the effects were, at most, additive[M30]. Schmahl and Kriegel irradiated mouse embryos
in utero at 11-13 days p.c. (1 Gy at each time) andinjected the pregnant mothers at 17 days p.c. with 0.5
mM/kg of ethylnitrosourea [S42]. Tumour development
was followed post-natally up to 18 months. Resultsfrom this series are shown in Table 2, with the interaction factor calculated according to equation (5). If
one considers total tumours as the end-effect, interaction appears to be antagonistic (w = 0.44). If one
takes each category of tumours separately, one may
conclude for at least one clear case of synergism andone of antagonism for leukaemia and hepatomas,
respectively. This appears to be a good example of achange in the tumour spectrum brought about by the
combined treatment. However. for more definitive
statements exposure-response curves within a broader
range of values for both the single and the combined
actions would be required.
141. Much work has been carried out on the skin, thetissue where the two-stage mechanism of carcinogenesis
was originally identified [818] and can be more easily
tested. Electrons or UV light in association with othercarcinogens usually result in a higher yield of tumours
than any of the agents administered alone. Thisapplies to methylcholanthrene [C14] and to
7,12-dimethylbenz(a)anthracene (DMBA) [E6, S28].
However, a recent report [B19] on this latter substancein association with 0.8 MeV electrons (5-25 Gy) in
respect to carcinogenesis of rat skin showed that thetumour yields were approximately equal to the sum of
the yields induced by the separate treatments, so thatprior irradiation did not appear to alter the susceptibility of rat skin to DMBA carcinogenesis.
142. The case of 4-nitroquinoline-1-oxide (4NQO) has
been particularly well analysed. When applied in
combination after beta rays from 9 0Sr-90Y (both agentsat doses that did not separately induce tumours) it
appeared to have a synergistic effect for skin tumour
induction in mice. Reversing the order of administration of the treatments led to a much smaller yield of

tumours by about an order of magnitude [H5]. Whenthe interval between beta irradiation and subsequentchemical treatment was made to vary between 11 and408 d, the tumour induction rate was found to bealmost at the same level for all treatment times,
indicating that the latent carcinogenic change inducedby skin irradiation may persist for a very long time andremain available for subsequent interaction with the
4NQO [H6J. Finally, caffeine was found to further
increase the incidence of malignant tumours in mouseskin when painted after beta rays and 4NQO treatment[H7].
143. Croton oil, a typical promoter of skin neoplasia
from which TPA is extracted. gives uncertain results
when combined with radiation: enhanced effects withUV [E2] and electrons [S29] or absence of any
enhancement [06, 820] have in fact been reported. It
may be said in very general terms that the concepts ofinitiation and promotion may be verified on the skinalso in the case of drug-radiation interactions.
However. the results of combined treatments on theskin could also be interpreted on different grounds andsome of the previously mentioned experiments [H5, E2.
E6] would in fact be regarded by others [N3] as clear
examples of co-carcinogenesis by chemical andphysical agents.
144. The situation with respect to other tumours or tosystemic leukaemogenesis is definitely more difficult tointerpret. In the case of the lung. urethane (which
specifically induces adenomas in mice) has been usedin association with x rays at various doses and dosages.
A reduction in the incidence of tumours (both aspercentage incidence and as tumours/animals) has
been obtained in one experimental series [F6]: cellkilling by the high radiation dose in the urethaneinduced tumours was held responsible for the effect.
Recalculation of these data by others [L21] led however
to a different interpretation. Additive effects ofradiation and urethane were reported in another series,
and the final outcome of the treatments was deemed todepend on two competing phenomena, cell killing andcell transformation, whereby, depending on the dose of
the two agents, any effect may become possible.
Immunological phenomena might also interfere in thiscase to make the picture very complex [C15].
145. Procarbazine (PCB). a drug used frequently inthe treatment of the Hodgkin's disease, is a known
carcinogen in experimental animals since it gives rise topulmonary adenoma and leukaemia in mice, mammary
tumours in rats and acute myelogenous leukaemia inprimates. Hybrid (BALB/c x DBA/2) F 1 mice were
given this drug and ionizing radiation at different times
to test for possible synergistic effects [A 7]. Singletreatment groups received 300 mg/kg PCB weekly for
four weeks. a dose effective for induction of pulmonary
adenoma and leukaemia; or 0.6 Gy/d of 300 kYp x raysfor five d. a dose which did not result in tumours of thelung. Combined-treatment groups received radiation
three days or three weeks before PCB or PCB threedays before irradiation at the above dosages. The experiments were terminated within 12 weeks with killing or
the surviving animals. Pulmonary adenomas in micereceiving both agents were significantly increased overthe level of induction by PCB alone. Thymomas werealso increased significantly in the animals given the
drug three days before or after irradiation. The authors
concluded for a synergistic effect of the combinationand hypothesized that an increase of the normal
tendency of mice to develop pulmonary adenoma
749

would be at the origin of the interaction. Immunosuppression combined with direct cellular damage mightalso be responsible for the effect.

of mitoses. This enhanced proliferation would be
essential for the expression of the radiation-transformed cells.

146. Among studies where combinations of chemical
carcinogens and radiation were tested, the experimentsof Metivier (M6] regarded the action of Pu02 given byinhalation, in combination with benzo(a)pyrene (BP) or
dimethylnitrosamine (DM NA), compounds which arewidespread environmental pollutants. Both carcinogens
were given after the exposure to the Pu02: BP (2 x 5mg) was administered intra-tracheally in association
with haematite 2-3 weeks al'tcr the nuclide: DMNA (2or 20 ppm) was given orally, added to the drinking
water. Tumours of the lung and of other sites, histological types of tumours, invasiveness and survival time
were the principal end-points investigated.

150. A biochemical approach to the study ofmechanisms of interaction between radiation andchemicals in the case of lung tumour induction was
followed by Queval and Bcaumatin [QI]. These authors
studied the correlation between the capacity by various
substances of inducing pulmonary enzymes and theirability to shorten the latency period of the lung tumoursin rats, following inhalation of radon daughters. The
research established that compounds such as benzonavone, methylcholanthrcne and benzopyrene arehighly effective in enzyme induction and capable. at thesame time, to shorten the latent period of tumour
appearance.

147. BP alone led to a small increase of tumour
incidence above the control level. PuOi (0.63 kBq)
produced similarly a slightly increased incidence. Bothagents combined produced an appreciable increase in
the number of tumours with an increased invasiveness.
Survival time renected closely the results on tumour
incidence, being essentially unchanged for the two
agents given alone and practically halved by their
combination. At least on qualitative grounds, a synergistic interaction was operating in these experiments,
the latency period of the tumours in the combined
treatment group being evidently shorter. For higher
levels of Pu02 (6.3 kBq) a synergistic action might also
be present, but its expression (particularly with regard
to survival time) is much less clear. In the case of
DMNA no synergistic action with respect to alpharadiation alone was found. At high concentrations (20ppm) the latter drug produced a subacute intoxicationand no synergistic effect. It was reported however that
inhalation of Pu02 in association with DMNA did
result in an increased tendency of liver tumours tometastasize into the lungs.

151. Large experimental series were carried out on the
combined effects of radiation and inhalation of
uranium ore dust and diesel oil exhaust fumes at the
Pacific Northwest Laboratory [Cl6]. The experimentson hamsters involved about 600 animals non-exposedor exposed to radon and radon daughters. uranium ore
dust and diesel engine exhaust, alone or in various
combinations. Squamous cell carcinomas developed inonly a few of the animals exposed to radiation and they
were always preceded by a squamous metaplasia of thealveolar epithelium. In general, however, the hamsterlung was found lo be rather refractory to the malignant
transformation and did not even develop lesions that
could be classified as pre-cancerous when exposed tolevels of the above agents which were regarded asrealistic for life exposure regimes. Thus, the hamsterlung under these conditions may not be a useful model
for pulmonary cancerogenesis in man.

148. In the experiments of Little et al. [LlO] the interaction between benzo(a)pyrene (BP) and alpharadiation of 210Po. was examined. The experiments
were performed on hamsters and the two agents wereadministered by intratracheal instillation, absorbed on
haematite particles or dissolved into physiologicalsaline. In a first series of experiments the two agentswere administered simultaneously in 15 weekly instillations (0.3 mg BP + 0.2 kBq 210Po/treatment). Under
these conditions the results were compatible with anadditive interaction of the two agents.
149. In a second series of experiments BP was given
15-18 weeks after the administration of a single dose of
1.5 kBq 210Po. While BP alone (2.4 mg in eight weekly
instillations of 0.3 mg) or 210po alone produced practically no lung tumours. the combinations of both agents
resulted in a clear synergistic effect. with 17% of theanimals developing frank tumours of the lung. Physiological saline and gelatine were mostly used as carriers.
When the administration of BP preceded the 2JOPotreatment no increase of tumour induction was seen. It
is remarkable that when the second treatment consistedof saline alone. without BP, a sharp increase of thetumour yield was seen. compared to the 2l0Po treatment
alone. The instillation of isotonic saline could act as anon-specific stimulus to cell proliferation [Ll 1] andsubsequent experimental work [L24) appeared to lend
support to this hypothesis. Autoradiographic experiments showed that after treatment by BP or by saline
the epithelial cells of the hamster lung undergo a wave
750

152. Knizhnikov et al. [K9] modelled another case ofindustrial exposure by a combination of shistose ash,benzo(a)pyrcne and 2IOJ>o. The mice were exposed toash alone, ash with BP or with 210Po, and to the triple
combination of the agents together. The yield of lungtumours and their latency period were studied and at
the levels used the yield was reported to increase from35% (ash only) to 61% (triple combination). The latency
period decreased in the same two groups from 300 to
200 days. An interaction factor may be calculated fromthese data of about 1.3. indicating some synergistic
interaction. Other control groups were included in thisseries.
153. The intragastric administration of 3-methylcholanthrene followed by x rays [S30) or fission neutrons[S4] produced no more than additive effects for
induction of mammary adenocarcinoma. However, thesame chemical applied locally on the brain, in
association with beta irradiation resulted in anantagonism which was proportional to radiation dose[M15].
154. There are many different experiments concerninga variety of other tumours. X rays alone or in combination with benzo(a)pyrcne produced the same
incidence of neoplasia [ K7]. Dibutylnitrosamine(DB NA) or 4-ethylsu Iphonyl-naphtalene-1-su lphonamide (ENS) combined with x rays showed no effect on
tumours of the urinary bladder but a reduction of the
mammary tumour incidence [F7). A synergistic action
on the production of liver and gastric carcinoma by
fission neutrons in combination with N,N'-2,7-nuorenylenebisacetamide (2,7-FAA) was reported, but nointeraction for intestinal tumours was found [V7].

Localized x-irradiation in association with the same
drug administered in the diet accelerated the inductionof hepatomas [N 1J and similar effects were reportedwith the association of x rays and o-aminoazotoluene[K8] and of I44Ce and dimetylaminoazobenzene (OBA)[M 16). Experiments on additive carcinogenic effects of
9 I O-dimethvl-1 2-benzanthracene or 1,2,5.6-dibenzanthracene in ~sso.ciation with chronic internal irradiationfrom 90Sr were also reported [Z 1, Z2J.
155. The mutagenic substance N-methyl-N'-nitro-Nnitrosoguanidine (MN NG) was tested in combination
with whole-body fission neutron irradiation for its
carcinogenic properties on the gastrointestinal tract ofrats. A high incidence of gastric and duodenal
carcinomas was found after the MNNG treatment but
the neutrons did not produce any tumour. Combining
the two treatments did not change the effect of MNNG
[V3]. Survival and tumour induction were tested inthree strains of rat following x-irradiation inassociation with urethane. In spite of some interesting
differences observed between strains, the overall effectof the joint treatment was not greater than the sum of
the separate effects at the dosage level studied [M 17].
156. In work by Sanders [S12], besides the combined
action of 239Pu0 2 and asbestos, the combined action of
239Pu0 2 with benzo(a)pyrene was also studied. both
agents being administered intra-peritoncally. !heaction of BP alone produced mostly abdommal
sarcomas. The combination of BP and 13.3 kBq of
239Pu0 2 resulted in an approximately additive yield of
sarcomas. Other tumours which are characteristic of theplutonium action were also produc~d. The admi_nistration of BP increased the translocat10n of plutonmm
to liver and Jung. which points to the need that possible
metabolic effects leading to different dose patterns invarious organs upon the joint administration of two
substances should be taken into account when
discussing the results of combined actions.
157. In conclusion. it appears that the evidence
reviewed is very connicting. The number of substancestested is large and the amount of information relating
to each of them very little. The pathogenesis of thetumour systems tested is complex and the conceptual
distinctions between induction and promotion cannot
be held in many instances. In some cases the application of chemicals after irradiation may enhance thetumour yield by comparison with the opposite order ofapplication. In other cases. the association of treatments may actually decrease rather than enhance theinduction of neoplasia when the toxicity of the
combined agents outweighs their additive carcinogenic
properties [U2]. No definite conclusions with respect toany class of tumours may therefore be drawn before thedose, the dosage schedule. the order of administrationand modalities of the combined treatments arc properly
and thoroughly explored, which is very rarely the casein the contributions that have been reviewed.
D.

THE SPECIAL CASE OF TOBACCO SMOKE
I.

General158. Tobacco smoking is a widespread habit of many
human populations in spite of a well documented
association between smoking and lung tumour
incidence. The relationship between annual death ratefrom this cause and number of cigarettes smoked per
day is reported to be linear with slope of about t ()-4 [05,D23] and with incidences of lung cancer rising fromabout 0.07 10-3 per year in non-smoking males to 3 10-3
per year for male persons smoking 35 or morecigarettes per day. The chemical composition of
tobacco smoke is very complex and includes more thanI OOO identified compounds [S9], a number of which arearomatic hydrocarbons that have been shown to act as
carcinogens. Smoke and tobacco tar also contain anumber of tumour promoting and co-carcinogenic
agents.
159. The two-stage nature of the carcinogenic actionof tobacco smoke was shown by a classical experimenton mouse skin by van Duuren et al. [VI]. The initiator,
1,2-dimethylbenz(a)anthracene (OM BA) acted in thiscase as an initiator and cigarette smoke condensate(CSC) as the promoter. Five weekly applications of
CSC after a single application of DMBA greatly
increased the rate of tumour appearance. by shifting thelatency period from 450 d (for DMBA alone) toapproximately 100 d. The initiating action of the tar
components is relatively low compared to that of
DMBA. In this particular case the initiator was achemical substance, but any other carcinogenic agent.
ionizing radiation in particular. could be effective in
combination with the promoters contained in thesmoke concentrate. This point was proven experimentally by McGregor [M27. M28) who treated rat skinwitn beta radiation and subsequently applied csc.
Rats treated with CSC only produced no tumours. A
two- to three-fold increase in the numbers of skintumours was observed in the groups under combined
action. as compared with the animals exposed to beta
radiation alone. It should. however. be realized that
only few agents can be considered as pure initiators or
promoters. the rule being that many carcinogenic
agents have the properties of both classes of substancesand sometimes to various degrees. according to thedifferent animal models tested.

2.

Experimental data

160. Various examples of interaction between
radiation and tobacco smoke have been reported inanimals [C5. C6. C16]. In experiments by Chameaud etal. [C5, C6] rats were exposed to radon inhalation in
special chambers. They developed respiratory canc_ers
as a function of exposure and exposure rate, startmg
from a control background incidence of practicallyzero. Similarities could be shown histologically between
these tumours and human lung tumours. Inhalation ofcigarette smoke in these animals did not result inmalignant transformation of the respiratory cells but
only in benign lesions of the bronchial epithelium andlung parenchyma [C7]. In interaction experiments the
exposures to radon daughters was chosen to be 100. 500
and 4000 WLM. because it was shown in previous tests
that the incidence of lung cancers of respectively 1-2.
5-10 and 30-40% would result from them [L8].
Cigarette smoke inhalation was carried out understandardized conditions for periods of 15 min ten times
per day. four days per week. for one year. No change in
the animals' life span was seen after this treatment. An
elaborate classification of the pathology was set up tofollow the spread of tumours at death.
161. For the highest radiation exposure (4000 WLM)
the incidence of lung cancer was 34% and it increased
to 68% in animals also exposed to smoke. At 500 WLM,
751

7% and 28°/i, were the corresponding figures and at 100
WLM. 0% and 3.3%. Since smoking was without effect,
equation (22) in a simplified form may be used toanalyse the data. Accordingly, the values of the interaction factors for the above groups are 2. 4 and oo.
Pathologically, tumours appeared to be more advanced
in animals receiving the combined treatments,
indicating that neoplastic lesions developed earlier inthese animals. Microscopically. the same tumour histotypes were found in the irradiated group and in thegroup with combined exposure. The authors pointed
out the similarity between these findings and those inuranium miners and proposed their rat tumour system
as a good model system for the human situation [C7,L8].
162. Another interesting aspect of the laboratory
experiments with rats which is in accordance with someresults from epidemiological studies on uranium miners
is the complex dependence of the lung tumour yield in
the animals on the exposure rate and on the level of
exposure. Human data strongly suggest that lungcancer may be produced more efficiently at low than athigh exposure rates [L6, Kl], in the sense that per unit
dose higher incidences of tumours are produced al low
than at high dose levels. It should be pointed out thatlow doses are usually obtained at low dose rates. In theexperiments of a French laboratory the incidence oflung cancer in the rat per 106 WLM changed from over
200 at cumulative exposures of around 175 WLM to 46
at 8000 WLM [L8].
163. The above mentioned French group investigatedin further experiments the temporal aspects of a
combined treatment in rats of radon daughters andtobacco smoke, by reversing the order of administration of radiation and tobacco smoke with respect tothe previously cited experiments [C7, C8]. Tn this caseradon exposure followed exposure to smoke [L9],without any enhancement of carcinogenesis. This observation is in keeping with the notion that tobacco smoke
has a promoting action. It was not possible to examine
the relationship between the level of exposure to smoke
and tumour incidence, since higher levels of exposure
led to a toxic action of some tobacco constituents and,on the other hand. lower exposures required an
excessive number of animals for statistical validation of
the data.
164. The effect or grading the exposure to tobaccosmoke may to some extent be studied by the use ofchemicals which are constitutents of tobacco smoke ortar, although it should be kept in mind that in this casethe mechanism of action could be rather different.
Morin et al. [MS] examined the effect of inhaled radon
daughters in combination with the I.P. administration(25 mg/kg/week for 13 weeks) of benzo-5, 6-flavone(BF). a substance which is not in itself a carcinogen.
Treatment with BF was started at three months after theend of radon exposure at 6000 WLM during about twomonths. One hundred percent of the animals developed
lung tumours (multifocal, invasive epidermoid typewith a latency period of 3 months), as compared withan expected 50% within 15 months after radiation
exposure given alone. When llF administration wasstarted 16 months after radon exposure, no difference
was seen with respect to the group receiving only
radon. This was taken as evidence that the promoting
action of BF was exerted during the period of latencyof the radon induced malignancies.
752

165. Grading the exposure to BF (25, 9, 3 mg/kg/
week) and to radon daughters (6000, 3000, 500 and 100
WLM) gave 12 possible combination groups [L9]. Preliminary data showed that the reduction of the latent
period was dependent on the product of the parameterscharacterizing exposure to each agent, as though alower dose of one could be compensated by a higher
exposure to the other in a multiplicative manner. Sucha dependence resembles to some extent the "relativerisk model" proposed by Lundin et al. [L6] to accountfor epidemiological data in uranium miners.
166. Modelling of chronic inhalation of radon
daughters and tobacco smoke simultaneously was
carried out on experimental animals at the Battelle
Northwest Laboratories [Cl 6]. The temporal aspect of
the administration of the combining agents differedfrom the experiments of the French group alreadyreviewed [CS, C6]. where exposure to smoke followed
the radon treatment. The experiments comprised
seventy beagle dogs: twenty of them were exposed toradon. uranium ore dust and cigarette smoke: twenty tosmoke only; and twenty to radon plus uranium ore
dust. The other animals served as the controls.
Exposure to tobacco smoke was performed throughspecial masks during several daily sessions.
167. Animals that developed lung tumours had in
general cumulative exposures to radiation in excess of
13 OOO WLM. This dose level is about two orders ofmagnitude higher than that reported to cause lungcancer in man. The possibility was therefore consideredthat the longer life span of the human species mightallow more tumours to appear while. for the same
tumour incidence, much of the exposure in dogs wouldbe "wasted", i.e., ineffective in producing additional
tumours. Differences in histotype between human anddog respiratory neoplasms were also noted. Cigarettesmoke had a reducing effect on the radiation lungcancers (2 cases out of 20 animals) as compared toanimals non-exposed to smoke (8/20). It was suggested-but in the absence of direct experimental evidencethat smoke through an increased production of mucus
might result in a lower dose of radiation to the target
cells; alternatively, smoke might stimulate mucociliary
clearance. Changes in the lung that were associated totobacco smoke were emphysema, chronic bronchitis
and bronchiolitis, lung fibrosis. The antagonistic effectof tobacco smoke on lung tumors induced by radon
daughters was confirmed in a very recent report ofthese experiments [C22].
168. ll may thus be concluded that reasonable doseresponse relationships for lung tumour induction inexperimental animals may be obtained for exposure toionizing radiation. The separate effects of tobaccosmoke may also be studied, but testing their combined
action poses serious problems. The temporal sequenceof administration is very important; there are probably
differences in target cells with respect to the two agents;
there may be other unknown factors complicating thepicture; the mechanisms of induction have not been
sufficiently clarified. It may be tentatively proposedthat a common feature of many experiments in animals(and of some epidemiological series in man) is apromoting action of the smoke (or some of its constituents). leading lo a shortening of the latency in tumour
appearance. Whether this might be due to a nonspecific stimulating action on the proliferation of therespiratory cpithelia or to a specific effect of somesmoke constituents is impossible to say at present.

3.

Epidemiological c,·idence

169. Uranium miners are exposed to radon and radon
daughters. They represent the first occupational groupon which extensive epidemiological surveys were madeof the effects of radiation in combination with tobaccosmoke. The exposure levels for this group of workers
are usually expressed in WLM: for the equivalence orthis operational unit with other radiation units, see
Annex D. In an epidemiological survey [L2) 3414
miners exposed to up to 104 WLM from the year 1950
were followed up to September 1967. Against 251
deaths expected during this interval of time. 398 deaths
were actually observed. the main causes for the excess
being violent deaths (120 observed versus 51 expected)
and malignant tumours of the respiratory tract
occurring ten or more years after beginning of work in
the uranium mines (62 observed versus 10 expected).
The time relationship and the increase in cancer
mortality as a function of radiation exposure indicate a
causal relationship between the two variables.
170. Information about the smoking habits of theminers were collected during the survey and also in anannual census of uranium miners which was started in
1963. Standardized mortality ratios of lung cancer bysmoking categories [H3] were used for calculation of
the expected death rate for respiratory cancer. The
reference population was a random sample of adultmales from the United States and the ten expected cases
mentioned above were calculated according to these
data. It was found instead that the cases expected
would be 16 for the same total population of 3414
miners if the lung tumour incidence among males offour Colorado plateau states in the United States would
be taken as the reference control. Table 3 shows the
distribution of observed and expected respiratory
cancer deaths between smokers and non-smokers. The
increase in the number of cancer cases is attributable toirradiation by inhaled radon daughters. The relative
excess of risk between smokers and non-smokers is thesame (3.9 against 4.0 for the two categories. respec-.
tively). If one calculates the increase in . cancer
incidence due to irradiation per person year at nsk, one
finds 1.7 10-3 for smokers and 1.7 1~ for non-smokers.
the difference being attributed by some to a 10-foldsynergistic increase of the risk for the smoking miners.
171. A more accurate analysis shows however that this
could be a misleading argument. It should be realizedthat the statistical significance to be attached to thenumber of tumours observed in the non-smoking groupis very low, owing to the small number of cases
observed. The estimate of the probability of tumour
induction obtained from this number is therefore
affected by a large error. A statement such as thepreceding one of a ten-fold increase in risk in thesmoking population. would be equivalent to using forthe assessment of the interaction factor the formula172. According to the reasoning presented in chapter
I of this Annex. when Po1 and P0 2 arc small, one calculates the interaction factor ro by the formula
(1)

=

P01 -

Po

(37)

Pol + Po2

Table 4 shows the results of separately analyzing thedata for the period 1950-1967 [L2] (A: top line) and forthe last four years of the same period, 1964-1967 [A I J(B: bottom line). As may be expected, risk estimates
based on the most recent period of observation are
higher. excluding re-evaluated estimates of spontaneousrisk and risk of smoking. The interaction factors are
however close enough to each other and indicate asynergistic interaction. In view of the low statisticalsignificance of the results. other indirect evidence may
be of great value.
173. Archer and collaborators [A 1, A3. L6] point out
some of this evidence. In a larger group of uranium
miners 207 lung cancers were identified; all of these
individuals except three were cigarette smokers [Al].
Since it is known that 71% of miners are smoking, it
is clear that in the above group smokers are overrepresented. Another observation relates to the age
at diagnosis: in the 207 people mentioned. 17 stopped
smoking eight or more years before diagnosis: 16
stopped between four and eight years; 19 smoked lessthan 15 cigarettes/day (light smokers). Controls werechosen to match as closely as possible the exposed
individuals in relation to age at the start of mining.
cumulative radiation exposure, years of hard rock nonuranium mining. All of them smoked 20 or morecigarettes per day and none stopped smoking morethan one year before diagnosis. The comparison
showed that non-smokers or those who stopped
smoking eight or more years before developing lungtumours had a mean age at diagnosis three years greaterthan smoking controls. Light smokers differed fromcontrols by a year and a half, and those who stopped
smoking between four and eight years before diagnosis
differed from controls by less than one year. The results
support the hypothesis that cigarette smoking acts inthese miners as a promoting agent [VJ] by decreasing
the length of the latent period. These conclusions werestrongly supported by an update of the earlier uranium
miners mortalitv studies in the United States [A3]. The
incidences of l~ng cancer between different categories
of smokers are shown in Figure XVIII [A3).

.,
VI

0:
IU If

>-I

z

0

"'ffi"
Q.
0
0

o,

~.
"'
z

(36)

:5
fr

.,...

1-

0

where the signs 1 and 2 refer to smoking and radiation,
respectively. In fact, this formula cannot be used under
the circumstances, because of the mentioned low statistical significance of the term P0 2 (the probability of
respiratory cancer death in the non-smokers) and of theabsence of the term Po1 from the numerator and denominator.

"'Cl.':::"'

Hon-smoker~.,.,
WORKING LEVEL IIONTttS

Figure XVIII. Mortality from respiratory cancer
as related to radon daughter exposure in different smoking groups (A3]753

174. High rates of lung cancer in smoking persons arealso observed in workers of industries that use known
carcinogenic substances such as chromates (0 I, L7] andasbestos [SlO]. In addition, among persons developing
lung cancers in the same groups of workers, smokers
were over-represented. These data may be taken toshow a non-specific promotive innuence of tobaccosmoking. At the present time there is by no means a fullunderstanding of these mechanisms in smoking
individuals. It could be that tobacco smoke contains
enough initiators and promoters to give the observed
yield of respiratory cancer. Alternatively, in case ofsmoking acting apparently alone. some environmental
factors may provide the initiating stimulus and the roleof smoking might be essentially promotive. The
chemical composition of smoke itself might even not be
the decisive factor in the promoting action of this agent.
As shown by experiments of Little et al. [LI OJ reviewed
previously, the irritation of the respiratory epithclia by
non-specific physical or chemical agents (instillation ofsaline solutioris, for example) could have a promoting
effect.
It could of course be debated if promotion as
175.
such is an effect to be included under the general
heading of synergistic. Two extreme situations may be
visualized in this respect. There could be, on the one
hand, a forward displacement in time of the tumours
appearing, but with a final yield of tumours notdifferent from the situation in which promotion is notoperating. Alternatively, a continuously increasing rateof tumour appearance might take place, leading finallyto an incidence higher than that in the absence ofpromotion. A variety of intermediate situations could
also operate between these two extremes. Clearly, if the
final tumour incidence would be taken as the reference
end-point, the first of the two silllations depicted would
not come under the definition of synergism, while thelatter would. But if instead, more correctly, the lengthof tumour-free life lost is taken to be the reference
parameter and it is assumed that smoking alone could
cause cancer, both situations. as well as all the intermediate ones, would be rightly described as synergistic
interactions.
176. In their 1979 paper Lundin et al. [L6] gave a moreelaborate quantitative treatment of respiratory cancer
death in uranium miners. A log-normal distribution of
the time elapsed from exposure to diagnosis based onother experimental and theoretical evidence [M4] wasassumed to apply. It was also assumed that this distribution would have a standard deviation of 0.17609 in log
t units, where t is the number of years elapsed after the
beginning of exposure. The choice of the standard
deviation was rather arbitrary and, according to criteria
developed in reference [M4]. somewhat below the range
expected usually. The parameter describing exposure(when the risk from earlier was added to that of later
exposures) was the Eff WLM (k) for the year k. definedas

Eff WLM (k) =l: w (k - j) WLM U)

(38)

J

where O<j<k; w(k-j) is the proportion of the area
under the log-normal distribution density curve which
is bounded by the interval from (k - j - 1 /2) years to(k - j + 1/2) years: and WLMG) is the exposure in
WLM during the year j.
177. Two alternative hypotheses were examined: thatthe increase in absolute risk might be proportional toradiation exposure, in which case the risk increase754

would be independent or the rate associated withcigarette smoking. aging or other environmental factors(a): that the increase in relative risk may be proportional to radiation exposure (b). In this case theincrease in risk should be proportional to that risk
which would have affected the miners in the absence ofradiation. The analysis of both models was carried out
based on the form of the temporal distribution of therespiratory cancer deaths.
178. Three temporal parameters were used, namely,
the age of the miners. the calendar year and the years
after the beginning of exposure. Computations were as
follows: (a) for the absolute risk hypothesis
nx = f1A X,ad

(39)

where Xrnd = av (Eff WLMx), In the above formula nx
is the predicted excess of lung cancer deaths amongminers in stratum x and the av (Eff WLMx) is averaged
over all the person-years at risk in the stratum; (b) for
the relative risk hypothesis(40)
in which nx is proportional to the product of the
expected number of lung cancers Ex in the stratum,
multiplied by the exposure X,ad, The symbols fLA andCf.R in the above equations are coefficients applying to
the two situations postulated. The sum of nx + E,. gives
the total predicted number of lung tumours for eachparticular set of parameters.
179. Calculations were made of the number of deaths
according to the age category and with assumptions ofmean latency times of 5, 10 and 15 years. The relativerisk model gives results which are closer to observations
and latency times of 10 to 15 years fit the data best. Oneof the parameters which is most strongly influencing
the expected number Ex is the smoking category (Table5). It may be assumed that Ex is proportional tosmoking exposure Xsm, Then the predicted excess of
respiratory tumour deaths will be proportional to both
smoke and radiation exposure as(41)
The probability of developing lung cancer per person
per year is shown in Table 5 for four smoking
categories. The last column of the table gives the corresponding interaction factors and shows that the highest
value is for former smokers and the lowest for heavy
smokers, (!) being intermediate for light smokers. This
result comes about through an insufficient increase in
Pot by comparison with Pt1 for heavy smokers, an observation which contradicts the previous conclusion about
the applicability of the relative risk model. The authorsinterpret this observation as evidence against a possible"synergism" defined as an increase in the total
radiation risk of lung tumour development. Such a risk
they consider to be approximately the same for all
categories of smokers and somewhat higher than for
non-smokers. They classify the observed increase in thelung tumour death as promotion. However, as alreadydiscussed before. there is good ground to describe it assynergism (see paragraph 175).
180. Lundin et al. [L6] do not exclude the possibility
that for longer time intervals the yield of lung tumours
in non-smokers might be the same as that of thesmokers exposed to radon daughters. One preliminaryreport on lung cancer in Swedish iron miners seems to
support this possibility [R 15]. In an other epidemiological study of metal and iron-ore miners in Sweden
carried out by Axelson and Sundell [A9] the risk for the
non-smokers was claimed to be higher. However. thesize of the groups analysed was rather small and thestatistical significance of the observed effects correspondingly low. Also, the methodology of the casecontrol study was not fully described in the publication[A9) and it raises some questions in the form presented.

181. Long latency time for lung cancer and theincidence dependence on the dose rate could also
obscure the final picture. Uncertainties in the distribution of miners between exposure categories could leadto distortions in the estimates of risk. The lower risk of
the American uranium miners could be justified tosome degree by their possible misclassification intohigher exposure categories [S 11. K 1]. It should also bementioned that for these miners the observation time
elapsed from the beginning of work in the uranium
mines is not much longer than 20-30 years. which might
be insufficient for the development of lung cancer
among non-smoking individuals. Another importantfactor could be the exposure rate which was lower for
the Swedish than for the miners in the United States. It
has already been mentioned that lower exposure rates
may bring about a higher total yield of potential
tumours. Different exposure rates can be met in
epidemiological studies with Czechoslovak [Sl 1, Kl]
and Canadian [H20] miners.
182. Enhanced mortality for chronic respiratory
diseases other than cancer resulting in pulmonary insufpulmonary
fibrosis.
ficiency(pneumoconiosis.
emphysema) and for acute conditions (pneumonia.
asthma) could also be the result of combined radiationand tobacco smoke exposure [A3]. An increase in therate of mortality from these diseases for uranium
miners in the United States was clearly observed whichmight be related to radiation exposure. It is interesting
to note that the rate is highest for light smokers. so thatat high exposure levels mortality is twice as high as forheavy smokers. Some possible interaction between
radiation and smoke is also evident for these diseases
but, at this point, the contribution of other ambientconditions like siliceous dust or diesel fumes should
also be considered, for which data are very scarce.
183. The epidemiological data discussed point to asynergistic interaction between tobacco smoke andradiation exposure in the sense discussed underparagraph 175. Non-specific effects induced by somecomponent of tobacco smoke could be responsible forthe results described. Thus, changes in the productionof mucus, a slower rate of clearance of radioactive
particles by the ciliary action and metaplasia of theepithelia might result in a higher dose delivered to thetarget cells in smokers than in non-smokers. Against
this general proposition is however the observation that
promotion by tobacco smoke is still found when smoke
is applied long after radiation exposure. Clearly. thesequestions cannot be settled now with the limited information available. Quantitation of the degree ofsynergism is also impossible with the necessary degreeof precision and significance, owing to the low numberof tumours observed, particularly among the nonsmoking individuals. and to the complex temporal
pattern of lung cancer development.

E.

OTHER DRUGS

184. It may appear somewhat artificial to separate inthis section substances which are utilized for theirpharmacological properties in clinical medicine fromother organic substances mostly developed for theirradiosensitizing actions. The separation may be madeon the ground that interaction with radiation may be
incidental in the former case but is pursued as a specificgoal in the latter. In spite of a widespread andincreasing use of many drugs in modern societies, it isdifficult to visualize situations where the combined
effects of any of them with radiation may pose significant problems in public health. The cases of interaction in the treatment of specific diseases where the
combined use of radiation and drugs might increase therisk of undesirable effects on the patient may be more
important. However. in most of the work reviewed
radiation doses were very high and, irrespective of thenature of the interaction (synergistic or antagonistic),
extrapolating the findings to lower levels may be very
difficult or impossible in view of the modification of
the form of the dose-effect relationships that mightoccur at low doses.
185. Antibiotics are widely used in clinical medicine
and some of them are also used in combination withradiation for cancer chemotherapy [P3. P4, PS, P16].
Among them. actinomycin D was shown to have asynergistic interaction with radiation on Chinese
hamster cells in culture. Elkind et al. [E3] related thiseffect to the ability of the drug to impair recovery ofsublethal damage. as shown by a reduction of theshoulder of the survival curves at low doses (2-5 10-3
~Lg/ml). Ten-fold higher doses given before irradiation
increased the exponential slope of the survival curves.
Time was also an important parameter in these experiments because no synergism was observed with treatments by actinomycin later than 10-12 h post-irradiation or when the drug was applied more than six hours
before irradiation [E4).
186. The molecular basis for the action of actinomycin D on cells is due to its proven ability to bind to
DNA and thus to create a steric hindrance to thesynthesis of RNA. Interesting studies on the combined
action of this drug and of another drug, cordycepin.
were reported on two cell lines in culture by Robertson
et al. [R13]. For actinomycin D interaction factors of
1.2 and 1.3 in the two cell lines were reported with xrays. The survival parameters affected were both theexponential slope and the shoulder of the survival
curve, but mostly the former. With cordycepin the interaction factors were 1.1 and 2.2. respectively, and themain parameter affected was the extrapolation number.
The nature of the differences described led the authors
to some hypothesis on their molecular basis.
187. Actinomycin D was also tested in preimplantation mouse embryos in tissue culture for its combined
effect with radiation [S15]. The concentration of the
drug was here several orders of magnitude lower (1~~Lg/ml) than in previously reported experiments byRobertson [R13] and the drug alone was ineffective atthese concentrations in retarding the development of
the embryos to the blastocyst stage. Combining the drugwith tritiated water led to a higher effect than that oftritiated water alone. with interaction factors between 2
and 4. depending on the tritium concentration in theculture medium. The lower values were observed at thehighest concentrations. This observation could be
explained by the more effective inhibition of the repair755

processes at low radiation doses. A re-analysis of thesedata in terms of isobolic diagrams [S48] showed that theresults of combined treatment fell clearly outside of theenvelope of additivity in the direction of synergism.
The possibility that the shape of the dose-response
relationships may be changed hy the combined treatments has been discussed in this context [S39). Other
experiments showing a radiosensitizing action of actinomycin D have also been reported [M7].

exposures to DHAQ was the end-point of this study.
DHAQ had a toxicity which was more pronounced
during the early phases of the cell cycle. After
combined treatment a synergistic effect was noted forcells in the S phase, but in all other phases of the cycle
additivity prevailed. 1n asynchronous populations
DHAQ enhanced the radintion-induced cell lethality
primarily by increasing the slope of the radiation dosesurvival curve.

188. In humans, Wara et al. [W2) studied the effect of
actinomycin D on the induction of radiation
pneumonitis occurring 1-3 months after the irradiationof Jung metastases in 41 patients. Doses of the order of
7.7 Gy were necessary to elicit pneumonitis in 5% of thepatients and these doses were reduced to 5.5 Gy (DMF
= 1.4) when an actinomycin treatment was given alongwith radiation. Similar DMF were obtained forradiation-induced intestinal
inJury,oesophageal
lethality and pulmonary lethality in LAF1 mice treatedwith this drug at the same time [PS]. The values of the
DMF obtained for a variety of chemotherapeutic drugs
in the above experimental series are given in Table 6
and a detailed review of experimental and therapeuticfindings related to this topic has been written by
Phillips and Fu [P3]. In patients treated for erythema of
the skin d'Angio et al. [06] reported a DMF of 3.4 bycombining x irradiation and actinomycin D.

192. Lucanthone (J'vliracyl D) has long been used in
the treatment of schistosomiasis. The drug has a heterocyclic ring structure resembling that of actinomycin D.
A synergistic type of interaction of this drug withionizing rndiation has been shown for HeLa cells. Thiseffect decreased with the time lag allowed between
radiation and the treatment with Jucanthone [84]. The
same publication refers nlso to increased 30-day
lethality in mice given 4 Gy of total body radiation anda simultaneous injection of 180 mg/kg of the drug.

189. Dritschilo et al. [D7] investigated the mechanismsof the combined action with radiation of actinomycin
D and adriamycin. Non-toxic levels of actinomycin
and minimally toxic levels of adriamycin produced
suppression of potentially lethal damage repair inplateau-phase Chinese hamster cells in culture. For
actinomycin this suppression persisted as long as the
drug was present in the culture medium, but as soon as
it was removed prompt repair took place. Thissuggested that suppression did not act through fixationof injury to a non-repairable state. Adriamycin was
different because cells exposed to it could eventuallyproceed to repair potentially lethal injury even in thepresence of the drug, after an initial delay of the repair
processes.
190. Redpath et al. [R4) studied the effect of
combining adriamycin (2 or 1 mg/kg, 5 daily fractions)and x irradiation (10 Gy/fraction. 5 daily fractions) on
mouse tissues. Enhancement of damage was seen forlung and foot skin damage, when the interval between
the beginning of the radiation and of the drug course
was within two to seven days. In another series theradiation sensitivity was studied after the single dose of
1 mg/kg intraperitoneally. No effect was found in thiscase. Experiments were performed in the same laboratory on the radiosensitivity of ICR male mice
irradiated whole-body with fast neutrons (mean energy
25 MeV) or photons (6 MeY). in combination with asingle dose of adriamycin (10 mg/kg) [C9]. The LDso/6
for photons was reduced from 13 to 10 Gy; that ofneutrons from 5.6 to 4.3 Gy. The RBE for gut damage
was unaltered by the addition of adriamycin. The data
indicated that for drug administration 16 hours before
or after the radiation exposure the interaction will be
the same.
191. A cell cycle dependence of the synergistic interaction of a drug with radiation was shown fordihydroxyanthraquinone (DHAQ), a potential cancer
chemotherapeutic agent similar to adriamycin andactinomycin D [KS]. The survival of x-irradiatedChinese hamster cells in combination with different
756

193. The influence of lucanthone in combination with
x-irradiation was also studied on Y-79 cells and on
spheroids [D3]. The treatment of asynchronous cellswith 5 ~tg/ml of the drug led to a progressive decreasein the proportion of cells in G1 and to an accumulationof S-phase cells. The toxicity of the drug was noted only
during this latter phase of the cell cycle. In general, thesurvival of the single cells after the combined treatment
was lower, owing to a reduced capacity of the cells toaccumulate and repair sublethal damage. For equallevels of drug toxicity, the radiation-modifying effect ofthe drug was greater in the spheroids. pointing to alarger interaction in the system which has greater
capacity for accumulation and repair of the sublethal
radiation damage.
194. Lucanthone has also been shown to be active inrespect to induction of developmental defects in mice.
Pregnant animals (8 days p.c.) were given 70 mg/kg of
the drug and treated one hour later with 0.5 Gy of xradiation. The treatment resulted in a distinct synergistic increase of the eye abnormalities of the embryos[M8]. The above studies were further developed with adecrease of the x-ray dose down to 0.01 Gy [M9].
Pregnant mice of the strains FI A and NMRI were
irradiated at eight days p.c. with 140 kVp x rays. with orwithout treatment with lucanthone. The foetuses wereobserved 4-5 d after irradiation for the presence ofmacro- and microscopic developmental defects (postimplantation loss. growth retardation. eye abnormalties. exencephaly. cleft palate and limb defects).
There was a strain specificity with respect to the sensitivity to lucanthone given alone, the NMRI mice being
more susceptible to lower doses of the drug. A dose of
0.01 Gy was reported to produce a statistically significant increase of the abnormalities and combination of
the two treatments gave rise to a synergistic interaction.
Some strain specificity was also found for the combined
effects, because the F/ A mice were more susceptible tothe joint action. Higher doses of radiation (0.5 Gy)
producing an approximately 4-fold increase of thecontrol abnormalities were also reported to produce
synergism. Other data on the enhancement of radiation
effects by antibiotics (ledermycin, reverine) were also
reported [M9].
195. Bleomycin was reported to potentiate theradiation damage in rat brain tumour cells of the line
9L [I-19). The drug enhanced cell lethality mostly
through an increase of the slope of the radiation dosesurvival curve, its D0 decreasing from 3.7 to 2.1 Gy inthe presence of the drug. There was also a more modest
decrease in the capacity for accumulation of sublethal
damage, shown by a decrease of the Dq from 3.2 Gy in
the absence to 2.9 Gy in the presence of the drug. Thiswas evidence for an inhibition of repair of the sublethal
radiation damage. Other authors observed an additiveeffect of bleomycin and x rays with small doses of theantibiotic and a synergistic action with high doses [1330].

196. In experiments by Lin t!l al. [L20] Chinese
hamster cells in culture were treated with the drug
diethyldithiocarbamate (DDC), a substance that has
been shown to inhibit the enzyme superoxide dismutase
responsible for eliminating the o; radical from thecells. The killing effect of the drug depended on its
concentration and exposure time. Eight to ten days of
incubation at concentrations of 1Q-9 M produced nochanges; while 10-4 M was a definitely toxic level.
DOC-treated cells later undergoing gamma irradiation
survived less well than cells treated with the same dosesof the two agents separately. The drug also significantly
enhanced the heat sensitivity of the cells.
197. Antimalarial drugs such as quinacrine and chloroquine or their derivatives are now also used for
diseases requiring long-lasting treatments such as
rheumatoid arthritis. Sensitization by these drugs incombination with radiation was shown on a variety ofsystems. E.coli K-12 rec+ incubated after irradiation
with quinacrine up to 0.4 mM showed a killing increase[FI]. Chinese hamster cells [V2] as well as tumour cells in
vitro [K2] were also reported to show similar effects. In
vivo Utley et al. [U3] showed a distinct effect on skinsensitivity, as judged by the hair loss in irradiated rats
treated intraperitoneally with daily doses of hydrochloroqui ne (52 mg/kg) for one week prior to irradiation.
A case of skin sensitization in a woman who had been
taking hydroxychloroquine daily (200-600 mg/day) for
years prior to and during radiotherapy for a breast
tumour was reported in the same paper.
198. Enhancement of x-ray induced cell killing bycaffeine treatment for 16-20 hours post-irradiation wasshown on a number of rodent and human cells in
culture. The effect was brought about by concentrationsof the drug causing less than 15% killing. It consisted
mainly in a removal of the shoulder of the survival
curve. without much alteration of its slope. The findings
indicated the existence in mammalian cells of processesof repair of radiation damage that are inhibited byexposure to the drug [WS]. Enhancement of the killing
effect of single doses of 60Co by caffeine was also
confirmed on human and hamster cells in culture atconcentrations of 2.0 - 2.5 rnM in the culture medium
for two days after irradiation [S23]. In split-dose experiments exposure to caffeine of the cells for 4 hours
between dose fractions did not result in any effect andit was therefore concluded that the sensitization bycaffeine was brought about through a modification of
expression of the potentially lethal. rather than of thesublethal. damage. The possible role of caffeine as asensitizer of the single-hit potentially lethal damage wasfurther confirmed experimentally and through ananalysis of the existing literature [S27].
199. The sensitization by caffeine on x-irradiated
HeLa cells was found to depend on the drug concentration [T6]. At post-irradiation levels of caffeine of 1
mM a synergistic effect was observed mainly on cells in
the G 2 phase, irrespective of where in the cell cycle they
had been irradiated. Increasing the caffeine level to arange of 7 to 10 mM brought about not only a higher

response of the G2 cells. but also some response of the
0 1 cells. Treatment at higher concentrations resulted indose-survival curves having smaller shoulders andsteeper terminal slopes. It is interesting to point out thatthe shapes of the time-survival curves measured for the
0 1 and G2 cells differed. The authors interpreted thisdifference to reflect two operationally distinct modes ofinteraction with the drug.

IV.

BIOLOGICAL AGENTS

A. GENERAL
200. Many biological conditions may influence thestate of health of a human population. Viral andbacterial infections. eating habits and the state ofnutrition, the conditions of living and working, the useof biologically active substances or drugs are known toaffect to various degrees the incidence and pattern ofdiseases in humans and therefore to alter the actuarial
characteristics of populations. There seems to be little
hard evidence that conditions adversely influencing thesurvival and disease incidence could also substantiallychange man's sensitivity with respect to late radiation
effects. This notion cannot however be dismissed ·or
excluded because the complex pathogenesis of theeffects of major interest in human radiation biology(tumour induction, genetic changes. developmental
abnormalities) leaves scope for combined actions inboth directions.
201. The Committee wishes to stress that the above
general notion is much easier to be entertained academically than to be experimentally demonstrated. The
agents that may be considered as possible candidates
for interaction with radiation are very many and diversified; their influence, already in the absence ofradiation, is often little known in respect to the effects
described above; and the data available arefragmentary. Therefore, to attempt a systematic
discussion is almost impossible. In spite of such limitations, the Committee has decided to gather the available
evidence on the effects of hormones and the effects ofinfections. Reference to existing epidemiologicalstudies will also be made.

B.

HORMONES

202. The inOuence of hormones on the radiation sensitivity of human populations with respect to cancer
induction can be predicted on the general notion thatmany experimental and human tumours arc known to
be variously susceptible to the action of hormones.
Mammary gland. prostate and thyroid tumours are very
hormone-dependent, while for other malignancies acertain degree of dependency may be postulated, forexample, on the notion of a different susceptibility
between sexes or on the effect of castration. As to thepractical significance of a combined action of
hormones and radiation, changes of the hormonal statetake place during physiological conditions (menarche,pregnancy. menopause, stress). Treatment of many
diseases requires prolonged use of hormonal preparations and an increasingly large part of the female
population use hormonal treatments (essentiallyoestrogens) for contraceptive purposes. Oestrogens arealso contained in commercially available cosmetic
preparations and some drugs used rather extensively(derivatives of Rauwolfia, phenothiazine, chlorinated757

hydrocarbons)
have hormonomimetic
act1vl!1es.
Finally, hormones themselves could be to some degree
carcinogenic [C4]. There appears. to be therefore sufficient ground for some analysis of their combined
actions with ionizing radiations.
203. Much general evidence about tumour induction
in animals has been discussed in the 1977 report of theCommittee, Annex I [UIJ. It was concluded that various
radiation-induced tumours arc differently affected by
the animals' hormonal balance during the course of the
carcinogenic process. The effects reported seemed to betumour-, strain- and sex-specific and it appeared likelythat the mechanisms of action (which are at present
almost unknown at the molecular or even at the cellularlevel) might have been very different under the various
conditions tested. Annex K to this report contains somediscussion of the innuence of the animal's sex on thelife shortening action of ionizing radiation. Thisappears mainly as a higher susceptibility to sex-specifictumours, particularly the mammary neoplasms and thetumours of the genital tract in the female.
204. Segaloff and Maxfield [S7] studied specificallythe inOuence of oestrogens on mammary carcinogenesis in the rat. Pellets containing 5 mg diethylstilbestrol (DES) and 15 mg cholesterol were implanted
subcutaneously into 8-week old A x C rats. The animals
were hysterectomized to prevent fatal oestrogen-induced
uterine infections. X-radiation was delivered only tothe left mammary chain by shielding the opposite one.
Spontaneous mammary tumours in this strain of rat are
essentiallynil.Radiationalone(about8Gy)producedonlya small numberoftumours{l.1 per chain at risk)appearing
late( median 80weeksat the a ppearanceofthe first tumour).
DES alone gave 1.7 tumours per chain with medianappearance times of 33 weeks. Combined treatments
resulted in an earlierappearanceofthetumours(26 weeks)
and in an increased incidence ( 5.6 turnou rs/ chain). Even acrude estimate based on final incidence would lead 10 aninteraction factor roof the order of 2, an estimate which(apart from its unknown statistical value) fails to takeaccount of the appearance time which is shortened by thehormonal treatment.
205. Shellabarger et al. [SS] irradiated with 0.43 MeV
neutrons rats of the strain A x C in doses of 0.096 Gy.
The carcinogenic response to irradiation was insignificant (3 adenocarcinomas in 33 rats); DES, on theother hand, produced some effect (182/25 rats).
Combining the treatments led to an earlier appearanceof tumours in much greater number (842/35 rats).
There were therefore strong indications of a synergistic
interaction and a crude estimation of o> is in the rangeof about 3. However. on Sprague-Dawley rats the samecombined treatment produced a negligible incidence oftumours (2/31 rats) in comparison with the action ofradiation alone (11/31 rats). DES in this case had nosynergistic but rather an antagonistic effect. The experiment is a good example that, depending on the strain
used, the same type of treatment may give rise to antagonistic or synergistic actions.
206. Most recent experiments by the same group [H26]
confirmed the effect of DES and showed, in addition, asynergistic action of 17-ethinyl-estradiol (EE2) in rats.
The complex of these data would imply that the synergistic interaction is not with the hormones examined
but rather with their oestrogenic activity. In otherstudies by Segaloff and Pettigrew fS5] graded doses ofradiation of 0.5, 1.5 and 4.5 Gy were used. Radiation
758

given alone increased the incidence of benign tumoursin proportion to dose. but the increase of malignant
tumours did not follow a statistically significant proportionality and resulted in a rather low number.
Combining radiation and DES produced a synergistic
interaction at 0.5 Gy (w == 1.4), but the increase intumour incidence was most pronounced at 1.5 Gy(crude w ""' 2.0) and somewhat less at 4.5 Gy (crude w= 1.6). The combined treatment led to an earlier
tumour development.
207. The role of prolactin in combination withradiation and with the chemical carcinogen N-nitrosoN-buthylurea was studied by Yokoro et al. [Y2] in
W /Fu rats. Prolactin was produced by grafting a
mammotropic pituitary tumour. Prolactin alone wasineffective in inducing mammary tumours. After dosesof 2 Gy of x rays two fibroadenomas were seen among
27 animals with mean appearance times of about 6
months. J>rolactin in combination with irradiation
accelerated tumour appearance and induced tumours in
60% of the animals at the dose of 2 Gy. There were
statistically significant differences in the tumour
incidence between animals receiving 0.5 or 2.0 Gy anda similar synergistic interaction of prolactin andradiation was also found in respect to 14 MeV
neutrons. Two interesting observations were made inthese experiments. First, delaying the pituitary graft aslong as seven months after irradiation still produced anenhanced effect, showing that the transforming lesions
induced by radiation could remain available forhormonal interaction for a very long time. Secondly,
most of the tumours produced by the interaction wereadenocarcinomas, while most of the spontaneously
occurring ones in this strain of rat are late appearing
fibroadenomas.
208. In a more recent study by the same laboratory[Y6] fission neutrons (2 MeV mean energy) mixed withgamma rays were given to W /Fu rats. Only 2% of theanimals developed mammary tumours after irradiation
alone (up to 0.2 Gy) but 42% did when prolactin wasgiven shortly after irradiation by grafting the prolactinsecrcting pituitary tumour. Delaying the prolactin
treatment up to 12 months produced 24% tumours,
which observation .supports the one previously reported[Y2]. A similar synergistic interaction of diethylstilbestrol (DES) and neutron irradiation in the productionof mammary, pituitary and hepatic tumours wasobserved in castrated male W /Fu rats [S41].
209. The above results suggested to Yokoro [Y2] thatin the previous studies the synergism between DES andradiation [SS, S7] could act via an increased productionof prolactin, At the same time, Shellabarger [S6] wasable to show that A x C rats (in which interaction withhormones was found) carried prolactin-secreting
pituitary tumours; on the contrary. the Sprague-Dawley
rats which did not show any synergism between
radiation and DES carried no such tumours. In recent
experiments by the group of Shellabarger {S44. H26] on
A x C rats a strong dependence was shown between theinteraction factor and the dose of DES and radiation.
The dependence on the DES dose appeared to be
mediated via the oestrogenic stimulation of prolactin
secretion. The higher and the earlier the levels of
prolactin in plasma, the greater was the yield ofindividual and multiple mammary adenocarcinomas.
210. Another oestrogenic hormone (polyestradiol
phosphate) and a corticosteroid (methyl-prednisolone)
were tested in combination with internal irradiation by

Nilsson et al. [N8] on CBA mice. Three doses of 90Sr
(0.925. 1.850 and 7.400 kBq/g) were applied, which led
to a maximum of 2% animals with pituitary tumours.
Polyestradiol alone produced 10% of such tumours.
Combining the treatments resulted in 44 and 37% ofanimals with tumours, for the first and the second doseof 90Sr, respectively. an increase corresponding to aninteraction factor of approximately 4. Combined
treatment also led to a decrease of the tumour induction
time with respect to the groups given the radionuclide
alone, close to that of the animals receiving only thehormone. Prednisolone in combination with radiation
was ineffective in increasing the incidence ordecreasing the induction time in comparison withgroups receiving strontium alone.

213. Although the most informative data on thesubject of combined action of radiations and hormones
can only come from epidemiological surveys, data inthis area are only indirect. It is known for breast cancer
induction that age at exposure is a major determinant
in all series available [T8, 1327. S47]. Taken together, thedata suggest that when the most profound hormonalchanges occur (menarche. menopause) the risk per unit
dose deviates most significantly from the mean risk for
the whole lire.

C.

INFECTIOUS AGENTS
I.

211. Modelling of situations in animals that may
operate in women taking contraceptive oestrogens wasundertaken in the Netherlands [B3, B5]. The complete
outline of these experiments calls for three different
strains of rat (Sprague-Dawley, Wistar Wag/Rij, Brown
Norway); four types of radiation (300 kV x rays and0.5. 4 and 15 MeV neutrons): a range of different doses(from 0.1 to 2 Gy, according to the radiation
employed); and various types of female animals (intact
or hysterectomized. respectively with or without
hestradiol-17-beta). The results of this series are still
incomplete. but some preliminary conclusions may be
drawn. For WAG/Rij rats the proportion of animals
surviving without tumours abruptly decreased startingfrom nine months of age after irradiation with 4 Gy x
rays and hormonal treatment. For animals receiving
only irradiation or hormonal treatment a 50% decrease
was observed after 22 months. The total yield of
tumours for the combined treatment group was also
higher. Considerable differences in the susceptibility totumour induction were found between strains. Brown
Norway rats having the lowest spontaneous incidenceof mammary tumours had an intermediate susceptibility to the radiation-induced ones. Pathological data
showed that malignant tumours were relatively rare in
the Brown Norway and in the Sprague-Dawley strains.
but were inste,1d quite common in the Wag/Rij rats.
amounting in the latter strain to nearly one-half of all
tumours. A synergistic interaction of radiation with theoestrogen treatment was manifested not only throughan increased proportion of rats with malignant tumours(from 0.43 to 0.83 in the Wag/Rij rats) but also throughan increased absolute incidence of neoplasia in
Wag/Rij and Sprague-Dawley rats. The minimum
latency period in untreated control animals could be inexcess of 22 months; in irradiated animals without
hormones this period decreased to 10-12 months and adecreased latency in the hormone-treated rats incomparison with untreated groups was observed as arule. The synergistic action of the hestradiol-17-beta isof the same type as the interaction between radiation
and DES.
212. Kennedy and Weichselbaum [K15] reported asynergistic interaction between cortisone and x rays fortransformation of C3H 10 T 1/2 cells in culture. The
end-point scored is of great significance since it relates
to tumour induction in vivo and the synergistic effectwas statistically significant at P < 0.001. However, thetransformation mechanisms in this particular cell line
are stiJI little understood [K 16) and it is not possible toquantitate the results in terms of transformation
frequency per surviving cell. It seems thus more
prudent to test for effects in vivo before accepting theconclusions as generally valid.

Viral infections

214. Viruses have a very important role in the pathogenesis of some radiation-induced experimental tumours
like the thymic lymphoma, the myeloid leukaemia andthe osteogenic tumours of different strains of mice. The
Committee has reviewed the relevant evidence inAnnex I of its 1977 report. It is difficult in fact in many
instances to separate the action of the virus from that ofradiation, because the interplay of the biological and of
the physical factors is in these cases so intimate that it
would not be possible to elicit the effect without thepresence of the two agents combined. To think of asynergistic effect under the circumstances would beinappropriate because none of the agents alone may be
active for the specific end-point. Moreover. the verticaltransmission of the viruses through succcssiYe animal
generations makes it a normal constituent of theirgenome, which is exactly the reason why some tumours
are specific to some strains.

215. Radiation enhancement of in vitro cell transformation by viruses has long been reported [P13. S45]. An
example was given of a combined treatment of \Vistar/
Furth rats with radiation and Gross mouse leukaemia
virus [Yl]. Animals aged 7-8 weeks were intraperitoneally inoculated with a standard dose of virus (0.4 mlof leukaemic filtrate) and none of the 15 animals
injected developed leukaemia. Whole-body x-irradiation (four doses of 1.5 Gy given at five days interval)
produced also no tumours in 12 irradiated animals. The
combination of both treatments gave rise to more than
50% leukaemias in 20 treated animals. ln view of thelack of effeccs by the separate treatments. the interaction factor would in this particular case be equal toinfinity. It \Vas suggested that radiation might haveacted through a modification of the physiological stateof the target cells by rendering them susceptible to theaction of the virus or through a modification of theimmunological response of the host.

2.

Bacterial infections

216. Environmental conditions have often beenreported to influence the induction of specific tumour
types in irradiated animals through their action on the
microflora. It is conceivable that the response to any
carcinogenic stimulus. including radiation, mayinterfere with expression of the carcinogenic damage bymodifying the number, susceptibility or turnover rate of
the target cells or by altering the immunologicalresponse against transformed cells. The most extreme
conditions under which to test such hypotheses arc
provided by the study of germ-free as opposed to
gnotobiotic or conventional animals.
759

217. Following irradiation of RF/Un mice myeloid
leukaemia is decreased in the absence of microbial nora
[W6], an effect which has been attributed to the reduced
myelopoietic cell proliferation in germ-free animals[W7, W8]. Radiation-induced lymphatic leukaemia is,on the contrary, unaltered by germ-free conditions inmany other strains of mice [P7, WI, W6]. Gnotobiotic
and conventional animals show no qualitative differences with regard to virus particles found with theelectron microscope [P7]. Induction of other solid
tumours in irradiated mice gives variable results [A JO,
W7] and radiation-induced malignant or benign
tumours are unaffected in germ-free rats. Thus. the data
essentially show that the pathogenesis of radiationinduced cancer is similar in conventionally reared or ingnotobiotic animals. It should be concluded that the
microbial flora as such has only a minor role in thedevelopment of haemopoietic neoplasms, perhaps via amodification of the immune system.

V.

CONCLUSIONS

218. The interaction between ionizing radiation andother agents represents a field of great potential importance in view of the ubiquitous nature of radiation andof the many situations of interaction that might occur inmodern life with a variety of physical, chemical or
biological agents. Yet, it is very difficult to define andsubstantiate the notion of interaction with even a
moderate degree of refinement. ~tany reports haveclaimed some kind of interaction but comprehensive
analysis does not show a sufficiently good conceptualbasis for the nature of the interactions. There is a lackof systematic treatment of any given case. particularly
with regard to the mechanisms of action. There isfurther a need to apply existing methodologies ofanalysis from other fields of the biological sciences tothe study of these problems.
219. The Committee has carried out a preliminary
analysis of the combined actions in the radiobiologicalfield. centered mainly around situations that may
possibly be of importance for risk assessments in man
and may therefore renect on the present foundations ofradiation protection. Available information on tumour
induction. genetic defects and developmental effects
was therefore scrutinized in the course of this analysis
for any evidence of combined actions. The conditionsof long-term exposure to low levels of the interacting
agents were reviewed in detail. although in most of thereports the levels of exposure were much higher thanthe environmental. Where possible, the accent was on
the results of epidemiological studies in humans,
although the bulk of the information relates to animals.
220. The Committee proposes that two types of interaction may be considered. The first is one where boththe ionizing radiation and the other interacting agent(s)are capable of producing some effect. Additivity.
synergism and antagonism arc the three possible conditions of interaction. The second type of combined
action is that between ionizing radiation and otheragents which are, when given alone, inactive. Protection
or sensitization are the terms that apply in these cases.
when reduction or enhancement. respectively, of theradiation effect are the end-results of the interactions.
Such classification is not an absolute one because thedoses of the interacting agents and the types of effectmay inlluence profoundly the nature and degree of theinteraction.

760

221. The concepts of exposure. dose and response
may be applied to the special case of the combined
action with ionizing radiation. The existing methodologies of analysis (isobolic diagram, envelope of
additivity, surface of response) allow the assessment, atleast on a semi-quantitative basis, of the results ofcombined treatments. These analyses may be further
extended to generalized probabilistic treatments of theexperimental results, taking into account the variabilityof the biological systems under study and leading to amore quantitative and satisfactory description of theinteraction factors.
222. The applicability of these rather abstract notions
to practical situations. particularly in the presence ofcomplex biological effects, has been discussed. The
need to define the effects with precision and to explore
the full exposure-response ranges to all agents. acting
separately or jointly, is a necessary prerequisite tomeaningful studies. Also, pitfalls have been identified
which may simulate conditions of interaction. In
relation to important biological end-points such as theinduction of tumours, the need to combine pathologicaland actuarial observations for a complete descriptionof the phenomena has been underlined.
223. The temporal pattern of the exposure (contemporaneous or sequential. chronic or acute. single or
fractionated) as well as the order of administration
appear of decisive importance in respect to the
production of a given type or degree of effect and havealso been examined in the Annex. All these conditions
relate to practical situations. even though they may tend
to blur the clearly defined notions of additivity,
synergism and antagonism. A detailed knowledge ofthe nature of the effects, their relationships to time andto the full range of doses of the interacting agents.
including the zero-dose condition, is important. In
many papers these basic conditions were imperfectly
described. In other cases. the statistical significance ofthe results was too low for a complete assessmen1 ofinteraction. Thus. the present conclusions should only
be considered as preliminary.
224. An instance of interaction could be that between
two different types of ionizing radiation. usually a
combination of high- and low-LET radiation. Uncertainties exist as to the degree of interaction, owing to
the essentially unknown nature of the primary radiation
lesions and their repair systems. Even in cases where
the yield of effect per unit dose of the two radiations
differs by an order of magnitude. the interaction iswithin the limits of hetero- and iso-additivity. The studyof the combined action of UV and ionizing radiation
may be very valuable for the analysis of primary lesions
and repair mechanisms. Experiments on survival of
mammalian cells point to simple additivity. The
important practical case of skin cancer induction, whentested in the animal, produced no evidence of interaction.
225. Examples of synergis1ic effects have apparen1ly
been reported in workers exposed jointly to ionizing
radiation and microwaves in the radiotcchnicalindustry. Functional disturbances of the nervous systemand subjective symptoms of discomfort were mainly
found in these workers. The nature of the symptoms,
the difficulties of their quantification, the frequently
uncontrollable conditions of exposure and the unsatisfactory dosimetry. the incomplete statistical evaluation,
are all reasons for which these reports should be
regarded with some reservation.

226. The combined action of suboptimal temperaturesand radiation has given evidence of interaction in both
directions, synergistic or antagonistic. depending
perhaps on the type of effect, order of administrationand level of exposure to the interacting agents. It would
not be expected that any such effect would normallyplay any important role in higher animals, in view of
their highly developed system of body temperature
regulation. High altitude, metabolic or physical stress,
mechanical damage, magnetic fields and ultrasoundwere also considered for a possible interaction withradiation: the results were variable but there was noevidence of significant synergistic interaction. ln allthese fields. the data are very few. the effects nonspecific and the mechanisms too obscure to allow any
delinitive statement.
227. The combined action of radiation. given internally or externally, with various types of dust showsunder repeated testing. particularly with regard totumour induction in the respiratory system, synergistic,
additive or antagonistic effects. Considering the uncertainties and limitations of the data, the synergistic effectof the combined treatment did not exceed a factor of
about two and the inhibitory effects a factor of about
four, compared to situations where radiation was
administered alone.
228. A variety of inorganic chemical compounds
containing lead, silver, cadmium, calcium. beryllium,
platinum. chlorine and fluorine. were also tested inexperimental animals in conjunction with radiation for
their carcinogenic, developmental or generally toxic
properties. The results were once more extremely
variable. In many cases the experience was so superficial, the effects so·varied and the biological systems so
different that no conclusions could be offered. Some ofthese interactions may be of significance in working
situations and could profitably be explored further.
229. In this review, radioprotective and radiosensitizing substances were not examined in detail. sinceconditions relevant to the exposure of the population
were the main object of the Annex. High levels ofradiation and nearly toxic levels of these substances
have been used in the relevant studies. A great varietyof underlying mechanisms. complex relationships to thedose. to the radiation type, to the presence of oxygen
were described for these chemical compounds. Since
these substances are only utilized in the clinical field,
none of them would be expected to pose significant
problems of public or occupational health.
230. The possible combined action of radiation withcompounds known for their carcinogenic properties
has been the object of special attention. The substances
examined include many initiators and promoters but
the systematic information collected for each one ofthese substances is very incomplete. The evidence
reviewed is conflicting and no final statement may beoffered in regard to any substance or to any class oftumours before the dose, the schedule of administrationand the treatment modalities are analysed to a greater
depth. which is seldom the case in the experiments
available.
231. Regarding benzo(a)pyrene and dimethylnitrosamine, two compounds having a widespread diffusionin the environment, experiments on lung tumour
induction provided some evidence of a synergistic
interaction (expressed mostly through a shorter latencytime) for the former, but not for the latter substance.

Fairly elaborate experiments in the hamster on the
combined effect of radiation. uranium ore dust anddiesel oil exhaust fumes yielded no evidence of synergistic effects, but the animal tested could be rather
refractory to lung tumour induction. These studies
should be extended in view of their practical implications.
232. Experimental data in animals and epidemiological experience on occupationally exposed humanpopulations is available concerning the combined
action of radiation and tobacco smoke. Tumours andinflammatory diseases of the respiratory system havebeen studied in this respect. In humans it appears thatsmoke may act by shortening the time of appearance ofthe radiation-induced lung tumours. It is not yet clear if
such an action may be the result of promotion by somecomponent of the tobacco smoke or due to a nonspecific effect of the smoke on the respiratory epithelia.
The experience in animals is still insufficient for a firm
conclusion.
233. The precise evaluation of an interaction factor inhumans critically depends on the length of the observation period as well as on the age structure andexposure history of the populations under study. It is
impossible to say if the displacement in time of thetumour appearance will eventually result in an
increased final yield of tumours in the smoking as
compared to the non-smoking irradiated population.
However, even if the final incidence of tumours
between smoking and non-smoking irradiatedindividuals were the same, the effect should still be
regarded as a synergistic one. since it would effectively
lead to a reduction of the tumour-free life of thesmokers developing tumours. This appears to be theonly well documented case of a synergistic interaction
in humans and in this sense it is a special case.
234. Antibiotics and other drugs were also considered
for their possible interaction with radiation. Variable
degrees of synergistic interaction were described for
effects ranging from cell survival in vitro to tumour
induction in animals. The relevance of these findings toindividuals outside the clinical field is however difficult
to evaluate. particularly in view of the limited diffusionof these substances in the general environment and of
the high doses usually involved in the above interactions.
235. Possible cases of interactions with biologicalagents which were considered included those withhormones and with infectious agents. Regarding
hormones, there is evidence that a variety of tumours of
the experimental animal may be sensitive to theiraction. Diethylstilbestrol and oestradiol-17-beta wereshown to have synergistic interaction for the
production of mammary tumours in various strains ofrat. with interaction factors in the range of 1.5 to 4. This
type of synergism is also expressed through a shortening of the time for tumour induction. There is a large
variability between strains, such that the same treatment
schedule could produce potentiation in some strains
and inhibition in others. There is also variability inrelation to the tumour type. Epidemiological information in the human species is scanty and only
indirect.
236. It is difficult for many animal tumours which are
known to have a viral etiology (thymic lymphoma,
myeloid leukaemia, osteogenic tumours) to consider
their induction as the result of a synergistic interaction,
761

because the effect could not he elicited in the absence ofeither the virus or radiation. There is also no evidence
that bacterial infection may play a major role in combination with ionizing radiation in modifying the yield oftumors.
237. For humans in environmental circumstances theCommittee has been unable to document any clear caseor synergistic interaction between radiation and otheragents, which could lead to substantial modirications of
the risk estimates for significant sections of the
population. Presumably this is due to the fact that mostof the agents likely to act synergistically with radiation.
as judged by the results of animal experiments, are notfound in sufficient concentration in nature. A specificexception is the case of tobacco smoke, which raises
essentially problems of industrial hygiene in someworking environments. Further research in the field of
the combined effects is desirable because this area ofstudy is still in an early stage of development and could
profitably be pursued in a systematic way.

VJ.

RESEARCH NEEDS

238. An eminently practical research need is that ofmodelling experimentally situations encountered inliving or working environments to test for undesirable
effects. A second important and more basic researchneed is the identification of interaction mechanisms.
The first need is essentially descriptive, the second
essentially interpretative and both may interrelate tomutual advantage. There is also a third research needfor the monitoring of possible effects in human populations by epidemiological studies. This latter is the mostvaluable for risk estimates in man.
239. Experiments of the first type are usually to study
in experimental animals situations of practical interestfor humans. It should be recalled that results obtainedin a given animal species are not easily extrapolated toother species. In designing these experiments. exposurelevels should be kept as similar as possible to themodelled situation. In combined action work, theassumption that effects showing at a given dose may
exist to a lower degree at lower doses may not be true.
Numerous examples of changes in the interaction withchanging dose levels of the combining agents exist. The
order and rate of administration of the agents should
ideally mimic the real situation. although this may be
impossible for chronic exposures of interest in practice.
Long-term chronic rather than acute end-points should
be focused upon. Tumour induction. effects on pre- andpost-natal development after exposure in utero andgenetic effects are the most significant classes of radiobiological end-points for further studies.
240. In the more basic studies, frequently involving
experiments at the cellular and sub-cellular levels, there
is considerably more latitude for research because therange of end-points is wider and the experiments financially less demanding. Good planning requires thecareful choice of experimental end-points and of
exposure level.
241. Epidemiological studies should have highpriority under the existing circumstances. The inherent
lack of control over many of the exposure variables
should be compensated by the best possible definitionof the exposure conditions, by the quantitation of the
responses and by adequate statistical treatment of theobservations. A conceptual and practical distinction
762

should be made between interactions of relevanceunder special working environments involving possible
problems of occupational medicine and large-scale
exposure situations which could change risk estimatesand could pose therefore more difficult problems of
public health.
242. The use of a standardized nomenclature in thefield of combined effects is highly desirable, because
too often misconceptions are made possible hy
inaccurate terminology.
243. Considering the main technical requirements forexperimental investigation of combined effects:(a) Efforts should be made to report biological data as
some function of the exposures in the target structures. For radiation. this problem is relatively
simple and studies of energy deposition arereasonably advanced. In other cases (physicalagents) this may simply require development ofbetter dosimetric techniques and apparatus, but inmost cases (particularly for chemical substances) it
will imply detailed studies of the intake. metabolism, concentration and excretion of the interacting substances when a direct measure of theirconcentration at the level of the target structures is
not possible;(b) There is a need to define clearly and specificallythe effects to be studied, especially when they arecomplex ones. For example, overall tumour
induction may not in itself be a sufficientindication of a combined action because, even in
the absence of significant changes in the overall
interaction factor, changes in the spectrum ofdifferent tumour classes could take place. In thecase of tumours it is important to study the rate ofappearance, together with the final incidence,
because shifts of the occurrence in time mightreveal synergistic actions which would not beapparent otherwise. Also. actuarial and pathological observations should be combined and data
corrections for competing risks should be applied;(c) The variable "time" in the combined actions
should be given proper attention. in the sense that
contemporaneous and sequential treatments andreversal in the order of application should be
examined. These studies are particularly importantwhen the agents under examination have initiatingor promoting characteristics and the sequence of
their action is therefore decisive. Fractionated andchronic treatments could also be profitably
examined, depending on the specific modelsituation and on the time characteristics of theagents combining:(d) It is essential that appropriate methodologies ofanalysis of the interactions be used to avoid
mistakes in the interpretation or inaccurate reportsof the data. It is only through such objective
analyses that precise statements and quantitativeevaluations may be drawn. There is, more specifically, a need to refer any given interaction to
conditions of iso- and hetero-addition;(e) Appropriate control series should be set up to test
exposure-rcponse curves not only around theexposure levels of interest for the particular experiment but also for an extended range of exposures,
including the zero values. Different combinationsof exposures of the interacting agents should also
be tested;(f) It is important that the nature of the interaction
should be as much as possible resolved through ananalysis of the effects at various levels of

biological complexity, from the population level.
through the whole-body, tissue, cellular andmolecular levels. These studies allow generalizations and avoid misrepresentations of the interaction.

radiation. particularly for end-points which arc or
significance for practical purposes;(c) At the systemic and whole-body level studies ofcombinations of tobacco smoke, fibres and dusts,
organic and inorganic carcinogens and pollutants
with radiation would be of great value;(d) In human populations, further surveys or smoking
and non-smoking workers professionally exposed
to internal lung irradiations should be pursued.
Under special working conditions the study ofinteraction of radiation with chemicals and microwaves would also be appropriate;(e) For the population at large. the possible interaction of hormones and radiation, particularly inhuman females, should be tested, provided
suitable groups might be identified. The increasingly widespread use of contraceptive hormones
is of particular importance.
(I) Studies of combined effects in the treatment ofpatients (for cancer and other diseases) by
combined treatment with radiation and chemotherapy and hormones. leading to carcinogenesis
and to non-stochastic effects which may be"recalled".

244. The specific areas or work identified by theCommittee as particularly important for their basic orpractical implications are:(a) At the molecular and chromosomal levels, studies
on the interaction of chemical. physical and viral
agents on constitutive and induced processes
related to DNA replication and repair or radiation
damage (in simple as well as complex organisms)
and relevant to the understanding or themechanism of mutagenesis and to the estimations
or genetic risks to man. These studies should
concentrate whenever possible on low doses ofradiation and of exposure lo other agents and becorrelated to relevant biological end-points likegene mutations and chromosome abnormalities aswell as to cell differentiation (e.g .. the immune
system. developing organisms) or carcinogenesis;(b) Studies of the interaction of different types ofTa b 1 e
Lung tumours following neutron irradiationand crysot1 le treatment[L16]
Number of rats with 1ung tumours
Group

Number of rats

IrradiatedIrradiated +Crysoti le

Carcinomas

tlesothe 1 i omas

20

0

4

9

Table

3

2

Effects on tumour develo ment of renatal ex osure of mice to x ra s
an et ylnltrosourea UU or to either treatment a one'. S42
Number of affected animals a/
Type of tumour

EllU
x-irra·treatment
diation(3x1Gy) (0. 5 Til/kg)

Interaction
factorw

x rays+mu

Control(whenapplicable)

2 ( 2.3)

3.4

Leukaemia
Lung tumours
llepatomas
Pancreatic adenomas
Intestinal tumours
Ovarian tumours

3 ( 5.3)
3 ( 2 .4)
8 (14.3) 22 (17 .8)
2 ( 3.5) 6 ( 4.9)0 ( 0 ) 1 ( 0.8)
0 ( 0 ) 2 ( 1.6)
6 ( 7.0)0 ( 0 l10 ( 12 .6)

6 ( 7 .6)
2 ( 2.5)
2 ( 2. 5)0 ( 0 )
10 ( 11.2)

11 (12 .8)

1 ( 1.1)0 ( 0 )
0 ( 0 l0 ( 0 l0.23

Total tumour
incidence

19 (33.9)

34 (27.6)

30 (38. 0)

14 (16.6)

0.44

Tumour multiplicity(affected organs
per animal)

1.0

1.0

1.5

1.0

!lumber of tumours
standardized

34

28

57

17

1.6

1.1

!I The percentage of affected animals is shown in parentheses.

763

Ta b l e
in Colorado

3

Res pi ra tory cancer deaths (~CD lla teau urarn um miners in re at ion to smok i 11
'L2

Smoking
category

Person-years
at risk (PYR)

Observed RCD(0)Expected nco(E)

Smokers
Non-smokers

26392
9047

60
2

15.5
0.5

0 - E
-pyJf"""

3.9
4.0

1.7 10- 3
1.7 10- 4

Ta b l e
Interaction factors and robabilities xof res irator cancer deaths
er one erson- ear at r, s for s ,n e on com i ne ac ionof smo ing and irra iatio11<IJ

Spon"'"' taneous

Smoking

Irradiation

Combined action.0...."'
"'

Po

ptl

0

A

0.6

B

1.1

5.9
4.4

p

=

ol
ptl-PO

pt2

5.3
3.3

2.2
7 .1

po2;

Pot

Pt2-PO

23
42.2

1.6
6.C

6 Pobs=

Pot-PO2241. l

6 pexp;

6 PobsPol+Po2 ~

6.9
9.3

3.2
4.4

A: Derived from the 1950-1967 data base [L2].
B: Derived from the 1964-1967 data base [Al J.

Ta b l e

5Interaction factors and robabilities x 10- 4 of res irator cancer deaths
er one erson- ear at ris
or uranium miners
o d1f erent smoking categories!L6]
SponSmoking
taneous~~~~~~

Combined action

Irradiation
Pot

A

l. 7

B

1.9

C

1.3

D

1.1

l. 7
2.6
7.0
27. 7

0

6.5

6 p

obs
pot-PO=

6 Pexp=

6 PobsPol+Po2 ~

4.8
42.1
41.0
51.2

0.7
5.7
26.6

40.2

5.5

39. 7

10.5
31.4

50.1

A r.on-smokers.
B Former smokers.
C Light smokers.
D Heavy smokers.
Table

6

Dose modif in factors Di-IF for combined treatment
y some chemotherapeutic drugs and ra iation[PS]

Dose modifying factor (Dr·IF)
Drug

Drug dose
mg/kg
Intestine
injuryActinomycin D
Adri amyc in
BCtiU

Bleomycin
Cyclophosphamide
Hydroxyurea
Vincri stine
Prednisolone

764

0.75
15
8.25
3
75
500
0.5
10

1.3
1. 7
1.1
1.1
1.0

Oesophageal
lethality(from
I.DS0/28)

Pulmonarylethality(from
LD50/160)

1.6

1.6

0.9
1.14
0.86

0.74
0.98
I. 3
1.03
1.18
0.84

7.3
3.8
i.6

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Bl

B2
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B4
BS

B6
B7
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