SOURCES AND EFFECTS
OF IONIZING RADIATION
United Nations Scientific Committee on the Effectsof Atomic Radiation
UNSCEAR 2000 Report to the General Assembly,
with Scientific Annexes
VOLUME II: EFFECTS

UNITED NATIONSNew York, 2000

NOTE
The report of the Committee without its annexes appears as Official Records of theGeneral Assembly, Fifty-fifth Session, Supplement No. 46 (A/55/46).
The designation employed and the presentation of material in this publication do not
imply the expression of any opinion whatsoever on the part of the 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.
The country names used in this document are, in most cases, those that were in use
at the time the data were collected or the text prepared. In other cases, however, the names
have been updated, where this was possible and appropriate, to reflect political changes.

UNITED NATIONS PUBLICATION
Sales No. E.00.IX.4
ISBN 92-1-142239-6

ANNEX H
Combined effects of radiation and other agents

CONTENTSPage

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
I.

IDENTIFYING INTERACTIONS AND COMBINED EFFECTS . . . . . . . . . . .
A. SCOPE OF THE PROBLEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Additivity and deviations from additivity . . . . . . . . . . . . . . . . . . . . .
2. Radiation effects and effects of other agents . . . . . . . . . . . . . . . . . . .
B. EXPOSURE ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Dose concepts for physical and chemical agents . . . . . . . . . . . . . . . .
2. Biochemical monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Gene mutation analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Cytogenetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

180
180
180
182
183
183
184
186
187
189

II.

MECHANISTIC CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. EFFECTS ON THE MOLECULAR LEVEL . . . . . . . . . . . . . . . . . . . . . .
B. EFFECTS ON THE CELLULAR LEVEL . . . . . . . . . . . . . . . . . . . . . . . .
1. Accumulation of (sub)lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Cellular repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Cytokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Toxicological analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. EFFECTS ON THE TISSUE/ORGAN LEVEL . . . . . . . . . . . . . . . . . . . .
D. DOSE MODIFIERS AND OTHER INDIRECT INTERACTIONS . . . . . .
E. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

189
190
190
191
192
193
193
194
197
197

III. SPECIFIC COMBINED EXPOSURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. RADIATION AND PHYSICAL AGENTS . . . . . . . . . . . . . . . . . . . . . . . .
1. Combinations of ionizing radiation . . . . . . . . . . . . . . . . . . . . . . . . .
2. Ultraviolet radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Electromagnetic radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Dust, asbestos, and other mineral fibres . . . . . . . . . . . . . . . . . . . . . .
7. Space flight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. RADIATION AND CHEMICAL AGENTS . . . . . . . . . . . . . . . . . . . . . . .
1. Genotoxic chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Non-genotoxic chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199
200
200
200
200
200
200
200
201
201
201
201
201

178

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS
Page

4. Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Mitogens and cytotoxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Antioxidants, vitamins, and other dietary factors . . . . . . . . . . . . . . .
RADIATION AND BIOLOGICAL AGENTS . . . . . . . . . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203
203
204
204
204

IV. COMBINED EXPOSURES IN CANCER THERAPY . . . . . . . . . . . . . . . . . . . .
A. MECHANISMS OF INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. SECONDARY CANCERS FOLLOWINGCOMBINED MODALITY TREATMENT . . . . . . . . . . . . . . . . . . . . . . . .
C. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205
205

C.
D.

V.

EFFECTS OTHER THAN CANCER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. PRE- AND POST-NATAL EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. GENETIC AND MULTI-GENERATION EFFECTS . . . . . . . . . . . . . . . .
C. DETERMINISTIC EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207
208
211
211
212
212

EXTENDED SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
FURTHER RESEARCH NEEDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Appendix:
COMBINED EFFECTS OF SPECIFIC PHYSICAL, CHEMICALAND BIOLOGICAL AGENTS WITH IONIZING RADIATION . . . . . . . . . . . . . . . .
A. RADIATION AND PHYSICAL AGENTS . . . . . . . . . . . . . . . . . . . . . . . .
1. Combinations of different types of ionizing radiation . . . . . . . . . . . .
2. Ultraviolet radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Low- and high-frequency electromagnetic radiation . . . . . . . . . . . . .
4. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Dust, asbestos, and other mineral fibres . . . . . . . . . . . . . . . . . . . . . .
7. Space flight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. RADIATION AND CHEMICAL TOXICANTS . . . . . . . . . . . . . . . . . . . .
1. Genotoxic chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Non-genotoxic chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Mitogens and cytotoxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Antioxidants, vitamins, and other dietary factors . . . . . . . . . . . . . . .
C. RADIATION AND BIOLOGICAL AGENTS, MISCELLANEOUS . . . . .
1. Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Viruses, bacteria, and genetic sequences . . . . . . . . . . . . . . . . . . . . .
3. Miscellaneous factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. COMBINED MODALITIES IN RADIATION THERAPY . . . . . . . . . . . .
1. Alkylating agents, nitrosoureas, andplatinum coordination complexes . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Antimetabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Antitumour antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Microtubule poisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Topoisomerase poisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Bioreductive drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

218
218
218
220
226
227
228
229
231
231
232
236
239
246
248
249
252
252
254
254
255
255
257
260
262
263
265

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

179

INTRODUCTION
1. Living organisms are exposed to numerous natural andman-made agents that interact with molecules, cells, andtissues, causing reversible deviations from homeostatic
equilibrium or irreversible damage. Many aspects of aging
and many diseases are thought to stem from exogenous andendogenous deleterious agents acting on key components ofcells within the body. Because of the worldwide proliferationof a number of man-made agents and the increasing release ofnatural agents due to human activities into the environment,
the assessment of toxicity, carcinogenicity, and mutagenicity
of a specific chemical, physical, or biological agent is, in fact,a study of combined exposures [G10]. Although this has beenrecognized for a long time, risk assessment is generally
performed with the simplifying assumption that the agent
under study acts largely independently of other substances.
Studies of interactions have indicated, however, that, at leastat high exposures, the action of one agent can be influenced
by simultaneous exposures to other agents. The combined
effects may be greater or smaller than the sum of the effects
from separate exposures to the individual agents. The action
at low levels of exposure, which are commonly encounteredin occupational and environmental situations, is less clear.
Continued, critical review of studies on the effects ofcombined exposures to radiation and other toxic agents isnecessary, particularly at the lowest levels of exposure, to be
sure that any modifications of the radiation effects caused byother environmental or occupational agents are recognized
and, as far as possible, taken into account in risk assessments.
2. In the UNSCEAR 1982 Report [U6], the Committee
discussed the problem of the combined action of radiation
with other agents. In reviewing the approaches and the many
reports in which synergisms were claimed, the Committee
noted that, in general, an adequate conceptual framework waslacking. Despite many reports showing the potential importance of interactions between different agents under specificconditions, mostly occupational, information on the mechanisms of action was largely missing, and the methodologies fordata analysis in different branches of the biological scienceswere based on different approaches. The UNSCEAR 1982
Report concluded that it was not possible to document clear
cases of interaction that could justify substantial modifications
to the existing radiation risk estimates. The Committee feltthat systematic investigations ofcombined effects were needed
to allow this field to move forward from its early stage ofdevelopment.
3. The objective of this Annex is to update the Committee's previous review of this subject [U6] and to reconsider
whether interactions of radiation and one or more other agentsshould be taken into account in evaluating radiation risks at
low doses. To achieve this objective, the following subjects are
considered:(a)the concepts of doses, targets, and detriments currently
used in risk assessments of radiation and chemical
agents;

(b)

(c)
(d)

(e)

recent developments from research on the possible
mechanisms of combined effects from low-level
exposures to radiation and other agents;
results and evaluations of data from experimental andepidemiological studies;mechanistic models applied to experimental and epidemiological results, with generalizations and extrapolations that might be pertinent to low and chronic
exposures;concepts and approaches in other areas of biologicalscience (for example, molecular biology and toxicology) that could suggest ways to develop databases
and to identify and assess the effects of interactions
important for human populations.

4.
Combined effects must be viewed in the light of theconsiderable insights gained from wider studies of cancer
induction (see Annex E, “Mechanisms of radiation oncogenesis", in the UNSCEAR 1993 Report [U3] and Annex G,“Biological effects at low radiation doses”), heritable defects(see Annex G, “Hereditary effects of radiation” in the
UNSCEAR 1993 Report [U3]), and DNA integrity (seeAnnex F, “DNA repair and mutagenesis”). Where necessary,
the following text refers to these and other Annexes.
5.
Since at low levels of exposure, the main endpoints
from ionizing radiation alone and from its interaction withother agents are stochastic in nature, this Annex will mainly
focus on this type of effect and consider cancer induction,
mutation and the possibility of prenatal effects. Several
specific areas where the combined action of high doses ofradiation and chemical agents are known to lead toconsiderable deviation from additivity will also be considered
but only in so far as they help to elucidate the mechanisms ofcombined exposures. These areas include the interaction ofchemotherapeutic compounds and sensitizers to enhanceradiation effects in clinical radiotherapy, the effects ofprotective agents on acute radiation exposure, and stimulatory
responses to radiation (reviewed in the UNSCEAR 1994
Report [U2]). The endpoints of interest in these situations ofhigh-dose exposure are deterministic effects.
6.
The Annex begins by introducing the problem ofcombined effects, considering the additivity or non-additivityof biological effects and the possible differences between
radiation and chemical carcinogenesis. This is followed byconcepts and definitions of physical and biological dosimetry
for radiation and other agents. Interactions of other agents inthe development of radiation-induced cancer are thenconsidered from a mechanistic point of view. A very
important part of the Annex is a review of data on the effectsof specific combined exposures on carcinogenesis. This isfollowed by a chapter on interactions in humans that produce
effects other than cancer. Finally, conclusions are drawn andrecommendations are offered. A detailed account of the
combined effects of radiation and specific physical, chemical,
and biological agents is provided in the Appendix.

180

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

I. IDENTIFYING INTERACTIONS AND COMBINED EFFECTS
A. SCOPE OF THE PROBLEM

1. Additivity and deviations from additivity

7. When discussing combined effects, it is of utmost
importance to provide clear definitions and terminology.
Multiple-agent toxicology uses many concepts the nomenclature for which is not unambiguous. Different names are
sometimes used for the same phenomenon, and sometimes thesame name is used for different mechanisms. The confusion
arises in part because the concepts were developed in different
disciplines, such as pharmacology, toxicology, biology, statistics, epidemiology, and radiation biology. Starting fromdifferent basic assumptions and with different aims in mind,
attempts are made to describe the effects of combined exposures to chemical and physical agents. The confusing terminology inhibits clear understanding and thwarts the comparisonof different investigations and results. In this Chapter somebasic problems concerning combined exposures are discussed.

8.
One of the basic questions surrounding the combined effects of two agents is the question of whether theeffect of a combined exposure to two or more agents isthe same as or different from the sum of the effects ofeach agent separately. Many terms and synonyms areused to indicate the result (Table 1). They are, in
general, based on deviations from the expected outcome(additivity). On a descriptive level, two classes of combined effects can be considered. In the first case, both
ionizing radiation and the other agent (or agents) aredeleterious on their own and combine to produce aneffect not directly predictable from the single exposures.
In the second case, only ionizing radiation produces aneffect, but its nature or severity may be modified by theother agent, which is non-toxic by itself.

Table 1
Terms and synonyms for combined effects
Effect smaller than anticipatedAntagonism
Antergism
Depotentiation
Desensitation
Inhibition
Infra-additivity
Negative interaction
Negative synergism
Subadditivity

Effect as anticipatedEffect larger than anticipated

Additivity
Additivism
Independence
Indifference
Non-interaction
Summation
Zero-interaction

Augmentation
Enhancement
Positive interaction
Potentiation
Sensitation
Superadditivity
Supra-additivism
Synergism
Synergy

9. On a mechanistic level, insights gained in more recent
years indicate that a much more refined classification may beneeded. The main classes of genotoxic and non-genotoxic
agents must be considered in relation to specific targets ofaction. For example, a chemical may act specifically at the siteof a radiation-induced lesion, modifying DNA repair fidelity,or it may modify cell growth, strongly influencing the clonal
expansion of precancerous cells. The many possibilities forinteraction are related to the complexityof the development of
the radiation effect and the many steps involved in
carcinogenesis. These steps are prone to the influence of many
classes of agents, both endogenous and environmental. The
multi-step process and the many levels of interaction to beconsidered are schematically depicted in Figure I. In view ofthis complexity, it is not surprising that many models, both
descriptive and mechanistic, have been developed to describe
the combined effects of exposures to different agents [B11, L2,
L8, L28, M16, S15, S16, S23, S25, Z1]. In the UNSCEAR
1982 Report [U6], the Committee reviewed these approaches.

possible among the undocumented (and sometimes unknown)
exposures that the individuals in these studies incur during
their lifetimes. In epidemiological studies, effects that may be
associated with exposures to specific agents or circumstances
may be the result of interactions among components of a
mixture of agents and may have resulted from, or been
influenced by, previous exposures. The emerging field of
molecular epidemiologymaybe able to address such questionsin the near future.
11. Most knowledge of interaction effects has been provided
byexperimental studies. These studies have an advantage overepidemiological studies: they retain control of
(a)

(b)
(c)

10. Although classical epidemiology is important in
identifying critical combined effects, it has little potential for
dissecting such interactions from the complex interplay

the population (e.g. selection of systems ranging from
DNA to intact animals and of species, strain, age,gender and previous exposure history);
the exposure (e.g. precise knowledge of the type,dose, dose rate and timing of exposure); andthe endpoints (e.g. selection of sampling time andfrequency, use of invasive and destructive tests,
consistency and completeness of health status
evaluations).

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS
10-15

Energy deposition
Excitation/ionization

10-12

Initial particle tracks

10-9

Radical formation

PHYSICAL INTERACTIONS

Diffusion, chemical reactions

TIME (sec)PHYSICO-CHEMICAL INTERACTIONS

Initial DNA damage

10-6
10-3

1 ms

100

1 second

103

181

DNA breaks / base damage

Repair processes
Damage fixation
BIOLOGICAL RESPONSE

1 hourCell killing

1 day

Mutations/transformations/aberrations

1 year

Proliferation of "damaged" cells
Promotion/completion

106
10 9

Teratogenesis

MEDICAL EFFECTS

Cancer
100 years

Hereditary defects

Figure I. Schematic development of the events leading to stochastic radiation effects.

12. Experiments with animals or cells have the disadvantage that the results and conclusions have to be extrapolated to humans. Additionally, conclusions drawn fromhigh-level exposures of animals and cells have to be extrapolated to the low levels of human exposures. The greatest
uncertainty is largely a problem of not knowing the shape ofthe dose-effect relationship at low exposure levels and whether
there are effect thresholds. A well balanced conclusion on the
combined action of two agents can only be given if the doseeffect relationships of both agents separately and of the
combined exposure are known and can be analysed using a(mathematical) model in which the interaction can beconsistentlyand quantitativelydefined. The majorityofstudies
on combined effects, including those with radiation, do notmeet these conditions.
13. For the basic case of a single agent acting on abiological system, the resulting effect will be dependent on thedose of the agent and will follow some kind of functionaldose-effect relationship. The effect level in the absence of theagent is termed the spontaneous or background effect. The
simplest relationship between dose and effect is linear. In therealm of linear dose-effect relationships, the three mostcommonlyconsidered types ofinteractions between twoagentsare additivity, synergism and antagonism, giving a combined
effect equal to, greater or less than the effects of independentactions, respectively (reviewed in [M16]).

14. For combined effects of agents with non-linear doseeffect relationships, the analysis is complicated, and more
precise definitions of the terms antagonism, additivity, andsynergism must be provided [S25, S49]. For example, for an
upward-bending dose-effect relationship (Figure II), an
additional increment of dose from a single agent will result ina non-linear increase in response, even in the case of additivity. The term synergism has sometimes been erroneously
used for such situations [Z3]. Although correct on a descriptive and mathematical level, such a broad definition would
render the term synergism practically useless in the study of
combined effects. With such a definition, different agents withthe same action spectrum, i.e. fullyindependent agents, would

SYNERGISM

Isoaddition

EFFECT

Moreover, in experimental studies one can relate exposures toeffects more directly than is typically possible in humanstudies. This is due, in part, to the fact that both the history of
the subject and the exposures under study are known andcontrolled, making cause-effect linkage easier. In addition,
experimental scientists can often determine that an exposure
actually results in a dose to the tissue manifesting an effect.

lo
ve
En

o
pe

d
fa

iv
dit

ity

Heteroaddition

ANTAGONISMEXPOSURE

Figure II. Interaction of two agents having non-linear
dose-effect relationships. Isoaddition results for mechanically similar agents, heteroaddition for independently
acting agents [I3, S49].

182

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

produce an apparent synergism in anycombination of concentrations as long as the single dose-effect relationships are bent
upwards, as is often the case in the dose range of interest.
From a mechanistic point of view, synergism can be defined
more narrowly to imply that agents combine by acting atdifferent rate-limiting steps of a multi-step process or atdifferent sites of a molecule, thereby enhancing the chance fora negative outcome, such as cancer, by different mechanisms[B23]. Such an assessment is often hindered by insufficientknowledge of the underlying mechanism of action andtherefore can rarely be made. Clearly, deviation from additivityis a poor indicator of synergism or antagonism, since nonlinear dose-effect relationships and threshold phenomena are
the rule rather than the exception for most endpoints inbiological systems, and interaction in the statistical-mathematical sense does not define an interaction in a biologicalmechanistic sense [B69].
15. In this Annex the term synergism will be used in anarrow sense. The most important question is whether data on
combined effects do show some modification of stochastic
radiation effects as a result of combined exposure with anotheragent. If not, no interaction will be assumed, and the resulting
effect is additive; if the result of combined exposure isdifferent, some form of interaction has to be assumed, and theresulting effect will be called sub- or supra-additive, depending on whether the effect is lesser or greater, respectively, than
the sum of the single-agent effects separately.
2. Radiation effects and effects of other agents
16. As far as carcinogenesis is concerned, the primaryeffects of ionizing radiation are on DNA, compromisingcell survival, cell proliferation, and proper physiologicalcell functioning. Although the deposition of energy along
the track of ionizing radiation can directly affect DNA,
most of the damage to DNA from low-LET radiation
comes from the formation of radical intermediates stableenough to diffuse several nanometers and interact withcritical cellular constituents (for details see Annex F,“DNA repair and mutagenesis”). Only a small fraction of
the radiation-induced molecular modifications occur in the
DNA of the cell nucleus, but practically all experimental
and theoretical evidence indicates that DNA, the main
carrier of genetic information in living matter, is thecritical target. Especially at the doses under considerationin this Annex, damage to structural and functional proteins
and lipids has not been shown to contribute noticeably tothe detriment from ionizing radiation. To protect the
integrity of the genetic information, most cells have highly
intricate enzyme systems to repair DNA damage efficiently
and effectively based on information contained within theundamaged complementary DNA strand. Despite that,
residual fixed damage may result even from low-dose
exposures, especially when both DNA strands aredamaged. Such damage may lead to reproductive cell
death, and therefore possible deterministic effects; tosomatic cell mutation, enhancing the risk of cancer; or to
mutations in germ cells, with possible deleterious effects inoffspring.

17. Longer wavelength radiation, such as ultraviolet(UV) light, although not ionizing itself, still acts mainly bymodifying DNA. The UV portion of the electromagnetic
spectrum covers the wavelengths between 200 and 400 nm.
Conventionally, a distinction is made between UV-C
(200280 nm), UV-B (280320 nm), and UV-A (320
400 nm). The effects of UV light depend on the wavelengthand the absorption properties of the target. Ultraviolet
radiation mainlycauses the formation of pyrimidine dimers
and 64 photoproducts, which may also lead to residual
DNA damage after repair. Apart from visible light up to
525 nm, which can still interact with photosensitizers togenerate reactive species and, subsequently, oxidative
damage to DNA, infrared, microwave, and low-frequency
electromagnetic radiation have no direct genotoxic effectsof their own. Indirect effects might arise from local heatingor from charge effects across membranes activating signaltransduction pathways and neurons. Such cellular changes
may be long-lasting or even be passed from one cell to its
progeny. Sugahara and Watanabe [S10] reviewed theepigenetic aspects of radiation carcinogenesis. Studies using
cell culture systems show that magnetic fields, depending on
their frequency, amplitude, and wave form, interact withbiological systems. Such effects have been seen on enzymes
related to growth regulation, on intracellular calcium balance,on gene expression, and on peripheral levels of the oncostatic
hormonemelatonin [H45]. These effects are potentiallyrelated
to tumour promotion. However, the considerable research
conducted thus far has not elucidated critical mechanisms or
revealed important health risks from non-thermal exposures.
Other than crude effects present only at high exposures, forexample strong irritations or protein denaturation, cellular
perturbations resulting from non-ionizing radiation cannot belabelled harmful per se.
18. Chemical agents may act as genotoxicants by, forexample, forming direct covalent links, by transferring
reactive molecular subgroups to DNA, by inducing DNADNA or DNA-protein cross-links, or by generating strand
breaks. The mode of action may be direct, by the formation ofsmall or bulky DNA adducts as well as strand breaks, orindirect, by the formation of radicals in the vicinity of DNA,leading to strand breaks or small adducts. On the epigenetic
or non-genotoxic level, chemicals may interfere with DNA
synthesis or repair or may prevent radical scavenging, thereby
promoting DNA damage. Non-genotoxic agents may also
influence a broad spectrum of other cellular events. Of
concern in cancer induction is any interference with cellproliferation, cell differentiation, cell senescence, andapoptosis or with the regulation of these processes.
19. Biological agents may also act at the genetic andepigenetic levels, i.e. they may be genotoxic or non-genotoxic,
respectively. Viruses are effective transport vectors for genome
fragments and may activate or block the expression ofendogenous genetic information. Viral involvement in many
animal tumours and also in human malignancies is wellestablished, e.g. the DNA tumour viruses of the papilloma
family in cervical carcinoma and the retroviruses HTLV-1 in
adult T-cell leukaemia (reviewed in [H13]). In addition,

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

biological-agent-induced influences on immune responses,
inflammation, fever, and endogenous radicals may lead tocytotoxic and/or growth stimulatory responses that are cocarcinogenic, as described later.

B. EXPOSURE ASSESSMENT
20. The most important prerequisite for a comparative
assessment of biological effects of different agents, and also of
their possible interactions, is the characterization of the
exposure or the dosimetry of both agents that may be related
to subsequent effects. Some of the main concepts used in
toxicology and radiation biology to convert exposures intomeaningful measures of dose and health impact are introduced in the following paragraphs.
21. The toxicity of an agent can be defined as its inherent
ability to adversely affect living organisms. The spectrum of
undesired effects is very wide, ranging from local, reversible
effects to irreversible changes leading to the failure of criticalorgan systems and then to death. The objective of dosimetry
is to relate the amount of agent presented to the organism ina way that is relevant to the effects observed and that ismeasurable in a physical, chemical, or biological manner.
Identification of processes occurring at the molecular level, i.e.
at a mechanistic level of the effect, would give the most basic
indication of a dosimetric measure. The present approaches
and possibilities are discussed below. In Section I.B.1,
dosimetry based on the measurement of physical or chemicalparameters of the agent itself, the physical or chemical
dosimetry, is considered. In Section I.B.2, measurement ofimmediate biological damage caused by the agent (biochemical monitoring) is discussed; this damage may or may not bedirectly related to the biological effect being considered.
22. Sometimes, when physical, chemical, or biochemicalmeasurements are not possible or cannot be made accurately
enough, certain biological effects may be detectable. Sucheffects may serve as indicators of the exposure to biologically
active agents. These “biological markers“ reflect damage
resulting from toxic interaction, either at the target or at an
analogous site that is known or believed to be pathogenicallylinked to health effects. A wide variety of biological markers
fall into this category, including gene mutation; alterations in
oncogenes and tumour-suppressor genes; DNA single- anddouble-strand breaks; and unscheduled DNA synthesis, sister
chromatid exchanges, chromosomal aberrations; andmicronuclei. None of these markers is highly agent- or
exposure-specific, and other factors (lifestyle and environment) that affect these endpoints can act as confounding
variables in molecular studies. Some possibilities for assay
systems to measure biological markers such as specific genemutations and cytogenetic damage in exposed humans arepresented in Sections I.B.3 and I.B.4, respectively.
1. Dose concepts for physical andchemical agents
23. Ionizing radiation exposure is generally measured interms of absorbed dose, i.e. the average energy deposited183

per unit mass. The unit of absorbed dose is the gray (Gy),with 1 Gy equal to 1 J kg1 [I3]. At the level of a cell or cell
nucleus, the minimal dose is determined by the ionization
density of a single track. Averaged over the volume of acell nucleus, a single event amounts to between one andseveral milligray (mGy) for electrons and about 300 mGy
for an alpha particle [U3]. Below these dose levels, theprobability of a cell being hit varies but the absorbed dose
per cell nucleus does not. For internally deposited radionuclides, their location and fate in the organism are used
to calculate the absorbed dose in the organs of interest, andusually the average absorbed dose in the organ is taken as
the relevant dose that causes the biological effect, assuming
a rather homogeneous distribution of energy absorption in
the tissue.
24. The definition of exposure or dose for non-ionizing
radiation and for most chemical and biological agents is moredifficult than for radiation. Ultraviolet radiation can penetrate
into tissue at most only for several millimetres, depending on
wavelength. The energy absorbed in the tissue of interest, andthus the effectiveness of UV, cannot be easily estimated.
Exposure to a toxic agent may be estimated by environmental
monitoring (referred to as external dose evaluation in
toxicology), internal monitoring (internal dose evaluation),and biochemical effect monitoring (tissue dose or biologicallyeffective dose determination) [E1].
25. For chemical and biological agents, the dose can bebased on the time integral of concentration, as for internal
exposures with radionuclides. However, in addition to thecommon important question of defining the critical cellulartargets, it is the activation and biodegradation of a chemical
agent in the different compartments of the organism that will
determine the degree of genetic damage or strength of anepigenetic signal. Although the local concentrations ofreceptors or reactants could possibly be estimated ordetermined, these vary considerably in their response toendogenous and environmental factors, which can lead todifferent sensitivities to the physical or chemical agent. This
may restrict the use of biochemical markers somewhat,
because their concentrations in body fluids will depend on themechanisms of uptake, the formation of reactive molecularspecies, and their breakdown. Somewhat like the dose concept
for ionizing radiation, exposure can be related to the numberof primary chemical events on DNA leading to the effectunder consideration. The above-mentioned quantitative link
between DNA alkylation and the product of concentration andtime for ethylene oxide may serve as an example [E3] (seealso paragraph 34). However, only rarely is the nature of such
events known or quantifiable.
26. To give exposure (or dose) its full biological meaning,
the concentration-time product at the level of the cellulartarget structure should be known. Even this is difficult todetermine owing to the many membranes and other barriers
to be crossed between the intake port and the place of action.
Many chemicals also undergo modifications by detoxificationin the liver, lung, and other organs, which change both their
toxicity and their biokinetics. One of the best known carcino-

184

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

gens, benzo(a)pyrene, becomes toxic only after metabolic
activation, leading to the ultimate reactive electrophilic carcinogen. Such transformations, called metabolic activation,
maydiffer considerablyamong species and even between maleand female subjects, making inferences from experimental
systems still more difficult. The induction of kidney cancer inmale rats by a group of chemicals (n-1,4-dichlorobenzene,
hexachloroethane, isophorone, tetrachloroethylene, andunleaded gasoline) may serve as an example. It took a great
deal of research [B29, E4] to show that the risk for theendpoint under consideration, namelykidneytumours in malerats, is a species- and sex-specific finding that relates to malerats but not to female rats, mice of either sex, or humans.
Mechanistic studies showed that male rats have a specificcirculating protein, alpha-2u-globulin, that binds the chemicals under consideration and leads torenal accumulation, withsubsequent kidney damage and the development of kidney
tumours. This protein was shown to be absent in humans.
Only such detailed molecular information allows a reasonablerisk estimate to be generated for humans [B29]. Unfortunately,
the species-specific detection and quantification oftoxic agents
formed in biochemical pathways are rarely achieved.
2. Biochemical monitoring
27. For chemical agents, internal dose evaluation involves
the measurement of the amount of a carcinogen or its metabolites present in cells, tissues, or body fluids. Analysis ofinternal dose takes into account individual differences inabsorption or bioaccumulation of the compound in question.
It may be relatively easy to measure the concentrations of thecompound in body fluids. However, doing so does not provide
data on the interactions of the compound with critical cellulartargets. Examples of this type of monitoring include organic
compounds or metals (e.g. lead) in the diet, cigarette smoke,or industrial exposures that can be detected in blood or urine[P3]. The binding of chemicals with cell constituents may bemeasured directly with radioactive labels. Even in vivo,
correlations between the administered amount of the toxicant,the number of molecules bound to critical targets, and thebiological effect can be established [P6].
28. From the energydeposition pattern of ionizing radiationin the tissue constituents and from some critical biochemicalparameters, such as oxygen pressure and the local concentrations of radical scavengers, the primary damage, i.e. thenumber of primary DNA lesions, can be estimated. A few ofthese parameters are even stable enough to be used as
biological indicators such as cytogenetic changes in peripheral
blood lymphocytes to assess exposures retrospectively.
29. In toxicology, the tissue dose or biologically effective
dose reflects the amount of carcinogen that has directlyinteracted with cellular macromolecules at a target site. It canbe assessed from the amount of DNA and protein damage(strand breaks, DNA adducts, protein adducts) in the target
tissue or by extrapolating from damage levels found insurrogate tissues, such as white blood cells. Experiments haveshown that, in general, DNA damage levels in target tissues
and non-target cells are proportional to the external dose. This

class of markers is more mechanistically relevant to carcinogenesis than internal dose, since it takes into account differences in metabolism (activation vs. detoxification) of the compound in question, as well as the extent of repair of carcinogen-altered DNA. Perera and Santella [P3] provided
examples of compounds and exposures that might be analysedusing this type of biologically effective dosimetry, as well as
the populations that have been studied.
30. DNA and protein adducts are measures of exposure tocarcinogenic compounds [E1]. They are mechanisticallylinked to cancer, as they cause DNA damage and mutationsin important genes, such as genes coding for growth control
or damage repair enzymes. Adducts have been used toestimate cancer risk by comparing their mutagenicity relative
to that of x rays. In the same way that the unit cancer risk of
x rays is defined, the relative mutagenicity is used to estimate
the cancer risk of a chemical exposure that causes adducts(gray-equivalent approach) [E1, E3].
31. In the case of agents binding covalently to different
cellular macromolecules, the degree of alkylation of proteinscan be used as a surrogate measure for their effects on DNA.
Ehrenberg et al. [E3, E10] showed in the mouse that the tissue
dose of ethylene oxide, i.e. the concentration of the alkylating
agent integrated over time, correlated well with the alkylation
pattern. In male mice, the authors were able to show with thismethod that the tissue dose for ethylene oxide, an agent
rapidly distributed to all organs after inhalation, was about
0.5 mM h per ppm h for most organs, including the testes. On
the basis of dose-effect curves of ethylene oxide and x rays inbarley, the same authors [E3] set a tissue dose of ethyleneoxide in humans of 1 mM h equal to 0.8 Gy of low-LET
radiation. Such an approach facilitates the comparison andcombination of risks of various agents.
32. Despite their relevance as dosimeters of biologicaleffects, the limitations of the current methods should be noted.
Most available assays provide information on total or multiple
adducts and are rarely capable of pinpointing the critical
adducts on DNA. Only for a few target organs, such as thelung or bladder, are epithelial cells available for routine
analysis. For other organs, DNA is not readily accessible;
many studies therefore use surrogate tissues (e.g. peripheral
blood cells and placentas). However, the relationship between
adducts in the target and those in surrogate tissues has notbeen well characterized in humans, although for certain
carcinogens it has been characterized in experimental animals[S17]. Again, it must be considered that there are species- andsex-dependent differences in the absorption and metabolismof chemicals in their various forms.
33. Bydefinition, all types of ionizing radiations generateions. Ionizing radiation can directly induce ionizations inDNA, causing direct damage. However, the majority ofdamage from low-LET radiation occurs in an indirectmanner via the formation of free radicals and H2O2, which
are precursors of oxidative damage [B8, S34]. When living
cells or organisms are irradiated, OH radicals are
generated in cells or tissue, which leads to many DNA

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

lesions, including oxidative DNA base products. Bothionizing radiation and oxidative stress generate free
radicals near DNA. Most of these radicals (R) interact
with oxygen, forming peroxyl intermediates (ROO) andfinal products (P). Most of the products are eliminated by
nucleotide excision repair and glycosylases [F10], while asmall fraction remain in the DNA [S35]. Critical arelesions leading to double-strand breaks or even more
complex local damage.

185

Numerous other products have been identified, and the
kinetics and mechanisms of their formation have been
described [B8, S12, S34, S36].

34. Free radicals are difficult to detect, identify, andmonitor because of their short half-life, particularly in livingorganisms. Such detection and monitoring can be achieved
only by detecting and measuring the products of their reaction
with endogenous bio-components or exogenous components
selectively added to a biosystem. Specific products of suchreactions or their metabolites may qualify as markers of aparticular process or specific free radical. In biosystems, theseproducts are called molecular markers, a subclass of biomarkers [G9]. For a product to qualify as a molecular marker,
there must be unequivocal proof of an exclusive origin of the
product. First, a comprehensive understanding of the kinetics,
energetics, and mechanisms of product generation is required.
Then other possible sources of the product must be excluded[S39].

36. On the basis of extensive studies in radiation chemistry
and radiation biology of the kinetics and mechanisms of OH
radical reaction with DNA components, it was suggested that
detection of thymine glycol, thymidine glycol, and 5-hydroxymethyluracil indicated endogenous OH generation in rats andhumans [C3, H18, W8]. Because thymine glycol can beabsorbed through the gastrointestinal tract and 5-hydroxymethyluracil may be generated by enzymatic hydroxylation ofthymine, these products may not always qualifyas biomarkers
for oxidative damage in organisms. Thymidine glycol is lessprone to such confounders and qualifies as one of the best
endogenous markers of OH [S39]. It was suggested that
8-hydroxyguanine could be another OH marker in biosystems[B12, F3, K1, R9, S30, W12]. Enzymatic hydroxylation of
guanine, however, has not been ruled out unequivocally.
Hence it is prudent to monitor more than one marker for eachspecific free radical under investigation. 8-Hydroxyguanosine
was analysed in the DNA of peripheral blood leukocytes ofpatients exposed to therapeutic doses of ionizing radiation[W12]. Radiation-generated oxidative DNA base products
were also measured in the DNA of irradiated cells [N2].

35. Although a molecular marker can be quantified bymeasurement in vivo, quantification of oxidative stress is
considerably more complex. The reactivity of all five bases,
adenine, cytosine, guanine, thymine and uridine, with OH
radicals is extremely high, whereas that of deoxyribose isabout five times lower [B34]. The distribution of damage will
therefore be governed by the relative abundance and reactivityof DNA and RNA components. Each DNA and RNA basecontains more than one site of attack. For example, OH adds
to the double bond of thymine at C-5 (56%) and C-6 (35%)
and removes hydrogen from the methyl group (9%) [J7]. The
5-hydroxythymidine intermediate leads to formation ofthymine glycol. The 6-hydroxythymidine intermediate is anoxidizing radical that gives rise to unstable hydroxyhydrothymine. The radical on the methyl group of thymine,
however, is a reducing radical that yields 5-hydroxymethyluracil as the final product (reviewed in [S39]). Addition of OH
to the C-8 position of guanine yields a well-known product,8-hydroxyguanine or 8-oxoguanine, which was discovered by
Kasai et al. [K1, K47] and described in detail [J4, S39].

37. The chemical reaction products in DNA are excisedfrom damaged DNA over a certain period of time by repair
mechanisms and eventually appear in the cell medium or
urine. Some oxidative DNA base products have beenmeasured in the urine of irradiated humans and mice. The
radiation yields of these markers, i.e. the increments per unitof energy (mass × dose), were obtained from the level one day
after irradiation minus the level before irradiation and areshown in Table 2. In contrast to the metabolic levels of thesemarkers, the irradiation yields per unit energy are the same forboth mouse and human, as expected, because the same
number of OH radicals is generated in both cases [S39]. The
metabolic rate plays an important role in the variability ofrelative rates of oxidative DNA damage. A high metabolic
rate, as in rodents, generates a high yield of urinary markers,
i.e. higher rates of DNA damage. The rate of DNA damage,
however, is not always proportional to the specific metabolic
rate because the efficacy of inhibition and scavenging ofoxygen radicals and peroxides as well as of DNA repair
systems varies in different species.

Table 2
Yield in urine of biological markers of oxidative DNA damage [B12]Metabolic yield(nmol kg-1 d-1)

Increment induced by radiation(nmol kg-1 Gy-1)

Specific
metabolic rate(kJ kg-1 d-1)

Thymidine glycol

8-Hydroxy-guanine

Thymidine glycol

8-Hydroxy-guanine

Human

100

0.3±0.1

0.3±0.1

3.1±0.8

6.7±1.5

Mouse

750

7.3±1

11±2

3.0±0.66.9±1.3

Species

186

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

3. Gene mutation analysis
38. The analysis and quantification of genomic changes areimportant steps in monitoring and in the elucidation of
mechanisms leading to critical health effects. Functionalchanges, i.e. changes in the phenotype of oncogenes andtumour-suppressor genes, may be of direct relevance to theprocess of carcinogenesis. The ultimate effects of ionizing
radiation and other genotoxic agents are genetic changes,
which are heritable, i.e. which can be passed in a clonal
fashion from one somatic cell to the following cell generations, or from a germ cell to the offspring. Therefore criticalstudies of combined effects should include gene mutation
analysis as one very important biological endpoint forstochastic health effects. Several methods are available for the
study of gene mutations arising in human somatic cells in
vivo. These methods allow determination of the frequency ofmutant lymphocytes or erythrocytes or characterization of
mutations at the molecular level in lymphocytes. The study oftypes, frequencies, and mechanisms of human somatic mutations in vivo is valuable in its own right and may also
improve the understanding of individual variation in sensitivity to environmental exposures, the influence of DNA repair
and metabolism, and the relationship between mutagenesis
and carcinogenesis [L7, M21]. Genetic changes are keyeventsin carcinogenesis. Most human tumours contain more or lessspecific mutations that are directly or indirectly related to the
carcinogenic process. A description of mutations in humantumours and the scientific background of manyof the concepts
and methods addressed in this and the following Chapter arepresented in more detail in Annex F, “DNA repair andmutagenesis”.
39. Early and probably single-step biological end points,
such as morphological changes in in vitro cell lines, mightalso serve as indicators of genetic changes. The developmentof cell culture systems has made it possible to assess the
oncogenic potential of a variety of agents at the cellular level.
Many assays for oncogenic transformation have beendeveloped, ranging from those in established rodent cell lines,
where morphological alteration is scored (e.g. loss of contact
inhibition in 10T½ cells), to those in human cells growing in
nude mice, where tumour invasiveness is determined. The
mutational changes involved are rarely defined. In general,
simple in vitro systems that deliver reproducible results are theleast relevant in terms of human carcinogenesis and humanrisk estimation. The most important potential of these systems
lies in the opportunity they offer to identify and quantify
factors and conditions that prevent or enhance cellulartransformation by radiation and chemicals [H11].
(a) Mutation frequencies
40. Five systems for biomonitoring humans exposed tocarcinogenic agents have been developed in which gene mutation is the endpoint. Two of these use as markers haemoglobin variants (Hb) [S18, T4] and loss of the cell-surface
glycoprotein glycophorin A (GPA) in donors heterozygous at
the MN locus in erythrocytes [L1, L3, L6]. The other three
involve detection of mutations in T lymphocytes in the

X-linked locus for the purine salvage pathway enzyme,
hypoxanthine phosphoribosyltransferase (hprt) [A5, A6, A8,
M22, R7, R8, T10], in the autosomal locus for humanleukocyte antigen-A (HLA-A) [J3, M18, T7, T8], and in theautosomal T-cell receptor genes (TCR) [K23, K24, N3, U15].
41. Mean background mutation frequencies in human cells
in vivo, as analysed by the five mutation assays, differ by
about four orders of magnitude. In summary, the relative orderof background mutation frequency values from normal adults
for the five markers are Hb (5 108) < hprt (5 106) < GPA(1 105) < HLA-A (>1 105 ) < TCR (>1 104) (reviewed in[C23]). For at least three of these mutation systems, sufficientnumbers of donors have been tested to show that, as a generalrule, the mutant frequency in normal, non-exposed donors is
low at birth, increases with age, is often elevated in smokers,
and is increased in people who have been exposed to known
mutagens and carcinogens. Despite the great variation inmutant frequency among individuals at each of the locistudied, these findings show the potential relevance of mutational analysis in the assessment of combined environmental
exposures. More recently, the polymerase chain reaction(PCR) has also been applied in the analysis of mutational
spectra. A fairly complete database has been compiled by
Cariello et al. [C51, C52].
42. The frequencies of hprt mutant cells in healthy adults
range from <0.5 to 112 106. In most cases the frequency of
hprt mutant cells is significantly increased after smoking[C17, C19, H2, T4, T11]. There seems to be no effect of sex
on the hprt mutant frequency. In most studies, an age-related
increase in mutant frequency is seen at the hprt locus,
estimated to be 1%5% per year in adult donors. Radiochemotherapy for various malignant disorders, including
breast cancer, hepatoma, other solid tumours, and lymphoma
increased the frequency of hprt mutant T cells by a factor of310 [D5, M20, N8]. Cole et al. [C18, C20] examined factory
workers exposed to styrene or to nitrogen mustard. In contrast
to styrene, nitrogen mustard significantly increased thenumber of mutant hprt cells in these donors. Tates et al. [T9]described a significantly increased mutant frequency in agroup of factory workers exposed to ethylene oxide.
(b) Mutational spectrum
43. The spectrum of mutational changes that arise
spontaneously or that may be induced by a physical orchemical agent in human cells is broad. At the DNA level it
encompasses, at one extreme, single-base events, and at theother, chromosomal rearrangements involving small to large
deletions or translocations. In addition, an important categoryof mutational events in humans involves losses or gains ofwhole chromosomes. The mutation spectrum in the
mammalian genome is reviewed in Annex F, “DNA repair
and mutagenesis”.
44. Many known mutagens form covalent DNA adductsthat are released from DNA either spontaneously or by biological repair processes [H16]. Mutations induced by a large
number of compounds, e.g. alkylating agents, arylating

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

agents, and radiation, have been scored and characterizedusing shuttle vectors. These experiments elucidate thesequence specificity of adduct formation and, subsequently,
the mutations and the mutational efficiencies of different
adducts [D9, I6, M10].
45. About 15%20% of hprt mutations in normal adults
result from gross structural alterations [A8, B31, H8, N7,
T10], as detected by Southern blot analysis. These include
deletions, insertions, and rearrangements. The break points
or alterations are distributed randomly within the gene,with no hot spots having thus far been identified [A8]. The
remaining 80% of the background in vivo hprt mutationsin adults consist of point mutations or small deletions,insertions, and frameshifts beyond the resolution ofSouthern analysis. Considering only the in vivo hprt
mutations (46 Lesch-Nyhan germinal, 51 normal adultsomatic, 86 exposed adult somatic), several hot spots ofpoint mutations were observed. In particular, four base-pairsites have been observed to be mutated in all groups [C13].
46. Ionizing radiation is known to induce gross structural
alterations in hprt and other reporter genes in cultured humancells. After exposure to radionuclides for diagnostic purposes,
an increase in the frequency of mutants with gross structural
alterations on Southern blots was observed to be 33%,
compared with 13% before receiving radionuclides [B31].
Mutations from post-radioimmunotherapy patients showed
clearly greater frequencies of gross structural alterations thanmutations from pre-radioimmunotherapy patients or normal
individuals. The latter two frequencies are quite similar,
suggesting that cancer per se does not produce this sort ofdamage at hprt [A9]. Taken in toto, the data from Albertini etal. [A9] on in vivo hprt T-cell mutations indicate thationizing radiation produces deletions, particularly large
deletions.
47. The yield of mutations caused byionizing radiation may
be influenced strongly by adaptive responses to othertoxicants or earlier exposures to the same agent. This topic
was reviewed in Annex B, “Adaptive responses to radiation incells and organisms” of the UNSCEAR 1994 Report [U2]. A
70% reduction in hprt mutant frequency in radioadapted
human lymphoblastoid cells has been reported, as analysed bySouthern blot analysis and multiplex polymerase chain
reaction assay [R10, R12]. The treatment was 4 Gy fromgamma rays alone or in addition to an adaptive dose of
0.02 Gy. The proportion of deletion-type mutations wasdecreased in adapted cells (42%) compared with that inmutants treated with the high dose alone (77%).
48. Using a shuttle vector system, Kimura et al. [K12]analysed mutational spectra of the human cDNA hprt gene, arecombinant DNA copy of the hprt RNA, arising spontaneouslyor induced bythe mutagens methylnitrosourea (MNU);
3-amino-1-methyl-5H-pyridol[4,3-b-]-indole (Trp-P2), a
tryptophan pyrolysate; and acetylaminofluorene (AAF). Most
mutations induced by MNU are G:C to A:T transitions. Thiscan be predicted by the major premutagenic lesion in DNA
produced by MNU, namely O6-methyl-guanine that specifi-

187

cally mispairs with thymine [S41]. Mutations that arise spontaneously or are caused by x rays, Trp-P2, or AAF give rise toa similar mutation spectrum of c-hprt. Base substitutions
account for about one third of all mutations. Mutations otherthan base substitutions make up some two thirds of all
mutations. The main mutational event in these cases is deletion. A noticeable feature of these deletion mutations is thefrequent presence of short, direct repeats at the site of thedeletion.
49. Mutational alterations in p53, a tumour-suppressor
gene, are mostly (more than 85%) missense mutations, while
those of APC, another tumour-suppressor gene, and hprt arelargely composed of nonsense, frameshift, deletion, andinsertion mutations, resulting in truncated gene products orloss of genes. The mutational spectrum in p53 is therefore
clearlydifferent from that of other genes. Mutations in the p53
gene detected in tumours seem to be the result of a functional
selection process for mutant p53 protein that gives growthadvantages to the cell. On the other hand, large deletions in
the p53 region may not be compatible with cell survival. Thissuggests that the mutations of p53 observed in tumours may
reflect only those mutations of the initial events that arecompatible with cell proliferation and may even reflect thosethat give the transformed cell a growth advantage over thesurrounding cells. Mutational selectivityin tumour-suppressor
genes is discussed in detail in Annex F, “DNA repair andmutagenesis”.
50. With respect to interaction mechanisms leading to
combined effects, present knowledge indicates that the
mutational spectrum found in tumours often reflects not theagent responsible for the primary DNA damage but rather
growth selection based on specific changes in the phenotype
or general chromosome instability emerging during carcinogenesis. Analysis of marker cells in peripheral lymphocytes
may overcome this problem, albeit at the expense of losing thedirect link to human disease.
4. Cytogenetic analysis
51. The main conceptual basis for using cytogenetic assays
for biological monitoring is that genetic damage in easily
available cells, such as peripheral blood lymphocytes, reflects
comparable events in target cells. The fact that chromosomal
abnormalities are often a characteristic feature in malignant
cells points to the direct relevance of such markers forclastogenic agents to be considered in combined exposures. In
addition, long-term follow-up of populations screened forchromosomal aberrations shows a clearly higher cancer risk
for the subgroup with an elevated level of chromosome
damage [B20, H5]. Microscopically recognizable chromosomal damage includes numerical aberrations and structural
chromosomal aberrations, in which a gross change in themorphologyofa chromosome has occurred. Chromosome andchromatid breaks, dicentrics, and ring chromosomes areimportant examples of this class of damage [N11]. The yieldof sister chromatid exchanges, which represent apparently
symmetrical intrachromosomal exchanges between the two
identical sister chromatids and which are already quite

188

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

frequent in unexposed cells, is also increased. Micronuclei
arising either from acentric chromosome fragments or from alagged whole chromosome with centromere [W15] are also
important markers, although the second production pathway
points to a mechanism driven partially by epigenetic factors.
(a) Chromosomal aberrations
52. Induced chromosomal aberrations can be divided into
two main classes: chromosome-type aberrations, involving
both chromatids of a chromosome, and chromatid-type
aberrations, involving only one of the two chromatids.
Ionizing radiation induces chromosome-type aberrations in
the G0 or G1 stage of the cell cycle (e.g. prior to replication),while chromatid-type aberrations are produced during the S or
G2 stage (e.g. during or after replication of the affected
chromatid segment). In peripheral lymphocytes, most ofwhich are in the G0 stage of the cell cycle, ionizing radiationinduces mainly chromosome-type aberrations.
53. Most chemical mutagens are S-dependent clastogens
and therefore produce mainly chromatid-type aberrations.
S-dependent compounds have no direct effect on the chromosomes of peripheral lymphocytes in vivo, because they
replicate only after stimulation in cell culture. Peripheral
lymphocytes can, however, carry unrepaired/misrepaired,
long-lived lesions that may lead to aberrations during
replication of DNA in vitro [S28].
54. The classical chromosome aberration assay formeasuring dicentrics is a reasonably good measure of dose
down to 100 mGy whole-body exposure [L31] or, withmuch effort, even lower. However, it is based on a genetic
change that considerably impairs the survival of indicator
cells and their stem cells, so that the signal fades withtime. Reciprocal translocations are considered lessdisruptive to the proliferative future of affected cells. It is
possible to score translocations with G-banding or FISH
(fluorescent in situ hybridization) techniques, with thelatter technique having a higher detection limit, about
500 mGy. In such systems, the preferential loss of affected
cells may still be a minor problem; in addition, clonal
expansion of cells carrying translocations conferring a
growth advantage may lead to an overestimation of thedose with time. Biological dosimetry using cytogenetic
parameters will be discussed later. It seems that all agentsthat apparently induce single-base changes (i.e. basedeletions, transversions, or transitions) also induce grosschromosomal changes that are visible under the
microscope. However, the number of agents clearly shownto induce cytogenetic changes in humans is still relatively
limited [A24, S31]. From known or suspected carcinogenic
agents, mixtures, or complex exposures to humans,
cytogenetic data are available for 27 compounds inGroup 1 of the IARC classification (known carcinogens tohumans), for 10 compounds in Group 2A (probable
carcinogens to humans), and 15 compounds in Group 2B(possible carcinogens to humans) [I1, I2]. Chromosome
damage in humans was found in 19/27, 6/10, and 5/15
cases in these groups, respectively.

55. Most of the informative data on induced chromosomal
aberrations in humans arise from high-exposure occupationalsituations. The comparisons of experimental animal data andhuman data for the endpoint of chromosomal aberration are
generally in good agreement. However, in a few cases there
are discrepancies between animal and human data. Highoccupational exposure to radon induces chromosomal
aberrations in humans. Animal experiments with comparable
exposures are negative. The most likely explanation is a
confounding by other clastogenic exposures in humans, e.g.
smoking.
56. Unlike radiation exposure, chemical exposures havebeen considered in very few cytogenetic follow-up studies.
Studies on the induction of chromosomal aberrations after
exposure to alkylating agents expressed in peripheral
lymphocytes show, like studies after radiation exposure, thatdamage can be conserved over several months or even years
after treatment [G3]. The persistence of chromosome damage,
however, varies with the type of exposure and the cytogenetic
endpoint examined.
(b) Sister chromatid exchange
57. The induction of sister chromatid exchange can beobserved in cells that have undergone two rounds of DNA
replication in the presence of bromodeoxyuridine (BrUdR),
which results in chromosomes having sister chromatids thatare chemically different from one another: one is unifilarly
labeled with BrUdR and the other bifilarly labeled. Such sister
chromatids stain differently from one another, and any
exchanges that occur between the sister chromatids can beclearly seen and counted [W7]. A number of studies
confirmed the ability of low-LET radiation to induce sister
chromatid exchanges in rodent cells [G4, L22, R5, U14] andhuman lymphocytes [G14]. However, in other studies, whennormal human lymphocytes in G0 were assessed for theirability to express sister chromatid exchanges following lowLET radiation exposure, they failed to do so, in contrast to thequantifiable induction of chromosomal aberrations [L21,
M28, P2]. This difference could possibly be attributed to thepresence of BrUdR, a known radiosensitizer, at the time ofirradiation in the rodent cell studies [L25]. Nevertheless, lowLET ionizing radiation and radiomimetic chemicals are notveryeffective at inducing sister chromatid exchanges, contrary
to S-dependent agents such as UV light [W11], alkylating
agents [T1, Y4], and cross-linking agents [S4]. High-LET
radiation (neutrons and alpha particles), however, induces
sister chromatid exchanges in normal human peripheral
lymphocytes exposed in G0. This suggests that the relative
biological effectiveness for sister chromatid exchange
induction is very large, since there is little low-LET response[A2, S11]. The induction of sister chromatid exchange as afunction of charged-particle LET in Chinese hamster cells
was recently described [G7]. At each LET examined there
was a dose-dependent increase in the frequency of sister
chromatid exchanges. In contrast to the majority of biological
endpoints, however, where relative biological effectivenessincreases as LET increases up to a maximum and thendeclines, it was found that sister chromatid exchange

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

induction already declined as LET changed from 10 to 120
keV mm1 [G7]. These observations can be explained on thebasis of repair differences for DNA damage induced byradiations of different LET, i.e. the faster the repair, the lesslikelihood there will be of unrepaired DNA damage at thetime of replication when sister chromatid exchanges areformed.
(c) Micronuclei induction
58. Micronuclei can be formed from entire chromosomes or
chromosome fragments [M36]. Theyresult from chromosome
breakage and/or damage to the mitotic spindle and are used asa measure of genotoxicity [H15]. Techniques to blockcytokinesis in mitogen-stimulated lymphocytes [F4, F5, M24,
P19] allow these micronuclei to be observed in binucleatedcells found after the abortive attempt of the cell to divide.
There is, however, a large and variable background frequency
of some 512 micronuclei per 103 binucleated cells [F5, Y2].
The background frequency increases with age from about 4
per 103 among those 20 years, to 8 per 103 for those 30
years, and nearly 12 per 103 for those 40 years [Y2]. The
increase is about 4% per year [F5]. The range of variability
increases with age as well. Farooqi and Kesavan [F18] also
found that the yield of radiation-induced micronuclei in mousepolychromatic erythrocytes was strongly influenced by small
conditioning doses (25 mGy). Micronuclei assays are fasterand have a greater potential for automation than the scoringof chromosome aberrations [M36].
59. Caffeinated and alcoholic beverages have no significant
effects on in vivo mean micronuclei frequency in binucleatedlymphocytes. Even the intraperitoneal (ip) injection of large
amounts of caffeine (15 mg kg1 body weight) did not induce
chromosomal aberrations in mice [F19]. However, theestimated number of diagnostic x-ray examinations to anindividual in the year prior to measurement was significantly

189

correlated to micronuclei frequency [Y2, Y5]. The effect ofage and x rays on lymphocyte micronuclei has been shown
repeatedly [A11, E2, F5, I1, I2]. Tobacco smoke and tobaccorelated exposures are listed in the IARC Monograph series[I1, I2] as micronuclei-inducing agents.
60. In an analysis of micronuclei frequency in survivors of
the atomic bombings, Ban et al. [B4] confirmed the age
dependency of background micronuclei levels in peripheral
lymphocytes. Females showed a somewhat higher frequencyof binucleated cells. Age and sex were independently acting
factors. There is no evidence for an effect of radiation dose on
present-day background micronuclei frequency in thesurvivors.
5. Summary
61. The primary molecular and cellular effects of the many
agents potentially involved in combined effects are extremely
diverse. No unifying concept of dose can therefore be applied.
However, comparisons of toxicity may be based on relevant
experimental and clinical endpoints with sometimes only
loose and enigmatic links to primary lesions and interactions.
A large number of quantitative and semi-quantitative
indicators of exposure are presently available. On the level ofgenotoxicity, DNA damage can be measured up to thefunctional level of single genes, thus allowing a comparisonof the biological activity of different agents and an assessmentof possible interactions on a directly relevant level. The
accessibility of critical cells and tissues to standard analysis
remains a problem. Qualitative and quantitative monitoring of
biological effects at the different levels of organization, frommolecules to organisms, not only might allow an assessmentof the exposure to the different agents involved but could also
form the basis for a better understanding of the mechanismsof combined effects and for the elucidation of dose-effectfunctions for cellular and clinical endpoints.

II. MECHANISTIC CONSIDERATIONS
62. In view of the many different agents that may be
involved in combined exposures with radiation and thecomplexity of the possible interactions, it is necessary to gain
some insight from the mechanistic point of view. This
Chapter will give a qualitative insight into the interaction
processes by describing important steps in the development of
the radiation effect and by suggesting how the radiation effectmight be influenced by other agents. For a quantitative
insight, various models have been developed to describe thebiological effects. Examples of such models will be briefly
discussed, in so far as they serve to improve understanding of
the mechanisms involved in combined effects. However, it
should be kept in mind that models have limited applicability,
and agents do not always have only a single mode ofinteraction.
63. Since cancer is the most important health effect forradiation at low doses, the review presented in this Chapter

deals mainly with mechanisms that are central to theemergence of malignant growth. An in-depth review of thescientific background of some of the concepts discussed here
was presented in Annex E, “Mechanisms of radiation
oncogenesis”, of the UNSCEAR 1993 Report [U3], Annex F,“DNA repair and mutagenesis” and Annex G, “Biologicaleffects at low radiation doses”.
64. The timescale of events for the various stages ofradiation-induced cancer ranges from less than a second totens of years. Schematically, three crude time-scale-based
phases can be defined on the molecular, the cellular, and thetissue/organ level. The molecular phase ranges from the earlyinteraction of the radiation track until initial damage inbiologically important molecules has occurred (of the order of
seconds). The cellular phase follows and lasts until thebiological reactions of the cells involved have occurred andbiological cellular effects are induced (of the order of a few

190

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTSdays). Ultimately, on the tissue/organ level, cellular damage
may progress in due time, with or without cooperation fromother damage, to clinicallydetectable cancer, which can occur
up to 40 or more years after the initial irradiation. These
phases are described below. A schematic representation of theprocesses is given in Figure I. The separation into thesephases is arbitrary; it is time-scale-motivated and serves here
only to describe the possible interactions of the radiation effectwith other agents. In reality, the processes are not separated
that rigorously, and interactions with another agent may occur
on more than one level or phase.
65. Radiation-induced effects other than cancer, such asdeterministic and teratogenic effects, involve similarphases in the development of the radiation damage. For
conciseness, these effects are not explicitly mentioned andconsidered here, but the data in humans are reviewed
briefly in Chapter V.

A. EFFECTS ON THE MOLECULAR LEVEL
66. Following the primary interaction of a radiation trackwith biological matter, an avalanche of events occurs, andvarious reactive species are left after passage of an ionizing
particle or photon: molecules are excited and ionized, radicals
are formed, and secondary electrons progress through thematerial. Most of these species are chemically very reactive
and produce other molecular species. These initial processes
develop in a very short time (of the order of microseconds)
and at short distances from the radiation track. The processes
are dependent on the physical and chemical characteristics ofthe material, the type of radiation, and the conditions in theimmediate environment of the target molecule, such as theavailabilityofoxygen, the presence of sensitizing or protecting
agents, the ambient temperature, and the ionization density of
the radiation. The processes involved in the interaction ofradiation at the molecular level are extensively studied inradiation biochemistry and microdosimetry, the concepts ofwhich have been described by the International Commission
on Radiation Units and Measurements (ICRU) [I7].
67. The biological effects of radiation arise mainly fromdamage induced in DNA molecules. Important types of DNA
damage are DNA single- and double-strand breaks, basedamage, intra- and intermolecular cross-links, and multiply
damaged sites (mds) (see Annex F, “DNA repair and mutagenesis”). A review of special models with emphasis on theimportance of the DNA damage is given by Goodhead et al.
[G17]. As far as epigenetic damage or modifications of othercell constituents are concerned, cytoplasmic changes andmitochondrial or membrane damage may also play a role incertain types of radiation effects, but the importance of thesefor radiation-induced cancer is disputed. Indirect effectmodifiers such as growth stimulation as a result of stem cellkilling may become important at higher doses.
68. The possibility of another agent interacting with theradiation effect in this early phase is dependent on changes inthe DNA environment. The direct environment of the DNA

defines the fate of radiation-induced reactive species, such as
water radicals, and the possibilityfor direct or indirect damage
to the DNA. Interaction leads to changes in the dose-effectrelationship for DNA damage and consequently to changes inthe dose-effect relationship for cellular effects (see Section
II.B). A well known modification of the radiation effect iscaused by a change in the oxygen content. Anoxic cells, ingeneral, are more resistant to radiation than well oxygenatedcells. Typical agents interacting with the radiation effect at
this level are electrophilic compounds, such as N2O, NO2, NO,
CO2, SO2, and SO3, and nucleophilic agents, such as cysteamine and cysteine [G17, O11]. For interaction with theradiation effect, the agents should, in general, be present in
the DNA environment during irradiation. They may modify
radiation effects by a factor of up to 3. More indirect effects
may result from vasodilators and constrictors modulating
oxygen pressure in irradiated tissue.
69. An important class of agents are hypoxic cellradiosensitizers, also called oxygen-mimetic agents, whichhave potential use in radiotherapy to enhance the effectiveness of the radiation treatment in anoxic or poorly oxygenatedparts of the tumour. These sensitizers must be present at the
instant of irradiation. The mechanisms are free-radical-based:
the compounds, in general, have increased electron affinity
and are believed to involve fast electron transfer processes in
DNA [A1]. Well-known agents include nitroheterocyclic compounds, such as metronidazole, misonidazole, and related
compounds, metal-based compounds containing Pt, Rh, Fe,
Co, and other metals, and nitro-compounds, such as nitrosoureas [S2].
70. Other chemicals protect healthy cells against theradiation effect. They may also be used in radiotherapy. Theseradioprotectors are mainly sulphur-containing compounds.
They act, in part, as radical scavengers and have to be present
at the time of irradiation to produce their protective effect. The
radioprotective effect is a factor of 3 or less. Typicalcompounds of this type are cysteine, cysteamine, aminoethylisothiourea (AET), mercaptoethylamine (MEA), and othersulfhydryl-group-containing agents [M4].

B. EFFECTS ON THE CELLULAR LEVEL
71. When the radiation has induced molecular damage, thecell reacts by attempting to remove the damage and restore
normal cellular function. The reaction depends on the type ofdamage. For simplicity, only damage to the DNA isconsidered here, which may be characterized as single-strand
or double-strand damage. Single-strand damage, such as
breaks or base damage, may be readily and effectively
repaired. Complex localized damage, such as a double-strand
break, is more difficult to repair and may lead to a biologicallydifferent behaviour of the cell. Repair depends on the cell’s
genotype. It takes place within a few hours after theirradiation. Some of the damage may be persistent and lead toa radiation effect at the cellular level. The most important
cellular effects are chromosomal aberrations, mutations andcell inactivation, killing, and apoptosis. Changes leading toANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

malignant transformation, which can be considered a specificclass of somatic mutations or chromosomal aberrations, areparticularly important for radiation carcinogenesis.
72. Attempts to characterize the initial biological effect of aradiation exposure and its dose-effect relationship have led tothe development of mechanistic biophysical models ofradiation action. The aim of these models is to present amathematical description of radiation action based on realistic
assumptions related to basic mechanisms [G12]. Broadly, acommon characteristic of these models is that they describe
the cellular radiation effect E(D) by a linear quadratic doseeffect relationship:(1)
E(D)  E  αD  βD 20where E(D) is the cellular effect from a dose D, E0 is theeffect without radiation (D = 0), α is the contribution to theeffect per unit dose and β is the contribution to the effectper unit dose squared.
73. The interpretation of the linear and quadratic doseterms depends on the underlying assumptions of the model.
The linear term has a single-track nature, sometimes called
intratrack damage. The quadratic term has a dual or
multitrack nature, involving the accumulation of sublethal
damage or sublesions [C16, K5, K8]. Some models do notaccount for repair; in other models, repair is consideredessential for development of the radiation effect. Most models
do not specify the initial type of damage [C16, K4], whileothers are more specific [C45]. In general, double-strand
breaks in DNA play an essential role in the radiation effect.
74. Equation (1) broadly describes the dose-effect relationships for exposures within one cell cycle and is generally used
to analyse cellular experimental data, such as chromosomal
aberrations, mutations, cellular transformation, andcell killing[I14]. The dose coefficients α and β depend on the effectconsidered, the cell type, the type of radiation, and thedevelopment of the radiation damage during the molecularphase [L11]. For instance, α is particularly dependent on thetype of radiation and, in general, is larger for densely ionizing
radiation than for sparsely ionizing radiation. The coefficientβ, in general, tends to decrease with higher-LET radiation. As
far as irradiation time is concerned, α hardly changes and ismostly invariable, but β changes markedly: it reaches amaximum for acute irradiation, decreases for lower dose rates,
and for irradiation times of more than a few hours is
negligible or zero. This implies that for chronic irradiation a
linear dose-effect relationship for cellular effects is anticipated.
75. The mechanism of interaction of another agent withthe radiation effect at the cellular level is broadly based on
three types of action: (a) the accumulation of sublesions
and lesions; (b) interference with cellular repair; and (c)changes in cell-cycle kinetics. All types of interaction are
most effective when the potentially interacting agent ispresent in the cell at the time of irradiation or within a few
hours later, roughly as long as the radiation effect is not
fixed and repair is still possible.

191

1. Accumulation of (sub)lesions
76. An important category of combined exposures involving accumulation of sublesions is that of combined exposures
to different types of ionizing radiation. For cellular effects
such as cell killing, mutations, and chromosomal aberrations,
it is well known that the combined exposure to two types ofradiation can lead to a larger than additive effect. Understanding how cellular damage produced by densely ionizing
radiation (high-LET radiation) interacts with that produced by
low-LET radiation is important both in radiation therapy andin evaluating risk.
77. With similarity in the underlying radiation mechanism,
interaction between different types of ionizing radiation can beshown to be, in general, of the so-called isoadditive type.
Modellers of cellular radiation effects tend to describe thelarger effect of combined radiation exposures in terms ofaccumulation of and interaction between sublethal damagedsites, which may lead to an extra contribution to the radiation
effect (increase of the quadratic term of the linear-quadratic
dose-effect relationship) [B35, C15, L10, Z14].
78. In general, if the (additional) radiation effect Ei ofradiation type i is linear-quadratic with dose Di,(2)

E i (Di)  αi Di  βi Di

2then the combined exposure to radiation types 1 and 2 willlead to effect Ec, given by
E (D , D )  α D  α D  ( β D  β D )2 (3)
c

1

2

1

1

2

2

1

1

2

2

In the absence of interaction, the effect would be given by
E a (D1, D2)  α1 D1  α2 D2  β1 D1  β2 D2
2

2

(4)

The extra effect is expressed in the difference between
equations (3) and (4) and can be calculated to be
E c (D1, D2)  E a (D1, D2)  2 β1 β2 D1 D2(5)Equation (5) indicates that the extra effect is dependent on
β1 and β2. Experimental evidence [B5, C6] shows that β ispractically independent of radiation type (i.e. low- or highLET radiation), so that interaction of sublethal damage canbe expected. Using this assumption, the radiation effect of
combined exposures of acute high- and low-LET radiation
could well be described by the equations given here [L10].
79. Considering this interaction process, one has to keep
in mind the following restrictions:(a) sublethal damage can be repaired by the cell, so thatwhen there is time between the two exposures, theextra effect will decrease;(b) the quadratic term for each radiation type separately
is dependent on dose rate, i.e. irradiation time, which
implies that the extra term for combined exposures

192

(c)

(d)

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

also vanishes for dose rates below a certain value (i.e.
less than 10 mGy min1);
the interaction process described here is, strictly
speaking, proven onlyfor exposures occurring within
one cell cycle. Deviations may be expected when
exposures occur over more than one cell cycle; andfor practical applications in risk analysis, deviations
from additivity are generally not very large, with themost significant deviations being expected for acuteirradiation exposures such as are used in radiation
therapy; additivity is virtually expected for combined
chronic exposures.

80. As Lam [L47] has shown, the interaction of two typesof radiation can also be described using the linear isobolic
relationship, which is usually used for the combined actionof two toxic agents. The reverse also applies: theinteraction with radiation of a toxic chemical that has asupralinear or quadratic exposure-effect relationship forcellular effects can be similarly described as the interactionof two types of radiation. As described above, if theradiation effect after a dose D is given by equation (1) andthe effect after an exposure X of a second agent is given by
E s (X)  σ X  ε X 2(6)then the effect of a combined exposure will be
E c (D, X)  αD  βD 2  σX  εX 2  ηDX

2. Cellular repair
82. The speed and fidelity of DNA repair is one of the main
determinants of the yield of fixed damage. Most moleculardamage to DNA is subject to a sequential series of enzymatic
reactions that constitutes the repair process. This topic has
been the subject of much recent study, and a spectrum of
analytical procedures, operative at both the molecular andcellular level, has been developed to monitor DNA repair[F10]. DNA damage may include altered bases, the covalentbinding ofbulkyadducts, intrastrand or interstrand cross-links
and the generation of strand breaks. Altered bases may begenerated by spontaneous reactions, most importantly deamination of cytosine to form uracil, of adenine to form hypoxanthine, and of 5-methylcytosine to form thymine. A rangeof alkylated products is formed in DNA as a consequence of
exposure to nitroso compounds and other alkylating agents.
Bulky adducts are formed as a consequence of the covalentbinding, to purines in particular, of polycyclic hydrocarbons,aromatic amines, aflatoxins, and similar substances. Twotypes of pyrimidine dimer are induced by exposure to UV
radiation: cyclobutyl pyrimidine dimers are most common,and the so-called 64 photoproducts are also produced. Crosslinking of DNA strands may occur following exposure tobifunctional alkylating agents and chemicals such as cisdiaminedichloroplatinum. Strand breakage may be caused byionizing radiation, heavy metals, chemicals such as
bleomycin, and endogenouslygenerated active oxygen species(reviewed in [S9]).

(7)This means that the effect of the combined exposure toradiation and the second agent is given by the sum of theeffects of the two agents separately and an extra effect(ηDX), which is proportional to the dose D of radiationand exposure X of the second agent. This extra term is theresult of the interaction of sublethal damage of radiation
with sublethal damage of the second agent.
81. This description of the effect of combined exposures canbe used for a number of compounds with radiation [L51]. In
this analysis it is assumed that the cellular effect of physicaland chemical agents can be described as a linear-quadratic
function of exposure X. Examples of such agents are ultraviolet radiation (UV) [L52]; alkylating agents such as thenitrosouric compounds ethylnitrosourea (ENU), 1,3-bis(2chloroethyl)-1-nitrosourea (BCNU), and 1-(2-chloroethyl)-3cyclohexyl-1-nitrosourea (CCNU)[L48]; benzo[a]pyrene (BP);ethylmethane sulphonate (EMS) [C7] and many more. The
conditions mentioned in paragraph 84 concerning theinteraction of two types of radiation should be kept in mind
for this interaction of radiation with another physical orchemical agent as well. Repair of the sublesions from thefirst agent before the other agent becomes effective canlessen the enhancement effect of the combined exposureand lead to an effect more nearly like additivity. In general,
the analysis can be applied to different cellular endpoints,
such as cell killing [L48, L50, L51], chromosomal
aberrations, and mutations [C7].

83. Efficient repair of DNA damage is necessary to retain
genomic stability and to prevent somatic and genetic diseasein humans and other organisms as well. There are several
modes of repair, and these may also be affected themselves bymutagenic agents. Failure of repair may thus be as much a
cause of disease as the initial DNA damage. To safeguard the
genome, cells are able to block cell-cycle progression in
response to DNA damage at specific transition points to allow
DNA repair. Most prominent are the so-called checkpoint
control mechanisms at the G1/S phase and G2/M phasetransition. The subject of DNA repair is reviewed in Annex F,“DNA repair and mutagenesis”.
84. Programmed cell death, known as apoptosis, obviates
the risks from error-prone repair in heavily damaged cells andis, accordingly, another important defence mechanism of thecell, preventing the survival of aberrant cells and, hence,
tumour development [D13]. Apoptosis can become activatedunder physiological conditions and also after damage to DNA[H44, T17]. p53 plays an important role in DNA damageinduced apoptosis [L32], so the clonal selection of cells with
non-functional p53 by hypoxia [G19] or by UV radiation [Z5]is potentially an important mechanism to increase tumour
yield. This was also shown for radiation teratogenesis in mice.
Norimura et al. [N18] found that p53-mediated apoptosis
strongly reduced fetal malformations after in utero exposure
to ionizing radiation (2 Gy), whereas p53/ strains displayed
a 70% incidence of anomalies. Such effects may lead to an
apparent threshold in the dose-effect relationship formalformations after in utero irradiation [N19]. Several other85. A second class of agents that can interact with radiationand cause changes in the radiation effect at the cellular level
are agents that modify the repair capacity of cells. Repair
inhibitors often influence the DNA structure and may be
immunosuppressive [S1]. These agents might have toxic
effects themselves. Examples are the intercalating agentsactinomycin D, adriamycin, and quinacrine. The xanthinederivatives (caffeine, theobromine, and theophylline) also
belong to this type of agent. The different effects reported forthese agents may be due to the different kinetics of repair inthe cell cycle and the presence of the drugs during different
phases of the cell cycle [B3, T16]. Depending on the drugs,
the repair of sublethal damage or of potentially lethal damage
might be involved in the interaction process.
3. Cytokinetics
86. Another important class of agents are chemicals thatchange the behaviour of the cells in the cell cycle. These
agents are indirectly related to those that interfere with repair,because cells that are irradiated tend to move more slowly
through the cell cycle in order to have more time for repair.
Some cytokinetic agents inhibit changes in the cell cycle.
Caffeine is an agent known to remove or alter the cell’s
capacity to induce a G2 block or shorten the S phase after
irradiation [S1]. The result is that caffeine enhances radiationinduced cell killing and chromosomal aberrations. Effects ofcytokinetic agents are investigated for different purposes,
among which is to study the mechanism of radiation-induced
cellular response and to answer questions such as, in which
phase of the cell cycle is the radiation damage fixed? These
effects are also investigated for their possible application inradiotherapy. Cytokinetic agents are not normally considered
important for environmental risks of stochastic radiation
effects. However, some chemicals, for example those withhormonal side effects such as environmental estrogens, havebeen shown to be effective even at environmental concentrations [S81].
4. Toxicological analysis
87. The cellular effects of combined exposures to radiationand other agents are part of the broad, classical field of
toxicology, in which the effects of exposures to two agents areanalysed using the isobolic method. The method is primarily
useful for agents with isoadditive effects, but it is used forother agents as well. It has been applied to radiation effects[L47, S1].
88. The use of an isobolic diagram to describe the
combined effect of two agents is shown in Figure III. The

193

LINEAR DOSE RESPONSE
1.0

ANTAGONISM

Line of additivity
0.5

SYNERGISM

0
0

0.5

1.0

*)
RELATIVE CONCENTRATION OR DOSE OF AGENT B (XB/XB
RELATIVE CONCENTRATION OR DOSE OF AGENT A (XA/X*
A)

types of cell loss or irreversible growth arrest occur inmammalian systems in addition to apoptosis; these include
terminal differentiation, senescence, and necrosis. Necrosis, incontrast to apoptosis, is not an orderly cellular process but
rather the disorganized death of a cell. Several recent reviewsof this topic have been published [S5, T17, W6]. Apoptosis isdiscussed further in Annex F, “DNA repair and mutagenesis”.

RELATIVE CONCENTRATION OR DOSE OF AGENT A (XA/X*
A)

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

NON-LINEAR DOSE RESPONSE
1.0

ANTAGONISM
ENVELOPE
OF ADDITIVITYSYNERGISM

0
0

1.0

*)
RELATIVE CONCENTRATION OR DOSE OF AGENT B (XB/XB

Figure III.
Isobolic diagrams for a given level of
response in two agents, both acting with linear (upper
diagram) and non-linear (lower diagram) exposure
response [U6].
The axes are normalized to values of 1.0 for each agent
acting separately, i.e. X*A and X*B.

exposures are indicated on the two ordinates, usually with thesingle-agent exposures yielding the same effect normalized toone. The case of additivity is described by a straight isoeffectline for any combination of two agents with linear doseresponse relationships for separate action (Figure III, upper
diagram). If the points deviate significantly to the left of theisobolic line, the interaction is synergistic. An antagonistic
interaction is postulated when the experimental points lie tothe right of the isobolic line. Even in such a simple theoreticalcase, to assess the combined action of two agents, several
combinations of the two agents leading to the given effect,
EAB, have to be tested. Although there are some importantbiological endpoints, such as frequency of point mutations,that show a linear or nearly linear increase after separate

194

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

exposures to genotoxic chemicals or radiation, the dose-effectrelationships for health impairments caused bycomplex multistage changes in biological systems are often better describedby exponential or sigmoid functions of dose. For these more
realistic circumstances, the line of additivity in the isobolic
diagram becomes curved and transforms into an envelope of
additivity (Figure III, lower diagram). In general, the order of
exposure to agents with differing dose-response relationships
then becomes important as well [R4].
89. Such isobolic analyses are important tools, for example
in optimizing combination therapy [L16, R4], but are of lessvalue in evaluating the effects of chronic exposures in theworkplace and in non-occupational settings [B69]. An
extended review of this approach and its mathematicalbackground was presented in the UNSCEAR 1982 Report[U6]. This approach is based on producing equal effects withdifferent combinations of the two agents under restrictedconditions of time. Owing to the general lack of such rangesof exposures in human populations, this method is notapplicable in epidemiology.
90. The interaction at the cellular level is restricted in time,so that damage from one agent seldom interacts with damage
from a second one. For low dose rates of radiation and longterm exposures of other agents, the supralinear or quadratic
dose terms of the dose-effect relationships tend to diminish,
and only a linear dose-effect relationship remains. In thesecases, the possible interaction in combined exposure also
decreases, and additivity results. This implies that sinceinteraction at the cellular level during low-dose, long-term
exposures to radiation and other agents can be expected tohave a low probability of occurrence, it is therefore of limited
importance for carcinogenesis.

C. EFFECTS ON THE TISSUE/ORGAN
LEVEL
91. After fixation of the radiation effect at the cellular level,
which occurs within a few days, a much longer time is needed
before an effect at the organ level occurs, i.e. before astochastic radiation effect is evident. The period of occurrenceof a stochastic effect is dependent on the type of effect. For
example, hereditary defects may occur when a germ cell, after
having been irradiated, forms the origin of an organism of thenext generation. For radiation-induced cancer, it is the time
between the initiation event, or possibly one of the following
steps of the carcinogenesis process, and the detection of atumour. Full consideration of the mechanistic aspects ofcancer development is given in Annex G, “Biological effects
at low radiation doses”. The events occurring after the
initiation event in the development of tumorigenesis are
considered to take place on the tissue and organ level and may
occur years or decades later.
92. It is generally accepted that carcinogenesis is a multistep process. The usual chain of events is considered to be
initiation of damage, tumour promotion, possibly withactivated proto-oncogenes or deactivated tumour-suppressor

genes, and malignant progression. Each ofthese processes canbe related to effects at the cellular level. The basic aspects ofthese processes were reviewed in Annex E, “Mechanisms ofradiation oncogenesis”, of the UNSCEAR 1993 Report [U3].
The concepts of multi-stage carcinogenesis have evolved overmany years of cancer research [A17, B10, B14, B15, C8, F7,
M27, R6]. Several lines of evidence that support the multistage model of cancer derive from studies of pathology,
epidemiology, chemical and radiation carcinogenesis in
animals, cell biology, molecular biology, and human genetics[K28, M5]. Germ-line mutations, somatic genetic events, andepigenetic stimulation by the host organism may all play
important roles in neoplastic development. The definition of
two broad classes of genes, proto-oncogenes with growthenhancing functions and tumour- suppressor genes withgrowth-inhibiting functions, brought a biological basis and aunifying concept to the multi-stage theory of cancer [V2].
Owing to the functional diversity of the products of thesegenes involving cell surface receptors, protein kinases,
phosphatases, and DNA-binding proteins, to mention only afew, this concept does not lend itself directly to a better
understanding quantitatively. However, in this area themodifications of the cancer process after exposure to external
agents may be investigated.
93. The number of genetic changes involved in theevolution of a specific malignant neoplasm is not knownwith certainty. In some cancers that occur early in life,
soon after exposure, or in genetically susceptible
individuals, there may be only one rate-limiting change
needed for malignant disease. Certain forms of leukaemia,
e.g. those resulting from reciprocal translocations [B64] orcancer induction in retinoblastoma heterozygotes, seem tofollow this course. Multi-hit models developed on the basisof specific incidence rates of solid cancers from epidemiological data often show an exponential increase in theincidence of specific cancers with the fifth to seventh
power of age [K11]. Most colorectal cancers have three ormore altered genes, [F6, V1, V2], and estimates of as manyas 10 or more mutational changes have been proposed tooccur in adult human cancers [B17]. Basically, all thesegenetic changes might be induced by ionizing radiation,other genotoxic agents, and the inherent instability of DNA
alone.
94. The distinction between proto-oncogenes andtumour-suppressor genes has important repercussions fordose-effect models, because the former class would
generally express its function dominantly, whereas thelatter could fulfill its protective function as long as one
allele is functionally intact, i.e. the tumour-suppressor
function would be a recessive trait. However, theprobability of developing cancer is in many cases higher inheterozygotes than a pure recessive trait would predict,indicating the importance of penetrance in the genetics ofthe different tumour-suppressor genes. Moreover, mutations in some tumour-suppressor genes like p53 and WT1
may be of the dominant negative type, in which themutated protein overrides the action of the suppressor wildtype allele [H4, M25].

194

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

exposures to genotoxic chemicals or radiation, the dose-effectrelationships for health impairments caused bycomplex multistage changes in biological systems are often better describedby exponential or sigmoid functions of dose. For these more
realistic circumstances, the line of additivity in the isobolic
diagram becomes curved and transforms into an envelope of
additivity (Figure III, lower diagram). In general, the order of
exposure to agents with differing dose-response relationships
then becomes important as well [R4].
89. Such isobolic analyses are important tools, for example
in optimizing combination therapy [L16, R4], but are of lessvalue in evaluating the effects of chronic exposures in theworkplace and in non-occupational settings [B69]. An
extended review of this approach and its mathematicalbackground was presented in the UNSCEAR 1982 Report[U6]. This approach is based on producing equal effects withdifferent combinations of the two agents under restrictedconditions of time. Owing to the general lack of such rangesof exposures in human populations, this method is notapplicable in epidemiology.
90. The interaction at the cellular level is restricted in time,so that damage from one agent seldom interacts with damage
from a second one. For low dose rates of radiation and longterm exposures of other agents, the supralinear or quadratic
dose terms of the dose-effect relationships tend to diminish,
and only a linear dose-effect relationship remains. In thesecases, the possible interaction in combined exposure also
decreases, and additivity results. This implies that sinceinteraction at the cellular level during low-dose, long-term
exposures to radiation and other agents can be expected tohave a low probability of occurrence, it is therefore of limited
importance for carcinogenesis.

C. EFFECTS ON THE TISSUE/ORGAN
LEVEL
91. After fixation of the radiation effect at the cellular level,
which occurs within a few days, a much longer time is needed
before an effect at the organ level occurs, i.e. before astochastic radiation effect is evident. The period of occurrenceof a stochastic effect is dependent on the type of effect. For
example, hereditary defects may occur when a germ cell, after
having been irradiated, forms the origin of an organism of thenext generation. For radiation-induced cancer, it is the time
between the initiation event, or possibly one of the following
steps of the carcinogenesis process, and the detection of atumour. Full consideration of the mechanistic aspects ofcancer development is given in Annex G, “Biological effects
at low radiation doses”. The events occurring after the
initiation event in the development of tumorigenesis are
considered to take place on the tissue and organ level and may
occur years or decades later.
92. It is generally accepted that carcinogenesis is a multistep process. The usual chain of events is considered to be
initiation of damage, tumour promotion, possibly withactivated proto-oncogenes or deactivated tumour-suppressor

genes, and malignant progression. Each ofthese processes canbe related to effects at the cellular level. The basic aspects ofthese processes were reviewed in Annex E, “Mechanisms ofradiation oncogenesis”, of the UNSCEAR 1993 Report [U3].
The concepts of multi-stage carcinogenesis have evolved overmany years of cancer research [A17, B10, B14, B15, C8, F7,
M27, R6]. Several lines of evidence that support the multistage model of cancer derive from studies of pathology,
epidemiology, chemical and radiation carcinogenesis in
animals, cell biology, molecular biology, and human genetics[K28, M5]. Germ-line mutations, somatic genetic events, andepigenetic stimulation by the host organism may all play
important roles in neoplastic development. The definition of
two broad classes of genes, proto-oncogenes with growthenhancing functions and tumour- suppressor genes withgrowth-inhibiting functions, brought a biological basis and aunifying concept to the multi-stage theory of cancer [V2].
Owing to the functional diversity of the products of thesegenes involving cell surface receptors, protein kinases,
phosphatases, and DNA-binding proteins, to mention only afew, this concept does not lend itself directly to a better
understanding quantitatively. However, in this area themodifications of the cancer process after exposure to external
agents may be investigated.
93. The number of genetic changes involved in theevolution of a specific malignant neoplasm is not knownwith certainty. In some cancers that occur early in life,
soon after exposure, or in genetically susceptible
individuals, there may be only one rate-limiting change
needed for malignant disease. Certain forms of leukaemia,
e.g. those resulting from reciprocal translocations [B64] orcancer induction in retinoblastoma heterozygotes, seem tofollow this course. Multi-hit models developed on the basisof specific incidence rates of solid cancers from epidemiological data often show an exponential increase in theincidence of specific cancers with the fifth to seventh
power of age [K11]. Most colorectal cancers have three ormore altered genes, [F6, V1, V2], and estimates of as manyas 10 or more mutational changes have been proposed tooccur in adult human cancers [B17]. Basically, all thesegenetic changes might be induced by ionizing radiation,other genotoxic agents, and the inherent instability of DNA
alone.
94. The distinction between proto-oncogenes andtumour-suppressor genes has important repercussions fordose-effect models, because the former class would
generally express its function dominantly, whereas thelatter could fulfill its protective function as long as one
allele is functionally intact, i.e. the tumour-suppressor
function would be a recessive trait. However, theprobability of developing cancer is in many cases higher inheterozygotes than a pure recessive trait would predict,indicating the importance of penetrance in the genetics ofthe different tumour-suppressor genes. Moreover, mutations in some tumour-suppressor genes like p53 and WT1
may be of the dominant negative type, in which themutated protein overrides the action of the suppressor wildtype allele [H4, M25].

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

195

MULTI-STAGE MODEL

Normal cellsµ (d)Stage 1 cells.......

µ (d)

1

2

TWO-STAGE MODEL

Stage K cells

µ (d)
k

β(t,d)

Normal cellsµ (t,d)1

Initiatedcellsµ (t,d)Malignantcells

2

δ(t,d)TWO-STAGE MODEL WITH DNA REPAIR
β (t,d)2µ (t,d)1Normal cellsρ (t,d)µ (t,d)3

Damagedcells

Initiatedcells

1ρ (t,d)Damagedcells

Malignantcells

3β (t,d)1

δ (t,d)β (t,d)3

2

Figure IV. Models of carcinogenesis.
Parameters: µ is the probability of transformation, βi is the birth or replication rate,δi is the death rate, ρi is the repair rate, i is the stage, d the dose, and t the time.

95. Multi-stage cancer models are described in Annex G,“Biological effects at low radiation doses”. The multi-stage
model proposed by Armitage and Doll [A16, A17, A18]
represents one of the first attempts to develop a biologicalmodel of carcinogenesis. Theypostulated that cancer develops
from a single cell that must pass sequentially through aparticular series of transformations to become a malignant
cell. The multi-stage Armitage-Doll model is illustrated in theupper portion of Figure IV. This model assumes that a normal
cell must pass through k sequential stages before becoming
fully malignant. This model has k + 1 types of cells: normal
cells, stage 1 cells, stage 2 cells, ..., and stage k (malignant)cells. The model supposes that at age t an individual has apopulation N0(t) of completely normal cells and that thesecells acquire a first mutation at a rate λ1(t). The cells with one
mutation acquire a second mutation at a rate λ2(t), and so on
until at the (k  1) stage the cells with (k  1) mutations
proceed at a rate λk(t) to become fully malignant.
96. The instantaneous tumour incidence rate h(t) at time
t in the multi-stage model is therefore approximately of theformdNk(t)/dt  λk(t)t xk1x2

N (x ) λ1 (x1) ...

m m m 0 1
0

0

0

(8)

λk1(xk1) dx1 ... dxk1
where N is the number of cells in the target tissue and λi(t) is
the instantaneous rate of the i-th cellular change (i = 1, ..., k).
For simplicity, it is often assumed that the transition rates arelinearly related to the dose di(t) of the carcinogen at time t for
the i-th stage. Therefore the transition rate from one stage tothe next is given by λi(t) = ai + bidi(t). Here, ai denotes thetransition rate in the absence of exposure and bi reflects theeffects of the carcinogen on the transition rate into stage i = 1,..., k. With similar values for spontaneous transition rates (aito ak), this model predicts that the age-specific tumour
incidence rate will be proportional to the (k  1)st power oftime and provides a good description of human cancer
incidence data with 2 < k < 6 stages [A20, A22].
97. To encompass the growing biological evidence that theprocess of carcinogenesis involves intermediate cells having

196

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

a growth advantage over normal cells, Armitage and Doll
[A17] modified their initial model. The initial model isgenerally viewed as not biologically plausible, because it does
not account for cell kinetics, more specifically the birth anddeath of cells. The modified model that includes cell kinetics
must sometimes assume very small and, in the opinion of
Armitage and Doll [A17], unlikely values for the growth rateof intermediate cells to fit the data.
98. Moolgavkar and Venzon [M29] and Moolgavkar andKnudson [M23] proposed a two-stage birth-death-mutation
model to describe the process of carcinogenesis in adults. By
incorporating both cell kinetics and tissue growth, this model
can be used to describe a broader class of tumour incidence
data than the classical multi-stage model. This model has
three cell types: normal cells, intermediate or initiated cells,
and malignant cells. The middle portion of Figure IV displays
the general two-stage model of carcinogenesis in which for anormal cell to become malignant, it must pass from thenormal state through the intermediate state and into themalignant state. The simplicity of this model allows
classifying external agents in three categories of carcinogen:initiators, which stimulate the first transition of a normal stem
cell into the intermediate stage; completers, which transform
an intermediate cell into a malignant cell by the second
transition; and promoters, which enhance cell division and thenet increase of intermediate cells with time [K46]. Ionizing
radiation and other genotoxic agents may be both initiatorsand completers.
99. The Moolgavkar-Venzon-Knudson model was later
extended to account for more than two mutational stages[L23, L24, M14]. For the generalized Moolgavkar-VenzonKnudson model it may be supposed that at age t there are N(t)susceptible stem cells, each subject to mutation to a type of cellcarrying an irreversible mutation at a rate of µ0(t). The cellswith one mutation divide into two such cells at a rate γ1(t). Ata rate δ1(t) they die or differentiate. Each cell with one
mutation can also divide into an equivalent daughter cell andanother cell with a second irreversible mutation at a rate µ1(t).
For the cells with two mutations there are also assumed to becompeting processes of cell growth, death and differentiation,and mutation taking place at rates γ2(t), δ2(t), and µ2(t),respectively. This continues until at the (k-1) stage the cell
will have accumulated (k-1) mutations. It will eventuallyacquire another mutation and become fully malignant.
100. With the advent of more sophisticated experimental
techniques and a growing understanding of the process ofcarcinogenesis, more refined mathematical models have beendeveloped and continue to be developed to embody the current
scientific knowledge and mechanisms of cancer. Mutation is
the result of DNA damage and the subsequent fixation andpropagation of the damage by DNA replication. This process
is included as a single rate constant in the models described in
the previous paragraphs. However, agents can affect DNA
damage rates, cellular replication rates, and/or the processes
of DNA repair. Kopp-Schneider and Portier [K19] expanded
the modelling of the mutation process to account explicitly forthe process of cellular damage to DNA, DNA repair, andDNA replication. The two-stage damage-fixation model has
five types of cells: normal cells, damaged normal cells anddamaged initiated cells both of which are subject to DNA
repair, initiated cells, and malignant cells (in which damage
has been fixed by replication). This model is shown in thelower portion of Figure IV.
101. It is clear that research on quantitative multi-stage
models is still in progress and that the complexity of the
carcinogenic process inhibits a choice of a universally
accepted and applicable model. However, it is also clear thatmulti-stage models have a biological basis and could describe
tumour incidence quantitatively and as such have a future in
improving radiation risk estimates and estimates of combined
effects. Always important is the question of complexity vs.
simplicity. The biology of cancer formation is so complicatedthat an ever-increasing number of parameters are needed tocover all possibilities of tumour formation mathematically. On
the other hand the available data are limited, so the number ofparameters that can be fixed is limited as well. As far as themathematics and statistics are concerned, it is preferable thatthe number of unknown parameters be as low as possible.
102. Most multi-stage models are used to describe the age
dependence of tumour incidence and the influence ofchemical carcinogens in animal experiments. In a few cases,
theyhave been used to describe radiation-induced tumours. As
far as human data are concerned, the induction of lungtumours by radon in miners [L9, M39] and the lifespanstudies of the Japanese atomic bomb survivors [H1, K45, L5]
were used to test multi-stage models. In general, ionizing
radiation acts mainly as an initiator, although it has someinfluence on other coefficients. An important conclusion oftheuse of multi-stage models for radiation carcinogenesis is thatradiation generally seems to affect only one step in the
carcinogenesis process; in other words, it is a co-factor of
background tumour incidence. This implies that the radiation
effect is dependent on background tumour incidence as wellas on other agents or factors that produce tumours or cancer.
103. The timescale for effects at the tissue or organ level islong and can last for years. The implication is that interaction
with another agent is possible even when the exposures of thetwo agents are separated in time for up to several years. A
comprehensive treatment of the carcinogenic effect of
combined exposures and the implications for dose-effectrelationships using a two-mutation carcinogenesis model isgiven in Krewski et al. [K46]. They classify carcinogenic
agents as initiators, completers, and promoters and conclude
that the joint effect of two compounds that both affect thesame stage in the carcinogenic process will be described wellby the additive risk model; however, the effect of combined
exposure to two carcinogens that influence different stages
will not necessarily result in a multiplicative model. Short
exposures that occur close together in time and do notoccur at either very young or very old ages can produce anearly additive relative relationship. Synergism would,
however, arise when the contribution to different transitions by different carcinogenic agents is large comparedwith the spontaneous rate and when the time course of

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

exposure is penalizing if the sequence of steps matters.
Brown and Chu [B9] concluded that the observation of amultiplicative relative risk relationship in studies of joint
exposure to two carcinogens is evidence of action at two
different stages of the carcinogenesis process. These
examples illustrate the importance of a full understandingof the timescale of exposure for both agents.
104. An overview of interactions for simple binary exposures
to agents with specific effects is given in Table 3. According
to the terminology used by Krewski et al. [K46], many
interactions leading to considerable deviations from additivity
are possible although hardly predictable. The effectiveness ofa carcinogenic agent depends not only on the exposure but
also on the time of exposure, age at exposure, time since
exposure, and duration of exposure. This time dependence iscompletely different from and should not be confused with the197

time involved in the cellular dose-rate effect. It is therefore notpossible to quantitatively predict the dose-effect relationshipfor tumour induction after combined exposure.
105. This assessment is based on the evaluation ofcarcinogenesis data using a two-mutation model. Deviationsof the carcinogenesis process in other ways, e.g. by disturbing
organ functions in a crude way, are ignored. The assumptionof otherwise undisturbed functioning of the organ or organism
is probably reasonable for low exposures but may complicate
analysis for high doses and exposures. The long-term development of tumours implies a long period of time over which theprocess can be influenced. For interaction in the genesis ofradiation-induced tumours, it implies that exposure to adifferent agent at a time that is completely separated from thetime of irradiation may influence the radiation effect in often
poorly predictable ways.

Table 3
Anticipated interaction response of two single-agent carcinogens a
[K29]

a

Carcinogen A

Carcinogen B

Interaction response

Initiator
Completer
Initiator
Initiator
Initiator
Initiator
Initiator
Promoter

Initiator
Completer
Completer
Promoter
Promoter and completer
Initiator and completer
Initiator and promoter
Promoter and completer

Additive
Additive
Multiplicative
Multiplicative to supra-multiplicative
Multiplicative to supra-multiplicative
Supra-additive to sub-multiplicative
Supra-additive to supra-multiplicative
Supra-multiplicative

See glossary for definition of terms.

D. DOSE MODIFIERS AND OTHERINDIRECT INTERACTIONS
106. Apart from the direct interference with the development of the radiation effect, further indirect interaction
mechanisms are possible when an agent changes theretention of the radioactive substance following inhalation
or ingestion and consequently changes the organ dose. A
well-known case is blockage of 131I uptake to the thyroid bystable iodine, which is used in nuclear medicine andenvisaged in future radiological emergencies to greatly
reduce the dose to the thyroid gland. Other drugs, such as
ethylenediaminetetracetic acid (EDTA), are used fortherapeutic reasons, to stimulate the metabolic transfer of
inhaled or ingested radionuclides and, consequently, toreduce the relevant organ dose. Natural chelators such as
citrate may also modulate the biological half-lives of metal
and actinide ions. Synergistic effects on this level are
known from the inhibition of mucocilliary clearance by asecond agent [F28]. Several examples of dose-modifying
agents are given in the Appendix, which covers specificinteractions. Mechanistic considerations indicate that theirritants and cytotoxicants implicated here generally
display an effect threshold and are therefore of little
concern for combined effects at low exposure levels.

107. Other modifications of the radiation effect are
possible when the physiological condition of the organism
is changed, either intentionally or by chance. Examples arechanges in hormone levels or in the immunological system.
Such changes may also be induced by radiation (e.g. UVradiation). Also, novel mechanisms of genetic change suchas radiation- or chemical-induced genetic instability, whichleads to new genetic damage many cell generations after
exposure (see Annex F, “DNA repair and mutagenesis”),
may be prone to more-than-additive effects. The results ofthis type of interaction are dependent on the conditions of
the change and are, in general, poorly predictable.

E. SUMMARY
108. Carcinogenesis and, consequently, also the development of radiation-induced tumours is a long-term process.
Mechanistically, three levels can be distinguished in thedevelopment of the radiation effect on cancer: the molecular, cellular, and tissue/organ levels. On each level, ionizing radiation induces changes and processes, and these
may be influenced by combined exposures to other agents.
A summary of the levels, processes involved in cancer
development, and examples of the many classes of substances and agent with a potential to interfere at different

198

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

levels of radiation-induced carcinogenesis is presented in
Figure V. The biological effect of combined exposures toradiation and other agents at low doses is, in general,
expected to be additive, especially in the case of chronic
exposures. Deviations from additivity may primarily be
expected from interactions on the tissue/organ level. In thisphase, exposure to other agents may take a long time, up totens of years, and for interaction to occur, the exposures toradiation and the other agent need not be simultaneous.
MOLECULAR LEVELDNA damage

CELLULAR LEVEL
Damagefixation

Thus, interaction can last a relatively long time, and theradiation effect can be influenced to a significant extent by
the interactions that take place during this phase. Studies
using multi-stage models show that classifying the agents
involved in terms of their action as initiators, completers,
and/or promoters may help to predict the result of theirinteraction with radiation and other agents. These studies
also indicate that the radiation effect depends on the
background tumour incidence.
Nucleophylic agents, cysteamine,
cysteine, sulphydryl compounds,
anoxia, vitamins
Gamma rays, x rays,
beta particles,
alpha particles, UV
Metals, platinum complexes,
actinomycin, bleomycin,
EMS
IUdR, BUdR, 5-FU,
actinomycin D,
adriamycin, quianin

Radical scavenging,protecting_

Interaction with
DNA sub-damage+

DNA repair stimulation_

DNA repair inhibition+

Cell-cycle
control change+/

Apoptosis inhibition+

Caffeine

Membrane damage+

Heat, mineral fibres,
dust, leadInteraction with
sub-mutation events+

Mutagens, tobacco,
ENU, BNU, DEN, DBE,
BCNU, CCNU, Me-CCNU

Progression
inhibition_

Vitamin A, E, K, selenium,
low-fat diet, DFMO_

Temperature,
microwaves
Caffeine, cyclophosphamide,
hyperthermia,
hydroxyurea
Cytokinetins, p53

Cell cycling

Initiation/
mutation
ORGAN LEVEL
Promotion

Conversion

Tumour growth

Metastases

Tobacco, asbestos,mineral fibres, hormones,
DES, TPA

Progression
promotion+

Conversion
stimulation+

Mutagens, asbestos, tobacco,
TPA, benzo(a)pyrene

Tumour growth
inhibition_

Tamoxifen, testosterone

Immune surveillancechange+/ _UV, Vitamin A, endotoxins,
dietary factors

Tumour growth
stimulation+

Tobacco, hormones(TSH, oestrogen, DES)

Cell cycling
inhibition_

Anoxia, selenium

Cell cycling
stimulation+

Mitogens, mechanicalirritation, dust

Mitogens, hormones,
DES, oestrogens,mineral fibres, dust

Cancer death
Figure V. Schematic representation of radiation-induced carcinogenesis and possible interaction mechanisms
with examples of agents having shown a potential for more (+) or less () than additive effects
for at least one tumour site.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

199

III. SPECIFIC COMBINED EXPOSURES
109. Recent data on combined exposures to radiation andspecific physical, chemical, or biological agents are reviewed
in this Chapter. For each type of interaction, data from epidemiological studies of the adverse health effects in humans
are generally reviewed first. Studies involving experimental
animal models are considered next. Lastly, various effects
observed in in vitro systems are reviewed. Carcinogenesis is
the principal endpoint ofinterest; non-neoplastic endpoints are
viewed mainly in relation to mechanistic considerations.

110. Only a minor fraction of the interacting agentsdescribed below are found in the human environment at
potentially critical levels. An overview of agents known orsuspected to affect human health on their own is given in
Table 4. Details of the experimental and epidemiologicalconditions and results of studies on specified combined
exposures may be found in the Appendix and aresummarized in Table A.1.

Table 4
Exposure conditions and characteristics of prominent environmental and occupational agents andsubstances that may produce combined effects with them
[B6]
Typicalenvironmental
exposure
(E)

Agent

Major healthendpoint

Ionizing radiation

1.5-4 mSv a-1

20 mSv a-1

CancerUV radiation[W31]

Noontime intensity:
UV-A: 40 W m-2
UV-B: 3 W m-2

350 nm:150 kJ m-2 in 8 h
300 nm:100 J m-2 in 8 h

Asbestos[I12]

aOccupational
limit for chronic
exposure(O)

Estimated
contribution tototal incidence/mortality of
endpoint (%)

Estimated
lifetime risk a(%)

Substance withknown orsuspected
combined effect-10 (O)

3-8 (E)

0.4-1 (E)
-4 (O)

Smoking,asbestos,
hormones,arsenic?

Skin aging
Skin cancer
Melanoma

Important (E)>50 (E)?

>20 (E)?

Phototoxicity,allergy with
UV sensitizers

Crocidolite:
0.2 fibers cm-3
Other forms:
2 fibres cm-3

Lung cancer
Mesothelioma

Low
>50%

Smoking

Benzene[M60, W34]

14 µg m-3 in indoorair

3.2 mg m-3

Leukaemia

2.5 (E)

0.01 (E)

Substrates for
activation/
detoxification
systems

Carbon tetrachloride [W34]3 µg m-3 in indoor air

65 mg m-3

Cancer

0.05 (E)0.01 (E)

Chloroform

Chloroform [I2]1.2 µg kg-1 d-1 fromtap water andshowering

50 mg m-3

Cancer

0.15 (E)0.03 (E)

Carbon tetrachloride

Dioxins/furans[F8]

1.3 µg kg-1 d-1
dietary intake

50 pg m-3

Cancer0.1 (E)

0.02 (E)

Ethylenebisdithiocarbamates(EBDCs) [L49]

n.a.

n.a.

Cancer, adverse
reproductive
outcomes

0.17 (E)0.034 (E)

Polychlorinatedbiphenyls (PCBs)[G8]

14 ng kg-1 d-1

1 mg m-3(42% CI)

Cancer

0.06 (E)0.01 (E)

Assuming 80 and 40 years of exposure for environmental (E) and occupational (O) levels, respectively. Risks are generally upper boundestimates based on linear extrapolations from high exposures.

200

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

A. RADIATION AND PHYSICALAGENTS
1. Combinations of ionizing radiation
111. Many experiments have been undertaken to investigate the cellular effects of combined exposures of two types
of ionizing radiations. In view of their potential radiotherapeutic applications, a wealth of data on the combined
action of neutrons, heavy ions, and gamma or x rays was
accumulated in the 1970s and early 1980s. This information was reviewed in Annex L, “Biological effects ofradiation in combination with other physical, chemical andbiological agents”, in the UNSCEAR 1982 Report [U6].
The results generally indicate additive effects of combined
exposures characterized by the so-called isoaddition ofthese agents at the cellular level [L10, L47], as describedin Chapter II. A few data [E5, L26] were reported onradiation-induced tumours; these, in general, include
exposure to internal emitters. The results indicate additive
to slightly supra-additive effects for combined exposures,
mainly because of the lower dose rates that are involved in
the internal exposure to alpha radiation in theseexperiments. For estimating the risk of carcinogenesis, theeffects of combined exposures to more than one type ofionizing radiation are expected to be isoadditive, i.e. to add
up in the same way as effects of increments of the same
agents, when at least one of the radiations is delivered at alow dose rate (chronic irradiation), as is generally the casefor occupational and environ-mental exposure levels.
2. Ultraviolet radiation
112. Ultraviolet (UV) radiation is recognized as the mostimportant initiator and co-factor for human skin carcinogenesis [S29]. It is mainly the skin that is exposed to UV
radiation. A study of combined exposures to gamma
radiation and UV radiation was presented by Shore, whoanalysed 12 studies on the incidence of skin cancer in
populations irradiated with known skin doses [S29]. In theabsence of a proper control (skin exposed to ionizing
radiation but not to UV), it was concluded that, at least for
combined exposures, the data are compatible with a linear
dose-response relationship for ionizing radiation [S29] butthat the interaction is unclear. The question of whether
relative risk or absolute risk models are more appropriate
remains open. From the mechanistic point of view,interaction at the cellular level may be expected, which
results in a more or less additive effect for low exposure
rates [L52]. The considerable variations in skin cancer
among different populations and subgroups seem to reflect
the large differences in UV exposures due to latitude andlifestyle and the differences in genetic predisposition toskin cancer due to skin type. The overwhelming dependence of skin cancer on extended exposures to UV prevents
conclusive epidemiological data on the interaction of UVwith ionizing radiation. Another important factor to take
into account in possible interactions is UV-induced
suppression of the immune system [B76, L58, N26]

3. Electromagnetic radiation
113. Neither low- nor high-frequency electromagnetic radiation have enough single photon energy to directly damage
DNA and therefore cannot be cancer initiators. However,strong electromagnetic fields may modify and stimulate
growth [K16, S33], and this has led to the hypothesis thatelectromagnetic fields may influence cancer development.
However, no straightforward inferences from experimental
results to exposure situations in occupational or environmental settings have been found at this stage for the combination of electromagnetic and ionizing radiation [B77, B78,
B79, U19]. Moreover, there is at present little indication froma mechanistic standpoint for potentially harmful interactions
between electromagnetic fields and ionizing radiation at
controlled exposure levels in the workplace or the clinic. The
possible modulation of radiation effects by heating produced
by strong electromagnetic fields is considered in the next
Section.
4. Temperature
114. Heat can kill mammalian cells in a predictable andstochastic way [D3]. Elevated temperature is used as amodifier of radiation sensitivity in many therapies to control
tumour growth. In combination with ionizing radiation, heatcan act synergistically on cell survival, cell proliferation, andcytogenetic damage by, for example, interfering with DNA
repair. However, extremely high temperatures, which are
generally not found in the workplace or in environmental
conditions, are needed in the cells at risk, so heat is notconsidered as potentially enhancing radiation risk.
5. Ultrasound115. Ultrasound has achieved widespread use in medical
diagnostic and therapeutic procedures. Studies have shownthat at the intensities used for diagnostic purposes,
ultrasound does not interact with ionizing radiation tocause cytogenetic damage in treated cells, although theyield of sister chromatid exchanges was observed to beslightly increased in one study [K20]. Because cavitationinduced mechanical damage by ultrasound shows highthresholds, this mechanism is of little concern forenvironmental exposures. Such damage has to be prevented
in other situations already caused by single-agent effects.
6. Dust, asbestos, and other mineral fibres
116. Mineral dust and fibres such as asbestos generally act
through non-genotoxic mechanisms such as mechanicalirritation and cell killing [B13]. The combination of radiation
exposure and exposure to dusts and fibres is quite common inindustrial settings and in the environment, and these agentsare reported in both animal studies and in vitro studies to act
synergistically at high exposures [B38, H11]. Silicosis wasshown to be a risk factor for human lung cancer in metalminers in the 1940s [H9] and is implicated as a modifier oflung cancer risk in radon-exposed underground workers[K49]. Combined exposure to phosphate ore dust, gamma

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

radiation, and radon daughter products also resulted inelevated lung cancer risks in earlier practices [B74]. Although
exposures and thus risks are considerablylower today, mineral
dust and fibres still deserve attention because they may
interact with radiation, including densely ionizing radiationsuch as alpha radiation in mining environments, to enhance
the risk of cancer.
7. Space flight117. A special form of combined exposure is experienced inspace flights, where a multitude of stressors act in combination on astronauts. This problem has been investigated using
animals [A13, A14, V3]. The most important environmental
parameter is microgravity. Space radiation effects werecomprehensively reviewed by Kiefer et al. [K54], the interaction of microgravity and radiation at the cellular level [H52,
K55]. No synergistic actions were found. A very important
aspect is a possible reduction of the immune response [S86],
which could have an influence on cancer development. Thechanges of many parameters that are normally stable inexperimental work on earth make well designed studies inspace potentially important in addressing combined effects ofphysical agents.

B. RADIATION AND CHEMICAL AGENTS
118. A multitude of natural and man-made chemicals withcancer initiating and promoting potential are present in thehuman environment and may interact with radiation.
Classification based on their mode of action is often
difficult, as many have more than one type of action, but atleast a crude separation can be made into substances thatmainly act by damaging DNA (genotoxic substances) andthose that act in other ways (non-genotoxic substances)[C48]. The former group includes chemically active
species, or substances that can be activated, bind to or
modify DNA directly, or indirectly via radicals. The nongenotoxic substances range from nonspecific irritants andcytotoxins to natural hormones, growth factors, and theiranalogues. They interact with the regulatory systems ofcells and organs and cannot always be considered toxic bythemselves. Some are clearly protective, e.g. they scavenge
reactive species before they interact with DNA.
1. Genotoxic chemicals
119. Numerous examples of combined exposures to radiationand chemical genotoxic agents can be found in the literature,including studies on the improvement of radiation therapy bysimultaneous treatment with a chemical (see Chapter IV). In
many cases, supra-additive effects are reported, caused byinteraction in the cellular phase and by the high exposurelevels involved. The agents include 1,2-dimethylhydrazine(DMH) [S27], N-methyl-N-nitrosourea (MNU), butylnitrosourea (BNU), N-ethyl-N-nitrosourea (ENU) [H6, K15,
S13, S20, S21, S22], diethylnitrosamine (DEN) [M8, P26],
N-2-fluorenylacetamide (FAA), 4-nitroquinoline-1-oxide(4NQO) [H39], bleomycin [D6], and 1,2-dibromoethane

201(DBE) [L13]. The effects are dependent on the species,
exposure conditions, time of exposure, etc., and sometimes thesame chemical is involved in a supra-additive and a subadditive result. In general, for short exposures to highconcentrations and for low chronic concentrations, deviations
from additivity are small, if at all existent. In most epidemiological and experimental studies, effects exceeding a level
predicted from isoaddition have not specifically been
demonstrated.
2. Non-genotoxic chemicals
120. Many chemicals in the human environment or their
metabolites do not specifically attack DNA but influencecell proliferation and cell differentiation on an epigenetic
level. These include the tumour promoter 12-Otetradecanoylphorbol-13-acetate (TPA) [H3, L24], carbon
tetrachloride, and α-difluoromethylornithine (DFMO)[O12]. These agents act in combination with radiation on
the cellular and tissue level of cancer development and cansignificantly enhance the induction of tumours. Specificmitogens may interfere with regulatory mechanisms andcell-cell signalling, but many substances with a highchemical reactivity act as non-specific irritants or toxicants
via membranes or proteins. For example, toxin-induced
cell death will induce proliferation in neighbouring cells,
which may enhance the progression of premalignant cells.
Substances acting in a non-specific manner, for example
lipophilic solvents, quite often show highly non-linear
dose-response relationships with apparent thresholds.
Other agents may interfere with critical cellular processes
involved in repairing damage to cellular constituents such
as DNA. The assessment of possible synergistic effects at
the exposure levels relevant for risk estimation remains
very difficult because of the high exposures used inexperimental systems and the apparent threshold levels.
One important group of chemicals, which includes cysteamine and mexamine, has radioprotective effects; thesechemicals scavenge radicals formed by ionizing radiation[M4]. A considerable number of agents may have both
genotoxic and epigenetic functionalities such as the baseanalogue 5-bromo-2'-deoxyuridine (BrUdR) [A15].
3. Tobacco
121. Given the large collective dose from radon and its decay
products in non-occupational and occupational settings andthe prevalence of active smoking, the combined effect of these
two exposures on human health deserves special attention[B27]. A large body of epidemiological evidence fromuranium miner studies allows, at least for higher radiation
doses, calculating risks and interaction coefficients directlyfrom human data [C46]. However, the fact that tobacco smoke
is itself a complex mixture of genotoxic and non-genotoxic
substances and even contains some natural radionuclides (thelong-lived radon progeny 210Po and 210Pb) makes amechanistic assessment difficult.
122. Because of the complex composition of tobacco smoke,the issues surrounding combined exposures to radiation and202

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

tobacco smoke are even more difficult to elaborate than forbinary combinations. Some 4,000 individual chemicalcomponents of cigarette smoke have been identified, and there
are probablya number of additional important but unidentified
components, for example, extremely reactive, short-lived
compounds or those present in very low concentrations [G1].
The complexity of tobacco smoke means that the action in
combined exposures with radiation can take place in both thecellular and organ phase of cancer development. Tobaccosmoke contains only relatively small amounts of DNAreactive carcinogens such as nitrosamines, polycyclicaromatic hydrocarbons, and pyrolysis products such ascarbolines. Hence enhancing and promoting factors, e.g.
catechols, other phenols and terpenes, are important.
Discontinuation of smoking progressively reduces the relativerisk of cancer development as time since withdrawal
increases, probably because of reduced pressure from theaction of promoters [W1].
123. In the last few years, joint analyses of original data sets
[C1, L18] and meta-analyses of published results [T14] haveyielded a detailed assessment of risk patterns and haveallowed investigators to test risk models. The most comprehensive and complete analysis of radon-induced healthrisks was published by Lubin et al. [L18]. The review contains
a joint analysis of original data from 11 studies of maleunderground miners. Data on smoking were available for 6 of
the 11 cohorts, but the assessments were limited byincomplete
data on lifetime tobacco consumption patterns and thesometimes exotic forms of tobacco use, such as water pipes in
the Chinese study [L18]. Single studies for which smoking
data could be analysed were generally not informative enoughto allow choosing between an additive or a multiplicative joint
relationship between radon progeny and smoking. The
Chinese cohort seemed to suggest an association more consistent with additivity, while the Colorado cohort suggested a
relationship more consistent with a multiplicative interaction.
For all studies taken together, the combined influence ofsmoking and radon progeny exposures on lung cancer was
clearly more than purely additive but less than multiplicativeand compatible with isoadditivity [B69]. The most recent
analyses of the BEIR VI Committee [C46], which were basedon an update of these data, suggested synergism between the
two agents that is statistically most consistent with a slightly
sub-multiplicative interaction. A best estimate from miner
data indicates that the lung cancer risk for smokers expressedin absolute terms is higher by a factor of at least 3. To further
characterize the association, more detailed data on tobacco use
would be needed. Age of starting to smoke, amount andduration of smoking, and type of tobacco were recognized asimportant determinants of risk. A further handicap of present
studies is that the sub-cohorts oflifetime non-smokers exposed
only to radon are very small. The statistical power of theconclusions on the radon-tobacco smoke interaction is correspondingly low. Data are available from a study by Finch etal. [F28] of smoke exposure and alpha-particle lung irradiation over the lifespan of exposed rats. The pulmonary retention of inhaled 239Pu was higher, increasing with the concentration of the 239Pu, in smoke-exposed rats than in shamsmoke-exposed rats. This effect on retention resulted inincreased alpha-radiation doses to the lung. Assuming anapproximately linear dose-response relationship between
radiation dose and lung neoplasm incidence, approximate
increases of 20% and 80% in tumour incidence over controls
would be expected in rats exposed to 239PuO2 + low-level
cigarette smoke and 239PuO2 + high-level cigarette smoke,
respectively.
124. Hypotheses on the mechanistic interaction between
tobacco smoke and radon were tested by applying the twomutation clonal expansion model of carcinogenesis of
Moolgavkar to data from the Colorado plateau miners [M39].
No interaction between radon and tobacco smoke in anyof thethree steps (the two mutation steps and clonal expansion) isneeded to fit the data, which are clearly supra-additive forradon and smoking combined. The model, however, shows asignificant dependence on age at exposure. Quantitatively
similar results were obtained byLeenhouts and Chadwick [L9,
L57]. A highlysignificant decrease in excess relative risk withtime since exposure is found in miner studies in contrast to
findings on lung cancer in survivors of the atomic bombings.
This may be explained by microdosimetric considerations. In
the case of high-LET alpha radiation from radon progeny, theminimal local dose from one single alpha track averaged overa cell nucleus is already in the range of several hundred
milligray, whereas one electron track yields a dose to the
nucleus of only 13 mGy. This means that even at the lowest
possible nuclear dose from alpha exposure, stem cells that are
hit carry a multitude of DNA lesions, which may considerably
impair long-term cell survival and maintenance ofproliferative capacity [B25, B27].
125. Smoking is also of great importance for nonoccupational radon exposures in the indoor environment.
Until now, little quantitative evidence has come fromindoor radon studies. Most of the case-control studies
published are inconclusive [A28, K53, P11]. Only one
larger study [P11] was indicative of an indoor radon risk
and its modification by tobacco that is comparable to what
is predicted from miner studies. It remains doubtful
whether the results from the many case-control studies
under way will in the near future allow narrowing theuncertainties that surround indoor radon risk and thepossible interactions with smoking. Emerging studyresultsfrom Europe based on much longer residence times may
offer better statistical power. Several large indoor casecontrol studies under way will narrow uncertainties in thenext few years. First results from the United Kingdom[D33] and Germany [K53, W35] are indicating a lungcancer risk in the range of ICRP projections. However,confidence intervals are relatively large and still include
zero risk in most analyses. Because of the limitation of theindoor radon studies, risk estimates based on miner dataremain the main basis for predicting lung cancer fromindoor radon exposure. A best linear estimate of the risk
coefficients found in the joint analysis of Lubin et al. [L18,
L35] for the indoor environment indicates that in the
United States, some 10%12%, or 10,000 cases, of thelung cancer deaths among smokers and 28%31%, or
5,000 cases, of the lung cancer deaths among never-ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

smokers are caused by radon progeny. About half of these
15,000 lung cancer deaths traceable to radon would then be
the result of overadditivity, i.e. synergistic interactions
between radon and tobacco. Based on the same risk model,
Steindorf et al. [S47] estimated an attributable risk forindoor radon of 4%7% for smokers and 14%22% for
non-smokers. Because of the many differences between
exposed persons and exposure situations in mines andhomes and the additional carcinogens such as arsenic, dust,
and diesel exhaust in mine air, these figures should beinterpreted with caution.

203

4. Metals
126. Toxic metals are important trace pollutants in thehuman environment (Table 5). They interact in many ways
with cellular constituents and may produce oxidative DNA
damage or influence enzyme activity at low concentrations,e.g. by competing with essential metal ions [H38]. Carcinogenic transition metals are capable of causing promutagenic
damage, such as DNA base modifications, DNA-protein
cross-links, and strand breaks [K7]. The underlying mechanism seems to involve active oxygen and other radicals arising

Table 5
Metals in the environment and effects on humans[M38, N14, S48]

Metal

a

Release a(109 g a-1)

Main sources of intake
and typical levels in the body

Characteristics affecting healthArsenic

31 (61)

Source: food (seafood up to 120 mg kg-1) anddrinking water
Concentration in body: 0.3 mg kg-1

Mutagenic, teratogenic, co-carcinogenic,
As3+causes skin cancer

Cadmium

8.9 (85)

Source: inhalation (2 µg cigarette-1) andfood (0.025 mg kg-1)

Mutagenic, teratogenic, co-carcinogenic,
causes cancer at multiple sites

Mercury

6.1 (59)

Source: metal vapours, food (up to 1 mg kg-1
MeHg+ naturally in fish), tooth fillings
Intake: by inhalation and ingestion
0.2 and 25 µg d-1, respectively

Mutagenic, teratogenic (brain damage), co-carcinogenic,
causes sarcomas and renal tumours

Nickel86 (65)

Source: food intake (0.2 mg d-1)
Concentration in body: 0.007 mg kg-1

Essential element; allergenic, comutagenic,
cocarcinogenic, causes nasal sinus cancer

Lead

12 (96)

Source: Food, dust, air (0.15 mg d-1)
Amount in body: steady increase
to about 200 mg at age of 60 years

Substitutes for Ca2+, neurobehavioural deficits (decrease infertility, abortifacient)
Low mutagenic and carcinogenic potential(may cause renal adenocarcinoma)

Antimony

5.9 (59)

Source: food and tobacco (0.005 mg d-1)

Mutagenic as Sb3+, organic antimony compounds used as
emetics

Vanadium

114 (75)

Source: food (0.01-0.05 mg d-1)

Essential element
Inhibits Na+/K+ ATPase and drug detoxification
enzymes at low concentration
Mutagenic, teratogenic, carcinogenicZinc

177 (66)

Source: food intake (10-50 mg d-1)

Essential element with small window of tolerance
Clastogenic (causes chromosome aberrations)
Causes growth of some tumours
at elevated concentrations

Global values; percentage of anthropogenic contribution is given in parentheses.

from metal-catalysed redox reactions. Cadmium, nickel,
cobalt, lead, and arsenic may also disturb DNA repair processes [H48]. Only a few data are available from combined
exposures of radiation and metals in human populations; nofirm evidence of interactions was observed. However, metals
and ionizing radiation have been shown to produce combined
effects in many other biological systems (see the Appendix).
Especially in underground mining, possible effects from theepidemiologicallyproven lung carcinogens arsenic, cadmium,
chromium, nickel, and antimony [M65] have to be assessed
together with high-LET radiation from radon. Arsenic in
particular is a major risk factor in combined exposures tomineral dust, radon, metals, and diesel fumes [K48, T5]. The
risk-enhancing effects of iron dust seem to be limited to very
high dust concentrations, leading to changes in lung function[B74]. The significance of these data for radiation risk
estimation at low dose levels remains unclear.
5. Mitogens and cytotoxicants127. Although many mitogenic and cytotoxic compounds
could have been considered above with genotoxic or nongenotoxic agents, they should be mentioned separately
because of their potential to interact with radiation,

204

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

principally by virtue of their ability to stimulate cellproliferation. From a mechanistic standpoint, they can be
expected to interact in the organ phase of radiation carcinogenesis, but the resulting interaction (sub- or supra-additivity)
is not always predictable. Examples of such agents include
N-methylformamide (NMF) [L15], caffeine [M11], theobromine, theophylline [Z4], 2-aminopurine, and tributyl
phosphate. Many studies assessing deviations from additivity
in combined exposures ofmitogens/cytotoxicants and ionizing
radiation are found in the literature (see Appendix), but thehigh exposure levels applied and the biological endpoint
studied generally do not allow directly transferring the results
to carcinogenesis in humans. However, any endogenous or
dietary levels of agents influencing stem-cell population sizeor kinetics will have the potential to modulate response toradiation.
6. Antioxidants, vitamins, and otherdietary factors
128. Diet can modify the effectiveness of chemical carcinogens, sometimes by a large factor, and interactions withradiation are found as well [B24, C26, H11, W29]. All classesof substances described in the five preceding Sections
III.B.15 are found in human food supplies. Actions ranging
from subadditive to supra-additive may occur, depending on
the specific agent. The radiation risk may be reduced whengrowth stimuli are reduced as a result of nutritional deficiency
or when repair possibilities are optimized. Synergism can be
expected where lower levels of radical scavengers or thecoenzymes needed for repair increase the yield of effective
damage from ionizing radiation or impair the speed andaccuracy of cellular recovery from damage. Some of theunderlying mechanisms ofspecific agents have been identified
in animal experiments. Tumour-incidence-enhancing effects
have been noticed with elevated consumption of, for example,
riboflavin, ethanol, and marihuana. Tumour-incidencereducing effects are found for low-caloric diets, vitamins A, C,
K, and E, retinoic acid derivatives (but enhancing effects ofartificial beta carotin in some smoker cohorts), selenium, and3-aminobenzamide. Very important in view of population
health are behavioural changes and a tendencyto malnutrition
in alcohol addicts, which may increase the susceptibility to
toxicants in the environment or at the workplace [U18]. In
general, the combined action is not specific for radiation but
is also found for other carcinogens, and the interaction isdependent on the dosage.
129. In summary, dietary factors are proven modifiers of risk
from diverse agents at levels found in human populations andprobably also influence the production and repair of endogenously arising lesions. Absence or deficiency of importantcoenzymes and nutrients on the one side and high levels of
directly or indirectly acting mitogens on the other interferewith molecular, cellular, and tissue responses to ionizing
radiation. A modulation in the radiation risk may occur insituations where growth stimuli are reduced or increased,
owing to nutritional deficiency or surplus or where thenumber of stem cells at risk is changed. Synergisms are also
to be expected where reduced levels of radical scavengers orcoenzymes needed for repair increase the yield of primarydamage from ionizing radiation or impair the speed andaccuracy of cellular responses to damage. In general, thesemechanisms apply to most deleterious agents in the humanenvironment.

C. RADIATION AND BIOLOGICAL AGENTS
130. Many hormones are potent growth stimulators, andthere is considerable evidence that they may modify cancer
risk. They include thyroid-stimulating hormone (TSH),
oestradiol-17 beta (E2), prolactin, diethylstilbestrol (DES), andandrogens in general. Their effect is dependent on tissue, typeof hormone, and dosage and is important enough to be kept inmind when analysing radiation risks. Tamoxifen, a synthetic
anti-oestrogen, has both cancer risk-enhancing functions(endometrium) and protective properties (breast), dependingon the organ [J9]. An important consequence of interaction
with hormones is the sex difference in tumour sensitivity,
mainly of organs of the reproductive system.
131. Viruses, bacteria and microbial genetic sequences havebeen shown to play an important role in the development oftumours. Cancer viruses may interact with radiation by mutation or translocation of dormant viral sequences. Experiments
so far give no clear indication of anyinteraction with radiationthat influences cancer development. Viruses may induce
genotypic and functional changes, i.e. they may act as highly
site-specific genotoxic agents in multi-step mechanisms.
Highly synergistic effects due to increased sensitivity may
arise for some endpoints. Little information is available atpresent on the mechanism of the induction of gastric cancer
by bacteria (Helicobacter pylori).
132. The interaction of several miscellaneous factors withradiation exposure and its role in carcinogenesis has been
investigated. Some of these factors are reviewed in the
Appendix. The role of others, such as psychosocial factors,
remains unclear and is outside the scope of this Annex.

D. SUMMARY
133. Combinations of different types of ionizing radiation
show mainly isoadditive effects. For decreasing doses andchronic exposure, the quadratic terms of the dose-effectrelationships tend to vanish and the linear terms to prevail,indicating additivity for low-level exposures. Also, for the
combination of UV radiation and ionizing radiation, additiveeffects are expected for low exposure levels. Temperature andultrasound are not considered to significantlymodifyradiation
risk. The temperature range and the ultrasound intensities
necessary for an interaction with radiation are too high to beof relevance for environmental or occupational settings.
Mineral dust and fibres, including asbestos, tend to show
supra-additive interaction with radiation at high exposurelevels. These levels were reached in workplaces in the 1950s
and earlier. Today the occupational exposures are lower, butthese agents still deserve attention for their potential toenhance risks after combined exposure.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

134. At high exposures, a wealth of supra-additive effects
between genotoxic chemicals (e.g. alkylating agents) andradiation were recorded. For low-level exposures, there isno mechanistic evidence of combined effects at the cellularlevel greater than those predicted from isoadditivity. Nor
are these agents expected to show a more-than-additiveeffect at the organ level. However, non-genotoxic agentswith mitogenic, cytotoxic, or hormonal activity may interact with radiation in an additive to highly supra-additive
manner. High exposures clearly have a considerable
potential for enhancing radiation risk during the organ
phase of radiation-induced cancer. Since most of thesesubstances show highlynon-linear dose-effect relationships
with sometimes considerable thresholds, the combined
effects with radiation at low concentrations could be ex-205pected not to deviate much from additivity, i.e. to be additive
to slightly supra-additive. Special attention has to be given to
the combined effects of radiation and tobacco smoke. Tobaccosmoke itself is a complex mixture of different genotoxic andnon-genotoxic chemicals. Combined exposures to radiationand tobacco smoke show clearly supra-additive effects. Heavy
metals and arsenic may generate free radicals or disturb DNA
repair mechanisms and therefore may also cause more-thanadditive effects. Many human cancers show considerable
dependence on lifestyle, nutrition, and other dietary factors.
Tumour- incidence-enhancing effects have been reported for
riboflavin, ethanol, and high fat diets and incidence-reducing
effects for low fat diets and some vitamins. In general, these
combination effects have been found not just for radiation but
also for other carcinogens.

IV. COMBINED EXPOSURES IN CANCER THERAPY

136. Central to cancer therapy is the relationship between thedesired and undesired effects of the therapies chosen. This
relationship is defined as the therapeutic index ratio or gain
[G21, H41]. The gap between the sigmoid curves of tumour
cure (tumour control probability) and dose-limiting toxicity tonormal tissue (normal tissue complication probability) is thetherapeutic index (Figure VI). The goal of cancer therapy is
to increase the therapeutic index by separating the two curves.
The therapeutic index is increased when the tumour control
probability curve is displaced to the left of the normal tissuecomplication probability curve. This can be achieved inradiotherapy by altering the exposure schedule. Important
techniques are hyperfractionation, accelerated fractionation,
split-course techniques, interstitial irradiation, manipulationof target volumes, shrinking field techniques and others.
Another approach to increasing the therapeutic index is tocombine radiotherapy with chemotherapy. Drug-ionizing
radiation interaction in therapy is useful only when it leads toa further separation of the curves, not just to their displacement [K43].
137. It should, however, be clearly noted here that the final
goal of tumour therapy is tumour control and therefore cell
death (apoptosis, necrosis) or blockage of cellular growth (lossof proliferative capacity, differentiation, senescence). These
effects are mostly deterministic and often mechanisticallydifferent from the stochastic radiation effects that are ofconcern in radiation protection. Emphasis is therefore placedon the mechanisms of interaction between the drugs andradiation that reveal possible mechanisms of interaction
between chemical agents and radiation under environmental
and normal occupational settings. Clinical results will bementioned only if mechanistic information with relevance forlow dose effects can be provided.
A

PROBABILITY

135. Many modern cancer treatment regimens combine
surgery, radiotherapy, chemotherapy, and/or immunotherapy.
Generally, combining the different treatments does not meanthat the different therapeutic agents interact in a mechanisticmanner. Central to the discussion of cancer therapy is, therefore, the distinction between non-interactive and interactive
combinations, with the latter being of interest in this Annex.
From the rapidly emerging understanding of the action andinteraction mechanisms of different agents in combinedmodality therapy, information relevant to possible interaction
mechanisms in environmental and occupational exposure
situations may be obtained.

B

Major complications

Tumour control

DOSE

Figure VI. Sigmoid curves of tumour control and complications [H41].
A: Dose for tumour control with minimum complications.
B: Maximum tumour dose with significant complications.

A. MECHANISMS OF INTERACTIONS
138. Publications on the mechanisms of interaction between
radiation and drugs are numerous but often lack precise andquantitative information. Factors on which the interaction ofthese two treatment modalities depends include the type oftumour and normal tissue involved, the endpoints studied, thedrug and its dose level, the radiation dose, dose rate, and206

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

fractionation, and the intervals between and sequencing of the
combined treatments. Chemotherapy could slow the processof cell repopulation after radiotherapy or it could synchronize
the cell cycle. Moreover, tumour reduction by chemotherapy
could improve tumour oxygenation, thus increasing the effectof radiotherapy. At the cellular level, inhibition of repair ofsublethal and potentiallylethal radiation damage byanticancer
drugs is probably the most important mechanism of radiosensitization. Exploitable mechanisms in combined chemotherapy and radiotherapy treatment can be described underfour headings, as was originally done by Steel and Peckham[S1, S23, S46]: spatial cooperation, independent cytotoxicity,protection of normal tissues, and enhancement of tumour
response.
139. Spatial cooperation describes a non-interactive combination of radiotherapy, chemotherapy, surgery, and othertherapeutic strategies that act at different anatomical sites. The
commonest situation is where surgery and/or radiation is used
to treat the primary tumour and chemotherapy is added as an
adjuvant to attack remaining local tumour cells and distantmetastases. There is an analogous situation in the treatment ofleukaemia, where chemotherapyis the mainline treatment andradiotherapy is added to deal with the disease in anatomicalsites, e.g. the brain, protected from chemotherapeutic attack by
vascular constraints or by blood barriers. Spatial cooperation
still appears to be one of the main clinical benefits of combination modality treatment. This mechanism does not
require interactive processes between drugs and radiation.
140. Independent cytotoxicity describes another form of noninteractive combination of therapeutic modalities. If two
modalities can both be given at full dose, then even in theabsence ofinteractive processes the tumour response should begreater than that achieved with either modalityalone. The costof this improvement on tumour response is that the patient has
to tolerate a wider range of toxic reactions in normal tissues(within and outside of the radiation field). As with spatial
cooperation, the mechanism of independent cytotoxicity does
not require interactive processes between drugs and radiation.
Independent cytotoxicity can even tolerate a subadditive interaction of the modalities and still produce an increase in therapeutic gain. The relative extent of reduction in toxicity tonormal tissue within the radiation field is the critical parameter of this mechanism.
141. The protection of normal tissues requires an antagonistic interaction of the combined modalities. Since two toxic
agents usually tend to produce more damage than either agent
alone, it would seem rather unlikely that chemotherapy inconjunction with radiation could reduce the damage to doselimiting normal tissue. However, there are well-documented
situations in which certain cytotoxic drugs increase the resistance of normal tissue to radiation or to a second cytotoxic
treatment.
142. Studies ofthis seeminglycontradictorymechanism haveconcentrated on the bone marrow and the intestinal epithelium. It has been shown by Millar et al. [M51, M52] that in
the bone marrow, the most effective cytotoxic agent, cytara-bine, does not modify stem-cell radiosensitivity; instead, it
stimulates enhanced repopulation by the surviving stem cells.
This phenomenon is highly dependent on the timing of thetwomodalities. Maximal radioprotection is achieved when the
drug is given two days before radiation. In the small intestine,
microcolonysurvival was increased when cytosine arabinoside
was given 12 hours before irradiation [P24]. Other cytotoxic
drugs with radioprotective action are cyclophosphamide,chlorambucil, and methotrexate [M51]. Recently, a topoisomerase II inhibitor (etoposide) was shown to increase theradioresistance of the bone marrow when given one day
before whole-body irradiation [Y1].
143. Normal tissue can be protected from radiation effects
by radioprotective agents. Increasing the differential
between tumour and normal tissue radiosensitivities would
give a therapeutic advantage. Radioprotectors can thus be
used as selective protectors against radiation damage tonormal tissue, allowing higher curative doses of radiation
to be delivered to tumours.
144. Chemical radioprotectorstarget the detoxifying mechanisms of the cell, in particular the antioxidant enzymes that areavailable for removal or detoxification of the reactive oxygen
species and their products formed by the action of ionizing
radiation. By far the most widely studied class of radioprotective agents is the thiols, and the most important nonprotein thiol present in cells is glutathione. Other classes of
agents conferring radioresistance to normal tissue are theeicosanoids, which are biologically active compounds derived
from arachidonic acid, the lipoic acids, and calcium antagonists (reviewed in [M51, M56]). The effects of biologicalresponse modifiers such as the cytokines IL-1 and TNFα as
radioprotectors in normal tissue have been discussed in recent
reviews [M53, M54, N12, N13, Z11, Z12].
145. Relative enhancement of tumour response is commonly
perceived to be the principal aim of adding chemotherapy toradiotherapy. A wide variety of biological mechanisms havebeen proposed to explain interactions between radiation andtherapeutic agents. In the context of this Annex, this kind ofinteraction is the most important mechanism with respect toenvironmental and normal occupational settings.
146. DNA adduct repair regularly involves strand scissions
by repair enzymes. Conversion of repairable into lethal DNA
damage may occur if a DNA-repair-associated single-strand
break combines with a radiation-induced single-strand break
to produce new DNA double-strand breaks. This mechanism
has been suggested for the interaction of cisplatin andradiation. A similar mechanism, the production of doublestrand breaks by combining single-strand breaks, may occur
when topoisomerase I or II inhibitors and radiation are
combined.
147. Many drugs inhibit the repair of radiation damage.
Antitumour antibiotics (e.g. dactinomycin and doxorubicin),
antimetabolites (e.g. hydroxyurea, cytarabine, and arabinofuranosyl-adenine), and alkylating agents and platinum analogues (e.g. cisplatin) have been shown to inhibit radiation-

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

induced DNA damage repair. Repair inhibition has been
detected in a number of ways, including removal of theshoulder on the cell survival curve, inhibition of split-dose
recovery, and inhibition of delayed plating recovery.
148. Cell-cycle synchronization exploits the fact that manycytotoxic drugs and radiation show some degree of selectivity
in cell killing at certain phases of the cell cycle. Antimetabolites show a maximum effect on cells undergoing theS phase. Radiation sensitivity is highest in the G2/M phase.
There is, therefore, an attractive possibility of complementaryaction between drugs and radiation. The most attractive
possibilityseem to be the interaction between microtubule andtopoisomerase poisons and primary DNA-damaging agents
such as radiation.
149. Activation of apoptosis by differential pathways
increases cell killing during tumour therapy and is therefore
another possibility for combined action of radiation andchemotherapeutic drugs. Ionizing radiation may activate theapoptotic process by a DNA damage-p53 dependent pathway,
whereas taxoids like paclitaxel may activate a pathway
downstream of p53 by phosphorylation of Bcl-2. There is,therefore, a possibility that radiation-induced cell killing canincrease, even in p53-deficient tumours. The involvement of
apoptosis in radiation-induced cell killing has recently beenstudied extensively [B75, D34, H44, H49, M69, O19].
150. Reduction of the hypoxic fraction by bioreductive drugs
targeted at hypoxic tumour cells increases tumour radiosensitivity. Most promising here is the development of dualfunction drugs specific to hypoxic cells and with intrinsic
cytotoxic activity (e.g. alkylating activity).

B. SECONDARY CANCERS FOLLOWINGCOMBINED MODALITY TREATMENT
151. The successful treatment of cancers involves radiation
therapy and/or multi-agent chemotherapy, each of which isused either as primary therapy or as an adjunct to therapy ofthe primary tumour, and it often includes surgery. Withfurther improvements in modern cancer therapy, the durationof survival and the curability of many patients has increased
up to 45%. However, along with this progress has come arecognition of the long-term complications of therapy, such as
secondary cancers (reviewed in [T30]). Although otherclinical consequences in non-target tissues are known, themain focus in this Section is on secondary cancers after
combined modality treatments. Secondary cancers resulting
from the combined effect of radiotherapy and tobacco smoke
are discussed in the Appendix, Section B.3.
152. No one specific type of secondary cancer is seen after
therapeutic irradiation. Secondary cancers can occur after
any initial cancer, when survival surpasses the latent
period. Radiation-induced leukaemias begin to appear after35 years. Solid cancers typically emerge more than 10
years after treatment but may occur earlier in particularly
susceptible individuals [F9, G32, T31, V10]. When the risk

207

of secondary solid cancer is elevated, it rises withincreasing radiation dose to the site and with increasingtime since treatment and persists as long as 20 years.
153. The predominant secondary cancer associated withchemotherapy is acute non-lymphocytic leukaemia (ANL).
Most ANLs have occurred after treatment with alkylating
agents or nitrosoureas. The findings are similar for Hodgkin’s
disease, paediatric cancers, ovarian cancer, multiple myeloma,
polycythemia vera, gastrointestinal cancers, small-cell lungcancer, and breast cancer [B22, B36, B63, B65, C14, F9, G32,
G33, R22, T31, T32, V10]. The risk for leukaemia rises withincreasing cumulative dose of the alkylating agent or nitrosourea. A few ANL cases were reported following combination chemotherapy, including teniposide or etoposide. The
leukaemias differ from those that follow alkylating agents inthat theyoccur sooner and that specific chromosomal abnormalities are induced [P9, P29, P30, W32]. Few solid tumours
have been linked to chemotherapy. Bladder cancer has been
associated with cyclophosphamide treatment, and risk isdependent on the cumulative dose of cyclophosphamide [P4,
T33, T36, W33]. Excess bladder cancer risk following
treatment with both radiotherapy and cyclophosphamide was
as expected from a summation of the individual risks. Bonesarcomas have also followed treatment with alkylating agents[T34]. In general, the risk for solid tumours after chemotherapy alone has been difficult to evaluate because too few
patients survived long enough after treatment by chemotherapyalone. At present, several cohort studies are under way
to assess this risk.
154. Earlier reports indicated a distinctive pattern ofsecondary cancers after treatment of childhood malignancies[M1]. The most common secondarycancer was bone sarcoma,
followed by soft tissue sarcomas, leukaemias, and cancer ofthe brain, thyroid, and breast. The cancers showing thehighest increases compared with the usual distribution ofchildhood cancers were retinoblastoma, followed by
Hodgkin’s disease, soft tissue sarcomas, Wilms’ tumour, andbrain cancer. This difference may reflect both the genetic
predisposition to develop multiple tumours in the case ofheritable retinoblastoma, soft tissue sarcoma of Li-Fraumeni
syndrome, and possibly the immune dysfunction associatedwith Hodgkin’s disease. To summarize the findings fromrecent study results [B68, O17, S6, S7], there is little
indication that heritable sensitivity to treatment is asignificant component of secondary cancer, but intensive
multiple agent therapy used in childhood cancer treatment
acts as an independent aetiological factor for a second
tumour. The risk for a second malignant neoplasm aftercancer in childhood is considerable. Absolute risks up to 7%
over 15 years following diagnosis of the primary cancer were
found for Hodgkin’s disease [B68]. This amounts to an excess
relative risk (ERR) of about 17, with breast cancer
contributing most. A follow-up study in the Nordic countries
showed a significant increase in the ERR from a low of 2.6 inpatients first diagnosed in the 1940s and 1950s to 5.9 forcohort members included in the late 1970s and 1980s,
indicating that newer treatments are not only more successful
but also carry a higher long-term risk [O17].

208

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

155. In patients with bone sarcomas as the secondary tumour
following childhood cancer therapy, the effects of radiation
therapy, chemotherapy, and combined modality treatment
have been analysed [H43, T34]. The risk for bone sarcoma
rose dramatically with increasing doses of radiation with alinear trend. Patients with heritable retinoblastoma had amuch higher risk for secondary bone sarcoma, but their
response to radiation was similar to that of patients with otherchildhood cancers. In addition to the radiation dose, theexposure to chemotherapy was evaluated. There was anindependent effect of exposure to alkylating agents in the risk
for bone cancer, i.e. radiation and alkylating agents acted
additively. The risk rose with increasing cumulative dose of
the alkylating agents. The effect of alkylating agents wasmuch smaller than that of radiation, and in the presence ofradiation at the site of the bone sarcoma, the alkylating agentsadded little to the risk.
156. Thyroid cancer risk after treatment of childhood cancer
is increased 53-fold compared with general population rates[T35]. The risk for thyroid cancer rose with increasingradiation dose. There was no increased risk of thyroid cancer
associated with alkylating-agent chemotherapy.
157. There was a sevenfold increased risk of secondary
cancers after treatment of acute lymphoblastic leukaemia(ALL) [N22]. Most of this risk was due to a 22-foldincrease in brain cancers. The brain cancers occurred inpatients diagnosed with ALL before the age of five years
and who received cranial or whole-body irradiation.
158. Among 29,552 patients with Hodgkin’s lymphoma, 163
cases of secondary leukaemia after treatment of the primarydisease indicated a considerable risk [K44]. There was nodifference in the relative risk of secondary leukaemias fromchemotherapyalone (MOPP regimen) and chemotherapyplus
radiotherapy. A relatively small risk for leukaemia was seen
after radiation alone, and this risk increased with radiation
dose. The risk did not vary significantly or consistently acrossradiation doses for any given number of chemotherapy cycles
but increased consistently with more cycles of chemotherapy
in each radiation dose range.
159. Significantly elevated risks for secondary solid tumours(lung, non-Hodgkin’s lymphoma, stomach, melanoma, bone,
and connective tissue) were reported in patients treated for
Hodgkin’s disease [T31]. The pattern of secondary tumours
was distinctive and was similar to the distribution of cancers
seen in immunosuppressed populations, such as renal transplantation patients or patients with non-Hodgkin’s lymphoma. All cancers of the stomach, bone, and connective tissue
occurred within areas previously treated with radiation
therapy. All those who developed lung cancer had received
radiation therapy and smoked. For breast cancer, a fourfold
elevated risk was reported in Hodgkin’s disease patients after
15 years of follow-up. The highest risk was in women
irradiated before the age of 30 [H19]. Comparable results werereported from a Dutch study [V11]. These authors reported an
overall relative risk of 3.5 for secondary cancers after
Hodgkin’s disease. Significant increases in relative risk of

34.7, 20.6, 8.8, 4.9, 3.7, 2.4, and 2.0 were reported forleukaemia, non-Hodgkin’s lymphoma, soft tissue sarcoma,melanoma, lung cancer, urogenital cancers, and gastrointestinal cancers, respectively. Risk factors for leukaemia werechemotherapyand host factors; for non-Hodgkin’s lymphoma
they were combined modality treatment rather than single
modality treatment or host factors. For lung cancer the risk
factors were strongly related to radiation therapy, while anadditional role for chemotherapy could not be demonstrated.
160. Significant excesses of ANL followed therapy for nonHodgkin’s disease with either prednimustine, a derivative of
nitrogen mustard, or with regimens containing mechlorethamine and procarbazine, for example MOPP therapy,(nitrogen mustard, vincristine, and procarbazine prednisone)[T6]. Chlorambucil and cyclophosphamide were associatedwith smaller increased risk of ANL. In this study, radiotherapy did not add to the leukaemogenicity of alkylating
agents. This finding should be interpreted cautiously,
however, because of the small number of patients and thelarge number of parameters evaluated.
161. Few studies have evaluated the late effects of adjuvant
chemotherapy and radiotherapy in breast cancer. The interaction of alkylating agents with radiation in producing
leukaemia in women treated for breast cancer was investigatedin a cohort of 82,700 patients in the United States [C29].
Based on 74 cases, the risk of ANL was significantly
increased after radiotherapy alone (relative risk = 2.4, 7.5 Gymean dose to the active marrow) and alkylating agents(melphalan and cyclophosphamide) alone (relative risk = 10).
Combined therapy resulted in a more-than-additive relativerisk of 17.4. The most common solid cancer that occurs after
breast cancer is contralateral breast cancer, but fewer than 3%of these tumours could be attributed to radiation [B2]. The risk
was highest in women treated at young age (under 45 years).
The usefulness of such studies is still hampered by the factthat an important proportion of patients developing primary
tumours might already belong to a genetically more sensitive
subpopulation [E11]. In addition, combined treatments might
be more often used in more advanced stages of tumours
needing higher total doses or more cycles for cure.

C. SUMMARY
162. A large number of chemotherapeutic drugs are usedin clinical cancer therapy in combination with radiation.
The main ones in use or proposed for use are described in
the Appendix, with emphasis on the mechanisms ofinteraction between the drugs and radiation that may haverelevance for combined effects between chemical agentsand radiation even at the low exposure levels found incontrolled environmental and occupational settings. The
main findings on modes of action and combined effects are
summarized in Table 6.
163. The predominant secondary cancer associated withchemotherapy is ANL and, to a lesser degree, bladdercancer. No one specific type of secondary cancer follows

Replacement of uridine in RNA andthymidine in DNA

BUdR, IUdR (thymidine analogs)

5-FU, precursor for FUdR

FUdR (uridine analog)

Pyrimidine analogs andprecursors

Antitumour antibiotics

Anthracyclines (doxorubicin,
daunomycin, epirubicin,
idarubicin)

Depletion of nucleotide pools by
thymidylate synthase inhibition

Methotrexate

Antifolates

Hydroxyurea

Replacement of thymidine in DNA

Cisplatin [cisdiaminodichloroplatinum (II)], carboplatin[diaminecyclobutanedicarboxylatoplatinum (II)]Platinum coordination
complexes

Antimetabolites b

Free-radical formation and/or
topoisomerase II inhibition
Increased tumour oxygenation by
inhibition of respiration (doxorubicin)

Natural products

Depletion of nucleotide pools by ribonucleoside diphosphate reductase
inhibition

Depletion of intracellular nucleotide pools

Bifunctional cross-linker

Bifunctional with two reactive
intermediates: isocyanate reacts with aminegroups (carbamoylation reaction) and
chloroethyl carbonium ion with
nucleophilic sites (alkylation)

BCNU, CCNU, MeCCNU

Nitrosoureas(Chloroethylnitrosoureas)

Addition of alkyl group to nucleophilic
sites in biomolecules

Critical target(s)DNA, mitochondriaDNADNA, RNA

DNA, RNADNA

DNA

DNA, RNA, proteins

DNA

DNA, proteins

Adduct forming agents a

Mode of action

Nitrogen mustards(mechlorethamine, melphalan,chlorambucil, cyclophosphamide)

Agent(s)Alkylating agentsClass of agent

Table 6
Combined modality treatments in tumour therapy

Protein-associated DNA
breaksInhibition of DNA synthesis
and repair

Impaired DNA repair andsynthesis, impaired
transcription

Depletion of dTTP pool

Impaired DNA repair,synthesis and transcription

Impaired DNA repair andsynthesis

Cross-links, DNA adducts

DNA adducts, cross-links,
glutathione depletion

DNA adducts, cross-links

Main effects

Additive for free radical formation;
supra-additive for topoisomerase II
inhibition;
supra-additive for respiratory
chain inhibition

Supra-additive

Supra-additive

Supra-additive

Supra-additive

Supra-additive

Supra-additive, double-strand breaks
result from the combination of strand
breaks associated with DNAplatinum repair and radiationinduced strand breaks

Additive to borderline
Supra-additive for alkylation
Supra-additive for glutathione effects

Mainly additive (isoadditive) toborderline supra-additive

Combined effects with radiation

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS
209

a
b

Metabolic reduction in anoxic tumour
cells to cytotoxic agents; additional
alkylating function in RSU 1069

Metabolic reduction in anoxic tumour
cells to cytotoxic agents withproduction of free radicals

Nitroimidazoles (metronidazole,
misonidazole, etanidazole,
RSU 1096)

Benzotrizine di-N-oxides(tirapazamine)

Effects cell-cycle independent.
Effects mainly cell-cycle dependent.

Metabolic reduction in anoxic tumour
cells to alkylating agents, mechanism
similar to mitomycin C

Quinone alkylating agents(EO9, porfiromycin)

Bioreductive drugs

Increased oxygenation of tumour

Hyperbaric oxygen

Inhibition of topoisomeras-I

Camptothecin, topotecan

OxygenDNA

Inhibition of topoisomeras-II

Epipodophyllotoxins (etoposide,
teniposide)

DNA

DNA

DNA

DNA

Spindle apparatus

Inhibition of tubulin polymerization,induction of microtubule disassembly

Topoisomerase poisonsIntercalates into DNA

Actinomycin D

Vinca alkaloids (vincristine,
vinblastine, desacetylvinblastine)

DNA

Bifunctional alkylating activity after
reduction of quinone entity;
highest activity in reducing environment

Mitomycin C

DNA

DNA

Critical target(s)Produces active oxygen species after
intercalating with DNA

Mode of action

Bleomycin

Agent(s)Microtubule poisons

Antitumour antibiotics(continued)

Class of agent

Table 6 (continued)

DNA damage by free radicals

Oxidative stress under aerobic
conditions, DNA-covalent
reaction products underhypoxic conditions

DNA adducts, cross-links

Blocking of replication forks(S-phase specific), singlestrand breaks

Protein-associated DNA
breaksCells blocked in mitotic phase

Inhibition of transcription

DNA adducts, cross-links

Radiomimetic drug

Main effects

Supra-additive

Supra-additive

Supra-additive when given before
radiation, pronounced cytotoxic
effect of the drug against radioresistant hypoxic cells

Additive to supra-additive

Supra-additive

Supra-additive when applied
together, repairable radiationinduced DNA damage istransformed into lethal damage

Additive to supra-additive
when given before radiation

Additive to supra-additive

Supra-additive when given before
radiation; pronounced cytotoxic
effect of the drug against
radioresistant hypoxic cells

Isoaddition

Combined effects with radiation

210
ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

radiotherapy. In general, there are independent effects of
exposure to alkylating agents and radiotherapy. For secondary
solid tumours, radiation is the main risk factor, while a rolefor chemotherapy has been demonstrated in some cases. For
lung cancer, an additional role of smoking was reported. Host
factors, for example, age of diagnosis and treatment for breast
cancer, are additional risk factors. For secondary leukaemias
the main risk factors are chemotherapyand host factors. Thereis only a small increase in this risk due to radiation. The effect211

on secondarycancers of increasinglyused adjuvant treatmentswith topoisomerase I or II inhibitors, microtubule poisons(discussed in the Appendix), and hormone treatment is as yet
unknown. In summary, secondary, treatment-related cancers
are observed increasingly because of the long-term success of
the initial treatments. At present, no important synergistic
effects between ionizing radiation and other agents are known.
Further investigations are needed to assess and to develop
strategies to reduce this potential complication.

V. EFFECTS OTHER THAN CANCER
164. Given that there is an overlap in the development ofradiation-induced cancer and other biological effects, such as
cellular effects, deterministic effects, and teratogenic effects(see Chapter II), results of combined exposures to radiationand other agents for these other effects might give some
information on the mechanistic aspects ofpossible interactions
for radiation carcinogenesis. In this Chapter effects other thancancer following combined exposures are reviewed with theaim of concluding whether the agents and low-level radiationinteract. It must be kept in mind that most deterministic
effects and many aspects of teratogenesis are a result ofcytotoxicityand cytolethality having apparent threshold levels
in tissue. Qualitatively, the results reported in this Chapter canbe considered as interactions occurring at the cellular andtissue/organ level of radiation-induced cancer. In view of themany data available and the aim of this Annex, only effects inhumans and mammalian organisms are reviewed.
165. Especially in earlier occupational situations, concomitant exposures to other agents may have caused pathologicalchanges in organs such as the lung, with considerable implications for exposure-dose conversion coefficients and possibly
also for target sensitivity towards stochastic effects fromionizing radiation. For example, in the studies of miners,
reduced pulmonary function and early onset of silicosis fromexposure to dust is also correlated with end points of interestin the context of this Annex [K21, K49, N25]. Although these
combined exposures have little relevance at present, they
contribute to the uncertainties involved in drawing inferencesfrom historic occupational risks and applying them to modernday working environments and non-occupational settings.

A. PRE- AND POST-NATAL EFFECTS
166. The effects of x-irradiation and hyperthermia at 43°C
both individually and in combination on mouse embryos were
investigated by Nakashima et al. [N1]. Cultured eight-day
B6C3F1 embryos were exposed to 0.32 Gyfrom x rays, 520
minutes of heating, or 5 minutes of heating and irradiation at
0.3, 0.6, and 0.9 Gy. Irradiation alone at 0.3 Gy showed noapparent effect on embryonic development, but irradiation at
0.62 Gy caused a dose-dependent increase in malformed
embryos. Heating alone for 5 minutes produced no malformed
embryos, while heating for 1020 minutes caused malforma-tions as a function of heating time. Combined treatments
produced higher frequencies (22%100%) of malformations
than would have been expected from considering the sum of
the separate treatments (0%42%). The malformations
observed were primarily microphthalmia, microcephaly, andopen neural tubes. The results indicate that in cultured mouseembryos irradiation combined with a non-teratogenic dose of
hyperthermia increases the formation of malformed embryos.
The interaction is most probably in the cellular phase ofeffects development (see Section II.B).
167. The interaction of exposures to heavy metals withradiation was studied during the pre-implantation stage inmice by Müller and Streffer [M3]. At this stage, placental
protection against chemical attack is lacking, and low cellnumbers limit replacement of damaged cells. Of the metals
arsenic, cadmium, lead, and mercury tested in micromolar
concentrations, arsenic showed no interaction with radiation[M59] and cadmium and lead showed supra-additivity only
for single endpoints: morphological development for cadmium
and micronuclei formation for lead. Mercury, however,showed considerable interaction for morphological development and cell proliferation. A classical construction of theenvelope of additivity in the range 03 Gy from x rays and08 µM of mercury chloride showed synergism, i.e. aninteraction effect exceeding isoadditivity. However, there wasno effect on micronuclei formation by mercury. Time factors
were shown to play an important role in these experiments[M57, M58]. For an enhancement of radiation risk, exposure
to mercury (3 µM) had to start immediately after irradiationand to last for an extended time period afterwards (112 hours).
The interaction is probably in the cellular phase, but the factthat a 24-hour exposure has little effect speaks against
inhibition of repair of radiation-induced DNA damage as the
only mechanism of mercury toxicity in this system.
168. The interaction of ionizing radiation with cadmium,
which at higher concentrations is teratogenic by itself, wasstudied by Michel and Balla [M37]. Metal exposure(2 mg kg1) on day 8 of gestation significantly increased
exencephaly and eye anomalies. They found considerable
antagonistic effects on survival, growth retardation, anddevelopmental malformations for combined exposures to
CdCl2 and x rays (0.5 and 1 Gy) in NMRI mouse embryos.
Since the metal exposure had to precede radiation for an

212

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

antagonistic effect, induction of maternal metallothionein
was proposed as a protective mechanism. However, application of metallothionein shortly before CdCl2 exposure
exerted no protective effect. HgCl2 alone induced a low rateof exencephaly, and combined treatment with x rays
resulted in additivity of single-exposure effects in the range
tested (0.5 and 1 Gy, 2 mg kg1). No conclusion on possible
implications for chronic, low-level exposures and radiation
carcinogenesis is possible.

interpret but are essential for a more accurate estimation of
the risks attributable to environmental agents. At the sametime they may contribute to an understanding of some of
the mechanisms underlying individual variability in thegenetic predisposition to cancer. With regard to combined
effects, no new or additional mechanisms are apparent for
genetic and multi-generation effects.

C. DETERMINISTIC EFFECTS
B. GENETIC AND MULTI-GENERATIONEFFECTS
169. An understanding of mutations in germ cells and ofcarcinogenic and teratogenic effects from germ-cell exposure
is of great importance for assessing health risks in future
generations [N19, N24]. The same holds true for potential
combined effects in these exposure situations. The hereditaryeffects of radiation have been considered in most previous
reports of the Committee. Experimental studies in animals
and emerging human evidence from epidemiologyconsideredhere deal mainlywith combined modalities in tumour therapy.
The combined effects of cytotoxic substances used in tumour
therapy on mouse stem cells and gamma-ray doses of 5 and9 Gy were studied using the spermatocyte test [D7]. Most of
the chemicals tested showed additive effects when combinedwith doses in the ascending part of the dose-response curveand potentiating effects when combined with doses in the
curve’s descending part. This has generally been consideredadditional confirmation that any kind of spermatogonia
depletion is sufficient to modify the genetic response of stem
cells. The chemicals mitomycin C and N,N',N"-triethylenethiophosphoramide (thiotepa) induced very low yields oftranslocations after single treatments. In combined treatmentswith a dose of 5 Gy, mitomycin C was found to have a
subadditive effect and thiotepa, an additive effect. Combinedwith a dose of 9 Gy, the compounds potentiated the effect ofradiation.
170. Based on the generally accepted hypothesis that mostcancers are multifactorial in origin, perinatal and multigeneration carcinogenesis should be considered in depth.
Nevertheless, the consequences of prenatal exposures andof prenatal events are often ignored [T15], partiallybecause it is not possible at present to quantify the role ofprenatal exposures tocarcinogens/mutagens in determining
or modulating the risk of cancer in humans. Tomatis [T15]listed prenatal events important to the occurrence of cancer
as the consequence of one of the following:
(a)
(b)
(c)

the direct exposure of embryonal or fetal cells to a
carcinogenic agent;a prezygotic exposure of the germ cells of one or bothparents to a carcinogen/mutagen before mating; ora genetic instability and/or a genetic rearrangement
resulting from selective breeding, which may favour
a deregulation of cellular growth and differentiation.

Because they involve both germ and somatic cells, studies
of prenatal carcinogenesis are sometimes difficult to171. Deterministic effects of ionizing radiation are theresult of exposures that cause sufficient cell damage or lossof proliferative capacity in stem cells to impair function in
the irradiated tissue or organ. For a given deterministic
effect, a large proportion of cells must generally be affected,
so that in most cases there are considerable thresholds in therange from tenths of a sievert to several sievert. Deterministic
effects were reviewed by the Committee in Annex I, “Latedeterministic effects in children”, of the UNSCEAR 1993
Report [U3]. Although deterministic effects are practically
excluded in controlled settings, side effects in tissue
adjacent to treated tumours and localized effects in skin,eyes, and lungs must still be considered when assessing
human health risks. Since loss of the ability to divide isalso a result of DNA damage, many of the molecularmechanisms that modulate combined effects incarcinogenesis also modulate deterministic effects. In thiscontext, the scavenging of radiation-induced radicals by
scavengers such as cysteamine will also exert antagonistic
effects for deterministic endpoints. Because this field is oflimited relevance for this Annex, only a few examples fromthis poorly explored field are given below.
172. There are suggestions from clinical findings that preexisting diabetes exacerbates radiation injury to the retinal
vasculature. Gardiner et al. [G2] studied this phenomenon in
streptozotocin-induced diabetic rats. In both diabetic andcontrol rats, the right eye was irradiated with 90 kVp x rays
to 10 Gy and the prevalence of acellular capillaries in trypsin
digests of the retinal vasculature was quantified 6.5 months
after irradiation. Diabetes as well as irradiation led to astatistically significant higher prevalence of acellular
capillaries. The net increase in acellular capillaries following
irradiation was much greater in rats with an eight-month termof pre-existing diabetes (180%) than in those that had been
diabetic for only three months (36%). These results suggest asynergistic relationship between pre-existing diabetes andionizing radiation in the development of retinal vasculopathy
that seems to depend on the duration of diabetes before
radiation exposure.
173. Ivanitskaia [I4] studied the reduction ofspermatogenesis
and of activities of key enzymes as a result of single or
combined action of ionizing radiation and mercury in rats.
The combined biological effects seemed to be close to the sumof the effects caused by the single agents.
174. Higher acute radiation doses are known to impair
immune functions at least temporarily. Generally, immune

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

deficiencies are the condition being considered. However,overstimulation of the immune functions or the emergenceof new antigenic sites as a secondary effect of radiation
damage have to be considered as well. A stimulation effectat high doses was described by Lehnert et al. [L14] ininbred C57BL mice. The mice were irradiated with 10 Gy
delivered to the thorax 24 hours prior to the induction ofgraft-versus-host disease by the injection of allogeneic
lymphoid cells (2 107 cells). In mice only irradiated or only
injected, survival was 100% at 250 days. In contrast, a
combination of the two treatments, graft-versus-hostdisease and partial-body irradiation, resulted in a mortalityof 83% and a mean survival time of only 29 days,
indicating strong synergybetween graft-versus-host disease
and partial-body irradiation. From histological studies ofthe lung, it appeared that about 40% of the deaths
occurring after combined graft-versus-host disease andpartial-body irradiation (PBI) treatment might be attributable to pneumonia. The cause of death in the remaining mice that received combined treatment is unknown.
Mice receiving combined PBI/lymphoid cell treatment also
develop a characteristic skin lesion that is not seen in nonirradiated mice and that is confined to the irradiated area.
A first indication of the mechanism involved is the factthat the amplifying effect of pre-induction partial-body
irradiation on the timing and severity of graft-versus-hostdisease is similar to the effect that would be produced by anincrease in the number of effector cells. Such a
proliferative response should display a highly non-linear
dose-response relationship with an apparent thresholdsimilar to immune deficiencies based on widespread stemcell killing. Therefore, no direct relevance of these findings
for much lower occupational or environmental exposures
is apparent.

213

175. Guadagny et al. [G18] described an increase in immunogenicity of murine lymphoma cells following exposure to
gamma rays in vivo. On the basis that mutagenic compounds
such as5' (3,3'-dimethyl-1-triazeno)-imidazole-4-carboxamide(DTIC) cause a marked increase in immunogenicityin murine
lymphoma cells in vivo or in vitro, they then conductedfurther experiments to test whether ionizing radiation would
be able to affect the immunogenic properties of cancer cells ina mouse leukaemia model. Male CD2F1 mice were inoculatedwith histocompatible L1210 Ha leukaemia cells and treatedwith 4 Gy of whole-body irradiation. A number of transplant
generations were carried out with leukaemic cells collectedfrom irradiated donors, generating a radiation-treated line.
The immunogenicity of radiation-treated cell lines increased
significantly compared with that of the L1210 Ha line as early
as after three passages in vivo. However, no strong transplantation antigens comparable to those elicited by treatment
with DTIC were found in radiation-treated cell lines, evenafter a number of transplant generations. The combination ofbis-chloroethyl-nitrosourea and the weakly antigenic radiation-treated cell line elicited a strongly synergistic immune
response of the host. Moreover, lymphoma induced withradiation-treated cell lines acquired strong immunogenic
properties after a single cycle of DTIC treatment in vivo.
Again, these results may well provide an experimental modelfor the exploitation of a radiation-induced increase of tumour
cell immunogenicityfor combined radioimmunochemotherapy
in cancer treatment, but no direct relevance for the risk ofradiation carcinogenesis is evident. As with other combinedmodalities in tumour therapy that also enhance and modulate
deterministic effects (described in depth in the Appendix), theabove examples do not indicate mechanisms leading tomarked supra-additivity for effects from low-level exposures
to multiple agents.

EXTENDED SUMMARY
176. In this Annex, the effects of combined exposures toradiation and other agents are considered particularly withrespect to the induction of stochastic effects at low doses.
A large amount of information on the combined exposuresof radiation and other physical, chemical, and biologicalagents is reviewed. In many situations agents can interact
with radiation and may significantly modify the biologicalprocesses and outcomes. The implications for radiation risk
assessment and limitation of individual and collective
health risks are considered.
177. For ionizing radiation, the main potential risk tohumans from exposures at low doses, i.e. at the level ofbackground radiation or a few times that level, is theenhanced incidence of stochastic effects, i.e. carcinogenesis
and heritable genetic effects. In this Annex the effects ofcombined exposures to radiation and other agents are
considered, particularly with respect to the possibility ofenhanced radiation carcinogenesis caused by chronic low
doses. Many radiobiological experiments, however, used

acute, high radiation doses and high exposures to the otheragent. It is usually not clear how these results might be
extrapolated to low and chronic irradiation conditions andto humans. Such data may, however, be informative on thepossible mechanisms of the interactions between various
agents and radiation, and in that sense they may be of
relevance for combined, low-dose exposures leading tocarcinogenesis.
178. For assessing the effects of chemical agents, thesituation is somewhat more complex than for ionizing
radiation. For genotoxic chemicals, the main biologicaleffects from low and chronic exposures are comparable toeffects from low levels of radiation, i.e. stochastic innature. However, as with radiation, most experimental data
are from high, acute exposures. The wealth of epidemiological data and risk estimates based on such information,is much greater for radiation protection than for toxicology. With only a few exceptions (e.g. asbestos, smoking,and arsenic) for which human data are available, chemical

214

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

carcinogenesis data are based solely on biochemical, cellbiological, and/or animal data. The general assumption about
the dose-effect relationships for radiation and genotoxic
chemicals in the low-dose region for chronic exposures andfor stochastic endpoints is, however, the same. In general, it
is assumed that these relationships are linear from high-dose
ranges with observed effects down to zero dose. For nongenotoxic chemicals, non-linear dose-effect relationships are
the norm, since higher order enzyme reactions are involved inmost cases (uptake of agents, incorporation, metabolization,
cell physiological reactions, etc.). These reactions are
dominated by sigmoidal dose-effect relationships withapparent thresholds, related to the biochemical MichaelisMenten kinetics.

181. In the early, molecular phase of the development of theradiation effect, interactions of chemicals with the primaryradiation process can occur that are important for the fixationof the primary molecular radiation damage. For an interaction
to occur, the active agents must be in close proximity to theDNA, which is the most important molecule for radiation
carcinogenesis, at the time of irradiation or during repair andin a sufficientlyhigh concentration. Molecular interactions arestudied particularly to investigate the early radiation mechanism, and changes in the radiation effect from interactions havebeen seen. However, because of the high concentrations
needed to observe a significant effect, the results from theseinvestigations are not of direct relevance for the low levels of
exposure found in occupational or environmental settings.

179. The starting point for an analysis of combined
exposures is to specify the doses of the agents at the site ofinteraction. For ionizing radiation, absorbed dose is thequantity most generally applied to characterize the exposure,and methods have been developed to calculate the dose intarget cells from the irradiation conditions. For other agents,
different methods are used, depending on the characteristicsof the agent involved. Unfortunately, the exposure of the cells
at risk for carcinogenesis is not always clear. No unifying
concept of dose exists. In this Annex, methods of biochemicaland biological monitoring and dosimetric evaluation are
reviewed. The conclusion is that physiologically based
parameters, such as concentration of toxic agents in blood orurine, are often not specific and sensitive enough to begenerally applicable for the analysis of the biological action ofa chemical agent. Therefore, biological endpoints at thecellular level, which are more directly related to stochastic
health effects, have been developed and used as measures forgenetic changes in somatic as well as germ cells. To these
endpoints belong biochemical parameters such as DNA
adducts, and gene mutation parameters such as mutation
frequency and spectrum, and stable chromosomal alterations.
A generally applicable method for analysing combined effects
is still lacking, but taking these endpoints as a measure of the
genotoxic burden of radiation or of chemical agents, aunifying risk concept based on genetic burden can beenvisaged.

182. An impressive amount of information concerninginteractions in the cellular phase can be found in theliterature. For acute exposures, many agents can interact withradiation in this phase, including physical (e.g. UV) andchemical (e.g. alkylating and other genotoxic) agents. Toxic
chemicals have been evaluated, using the isobolic method ofanalysis. Different interaction mechanisms are involved,
ranging from an accumulation of DNA (sub)lesions, sometimes enhanced by repair inhibitors, to modulation of cellcycle kinetics. The results of the interactions range from
subadditive to supra-additive; however, for interaction to
occur, the agents must generally be present during or shortly
after irradiation, and the interaction effects decrease at low
doses and dose rates. This mode of interaction may have
implications for radiation carcinogenesis, but at low doses andfor chronic irradiation, deviations from additivityare expected
to be small.

180. Carcinogenesis is, in general, a slowly developing
process extending over years and even decades. From amechanistic standpoint, different phases in the developmentof the effects of an agent can be considered. These may becharacterized broadly as changes on the molecular, cellular,
and tissue/organ levels. Agents can interact with radiation ineach phase to produce an effect. Radiobiological research hasturned up numerous agents potentially capable of influencing
the progression of early radiation effects towards adverse
health effects. General conclusions are hindered by themultitude and complexity of the possible interactions and thedependence of the combined effect on the sequence of the
exposures. More explicitly, because of the long time period
between the initial radiation event and the final effect, acombined exposure to radiation and another agent may occur
after simultaneous exposure but also from exposures hours oreven years apart.

183. In the organ phase of cancer development, interactions
in combined exposures can be significant. The long durationof this phase creates many opportunities for interaction withother agents. As is also concluded for chemical carcinogenesis, these interactions are potentially important, but only
a few data from human epidemiological studies are suitablefor quantitative analysis. Radiation has been found to interact
with physical agents such as UV radiation and mineral fibres;with chemical agents such as alkylating chemicals, tumour
promoters, dietaryfactors, arsenic, and heavymetals; and withbiological agents such as hormones and viruses. Well-defined
effects are summarized in Table 7. Observation and identification of combination effects in this phase is difficult, because
the duration of the interaction with the radiation damage may
be long. In view of the many possibilities, it may well be thatdifferent interactions in the organ phase are largely responsible for the variations in background cancer incidence
between populations.
184. A very important combined effect is the interaction ofsmoking and exposure to radon, although even in this casethere is still no unambiguous conclusion on the interaction
mechanism. Epidemiological data clearly indicate thatcombined exposure to radon and cigarette smoke leads tomore-than-additive effects on lung cancer. These results
warrant special consideration in estimating the radiation
risks because a large proportion of the world’s population

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

215

effect of radiation is dependent on the background tumour
incidence; they also show how the interaction of radiation
with other agents might influence carcinogenesis.

is exposed concomitantly to considerable levels of indoor
radon and smoking. The combined analysis of 11 minerstudies [L18] indicates that the effect of radon may beenhanced by a factor of about 3 by being combined withsmoking.

186. Information is scarce on combined exposures ofradiation and specific agents that might alter the radiation
health risks caused by ambient exposures in the humanenvironment. The possible relevance of the interaction ofother agents with the radiation effect is obscured by the
many sometimes poorly known or unknown sources of
uncertaintysurrounding radiation-induced carcinogenesis,
such as variations in background cancer incidences,
population characteristics and genetics, diet, and individual
susceptibility.

185. Since the Committee's previous review of this subject[U6], there have been advances in modelling the multi-stage
processes involved in carcinogenesis. The development andapplication of mechanistically based, multi-stage
carcinogenesis models promise to give new insights into theinteraction processes, especially because with these models it
is possible to analyse interactions at the tissue/organ level ofcarcinogenesis. The results indicate, for example, that theTable 7
Agents that interact with ionizing radiation of importance in radiation carcinogenesis
Interacting agent

Interaction

Endpoint

Physical agents
External ionizing radiation with internal emitters
Ultraviolet radiation (UV)
Alpha emitters with mineral fibres, including asbestos

Supra-additive
Possibly supra-additive
Supra-additive

Bone cancer
Skin cancer
Lung cancer

Chemical agents
Nitroso compounds, such as MNU, DEN, 4NQO
Tumour promoters, such as TPA
Smoking
Vitamins
Diet/fat
Arsenic

Supra-additive
Supra-additive
Supra-additive
Subadditive
Sub- to supra-additive
Supra-additive

Effects shown only in animal experiments
Lung cancer
Interaction dependent on comparing level
Extrapolated from chemical carcinogenesis

Biological agents
DES
Testosterone

Supra-additive
Supra-additive

Breast cancer
Prostate cancer

CONCLUSIONS
187. Combined exposures are a characteristic of life. The
environment in which organisms reside and the organisms
themselves are complex systems in which a multitude ofinteractions between physical, chemical, and biological factors
occur. The specific agents involved in exposures in theenvironment and in occupational settings vary widely, butalmost all physical and chemical agents, both natural andman-made, are capable of producing adverse effects undersome exposure conditions, although individual agents differ
considerably in their capacity to do so. In general, for many
agents essential for life, there is a spectrum of effects associated with exposure, ranging from deficiency throughsufficiency to adverse effects with increasing levels of
exposure.
188. Although both synergistic and antagonistic combined
effects are common at high exposures, there is no firm
evidence for large deviations from additivity at controlled
occupational or environmental exposures. This holds formechanistic considerations, animal studies, and epidemiologybased assessments. Therefore, in spite of the potential
importance of combined effects, results from assessments ofthe effects of single agents on human health are generally
deemed applicable to exposure situations involving multiple
agents.
189. With the exception of radiation and smoking, there
is little indication from epidemiological data for a need toadjust for strong antagonistic or synergistic combined effects.
The lack of pertinent data on combined effects does not
imply per se that interactions between radiation and otheragents do not occur. Indeed, substances with tumour promoter and/or inhibitor activities are found in the daily diet,and cancer risk therefore depends on lifestyle, particularly
eating habits. Not only can these agents modify the naturalor spontaneous cancer incidence, but they may also modify
the carcinogenic potential of radiation. Such modifications
would influence the outcome particularly when radiation

216

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

risks are projected relative to the spontaneous cancer
incidence.
190. The analysis of small effects of combined exposures ofother agents with radiation is also inhibited by the lack of welldefined and pertinent harmonized measures of the exposuresto radiation and the other agent. A generally applicable
method for use in the analysis of combined effects is stilllacking, but taking end points such as measures of the
genotoxic effects of radiation or chemical agents, a unifying
risk concept based on genetic burden could be envisaged for
combinations of genotoxicants. At this stage, the uncertainties
in the data permit the possible interactions from combined
exposures of radiation with other agents to be only
qualitatively recognized. A quantitative assessment of theradiation risks at low doses is not yet possible. In other words,
even possible deviations from additivityat lower exposures are
generally too low to show up in experimental studies orpopulation cohorts.
191. The extent to which the effects of combined exposures
can be elucidated is highly dependent on clarification of the
carcinogenesis process itself and its dependence on environmental and lifestyle factors. Interactions of agents with radiation can be broadly grouped into three different levels (molecular, cellular, and tissue/organ levels) of the radiation effect.
The molecular phase lasts only a fraction of a second until the
primary radiation damage to DNA has occurred. For interactions during this phase, the other agent has to be present
concomitantly and in a high enough concentration.
192. The cellular phase of the radiation effect lasts for one
or a few cell cycles until the primary radiation damage in
DNA has been repaired or the remaining damage has been
fixed into heritable genetic damage (mutation in somaticand germ cells). For low doses and dose rates of radiationand low doses and chronic exposures to genotoxic chemical
agents, the supralinear or quadratic terms of dose-effectrelationships tend to vanish, and the linear terms dominate
for single-agent effects. In the absence of target specificity,
this implies that interaction at the cellular level duringlong-term low-level exposures to radiation and chemicals
is of limited importance.
193. During the tissue/organ phase of radiation-induced
carcinogenesis, which lasts from the first fixed genetic
alteration to the clinically manifested tumour and which may
include several genetic and epigenetic changes, combined
effects can occur from exposures of two and more agents
spread over days or decades, giving a large potential for
combined effects. Besides genotoxic chemicals, many nongenotoxic agents may interact during the organ phase.
Tumour promotion, mitogenic stimulation, and hormonal
activation are a few of the important examples of processes
with the potential for more-than-additive effects. Also,radiation- or chemical-induced genetic instability, which leads
to new genetic damage after many cell generations, may beprone to more-than-additive effects. An overview of possible
interaction processes and groups of agents involved in theseprocesses is given in Figure V.

194. Within the framework of the multi-stage mechanismof carcinogenesis, the following general conclusions can be
drawn for the combined action of different carcinogenic
and co-carcinogenic agents:(a) genotoxic agents with similar biological and mechanistic behaviour and acting at the same time will interact
in an isoadditive or concentration-additive manner. Thismeans that concurrent exposures to ionizing radiationand other DNA-damaging agents with no specificaffinity to those DNA sequences that are criticallyinvolved in carcinogenesis will generallyresult in effects
not far from isoadditive. Isoadditivity at this point
includes “apparent synergisms” or “autosynergisms”
resulting from non-linear dose-effect relationships ofthe
single-agent effects. Supra-additivity of this quality
generally does not exceed the expectation value derived
from high-exposure, single-agent effects combined withlinear dose-effect models;(b) for genotoxic agents acting on different rate-limiting
steps of multi-stage stochastic diseases like cancer,
strong deviations from additivity might result. Deviation from additivity can depend on the specificity of theagents for the different steps, sequence specificity, andthe sequence of exposures. Highly synergistic effects
are, however, only to be expected in cases where both
agents are responsible for a large fraction of the totaltransitions through the respective rate-limiting steps;(c) in combinations of radiation and non-genotoxic agentsin which the second agent causes promotion, i.e. themultiplication of premalignant cells, highly synergistic
effects may arise. This combined effect is dependent on
the exposure schedule. Thresholds for such combined
effects are generally implicated from the highly nonlinear dose-effect relationship for the non-genotoxic
agent acting alone;(d) for agents acting independently and through different
mechanisms and pathways, heteroadditivity or effectadditivity is predicted. Apparent thresholds will notinterfere with each other, and possible conservatisms inlinear dose-effect extrapolations from high exposures
will not be affected;(e) in combinations of agents, in which one agent induces
adaptive mechanisms, e.g. increased DNA repair
capacityor increased radical scavenger function, and theother agent induces DNA damage, antagonistic effects
may arise. Owing to the generally short half-times ofadaptive mechanisms, the exposures have to occur
concurrently or nearly concurrently.
195. In summary, the following parameters need to beconsidered to address and assess potential combined effects:
the mode of action of the agent (genotoxic or non-genotoxic);
the shape of the dose-effect relationship for single-agent
effects; the dose or concentration involved (low or high); thetype of exposure (chronic or acute); and the sequence and timeinterval between exposures (simultaneous or before or afterradiation exposure).
196. There has been little systematic research on possible
interactions of radiation with other agents. An exception isANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

the use of combined modalities in tumour therapy, but thehigh doses and deterministic effects involved cannot beeasily related to stochastic, low-level combined effects.
Considerable progress in the biological sciences and themany radiological and toxicological disciplines involved
will be needed to allow predicting the potential presence ofcombined effects at low exposure levels and negative217

health outcomes. It can be stated, however, that theconclusion of the Committee’s previous review on combined effects [U6] still holds: except for radiation andsmoking, there is no evidence that low-level exposures tomultiple agents yield combined effects far from additivity,or above the estimates resulting from linear extrapolationof single agent effects to lower doses.

FURTHER RESEARCH NEEDS
197. The lack of systematic mechanistic understanding andquantitative assessments of combined exposures and theresulting possible interactions urgently needs to be resolved.
This elucidation of interactions of agents in combined effects
critically depends on both a qualitative (mechanistic) and aquantitative knowledge of the action of any single exogenousor endogenous agent involved. The basic tenets ofexperimental toxicology and radiobiology will have to be
applied in studies of the adverse health effects of combinationsof exposures. In view of convincing evidence that the critical
stochastic endpoint, cancer, is multifactorial, the many studies
concentrating on single carcinogenic agents and attempting toquantify cancer risks as if they were due to single factors haveto be supplemented and extended to address potential
modifications from joint effects.
198. Present knowledge of the many qualitatively different
interactions already found in biological systems speaks
against the emergence of simple unifying concepts to predictmodifications of risk from combined exposures. However,mechanistically based classifications of interactions may be
helpful in predicting effects. At present, relevant knowledge
is being gained on interaction mechanisms in different partsof the long process of radiation carcinogenesis. A better
understanding of how important these separate physical,
chemical, and biological interaction mechanisms are for theultimate endpoint will help to create a basis for risk
assessment, and a better understanding of the carcinogenesis
process itself and the rate-limiting steps involved may
contribute to an understanding of the interactions as well. The
development of mechanistically based cancer models could
greatly improve the estimation of quantitative risks.
199. Individual genetic susceptibility is already a concern for
the assessment of radiation risks. In addition to those parts ofthe genome susceptible to radiation-induced effects, e.g. repair
and proofreading genes, heterozygosity for oncogenes andtumour suppressor genes, many additional gene products
determining the biokinetics andbiotransformation ofchemical
agents will have to be considered in the individual response tocombined exposures involving chemicals.
200. Progress in the analysis of interactions between ionizing
radiation and toxicants is often hampered by a lack ofscientific data that quantitatively relates chemical exposure to
health risk or experimental endpoints. The implementation ofstandard protocols and dosimetry to harmonize reported
research is urgently needed to allow comparison of data fromstudies on different agents. Data will also have to be extended
over a sufficiently large exposure range to allow extrapolating
to the doses relevant in environmental health.
201. Epidemiological studies have alreadyrevealed important
combined effects for carcinogenesis, particularly for the joint
effect of cigarette smoke with either radiation or asbestos. New
tools in molecular biology point the way to the field of
molecular epidemiology and will provide investigators withmarkers of exposure and damage that are much more sensitive
than the cruder incidence measures of clinical diseases. Suchapproaches, if successful, can be expected to yield significant
new information on interactions between agents at the cellularand molecular levels. Markers of this kind can also be used in
more classical human epidemiological studies of cancer, someof which may help in probing potential interactions between
agents that may have induced the disease.
202. A mechanistic assessment of combined effects isdependent on progress in the scientific understanding of othergeneric issues, such as extrapolation from high to low levels,transfer of data from laboratory animals to humans, and age
dependence of the radiation risk, that are central to the generalrisk assessment process. Current approaches to the risk
assessment of complex exposures rely heavily on linear doseeffect relationships and additivity models. However, in thedose and concentration range of interest for human exposures,
dose-effect relationships other than linear (sigmoidal and evenU-shaped curves in the case of partially stimulatory or
essential agents) are reported as well. This issue must be fully
addressed in assessing the risks of combined effects.
203. Finally, a comprehensive approach for the study andquantitative assessment of combined effects must be
developed. The gap between different conceptual approaches
in the assessment of risk in chemical toxicology andradiological protection has to be bridged urgently.
Multidisciplinary approaches to research (radiobiology,
toxicology, cell and molecular biology, biostatistics,
epidemiology) have to be forged. In some instances, recasting
and combining results from recent studies and on-going work
into more refined models that take into account additional
mechanisms of responses and that take advantage of themultidisciplinary approach may improve the understandingand quantification of specific interactions. This, together withthe application of refined multi-stage models, will help toreduce uncertainties at the low exposure levels found in thehuman environment.

218

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

APPENDIX
Combined effects of specific physical, chemical, and biological agentswith ionizing radiation
1. Studies of the combined effects of specific physical,
chemical, and biological agents in association with radiation
exposure are reviewed in detail in this Appendix. The
intention is to provide an overview of the available literaturein support of the more general findings, summaries, andconclusions of this Annex. First, data from epidemiologicalstudies of the adverse health effects in humans of each groupof agents are reviewed. Studies involving experimental animalmodels are then considered. Finally, various effects observed
using in vitro systems are reviewed. Carcinogenesis is theprincipal endpoint ofinterest, but non-neoplastic endpoints arealso discussed. Only a minor fraction of the interacting agentsdescribed below are found in the human environment at
potentially critical levels. A few such critical agents alreadyknown or suspected to affect human health on their own arelisted in Table 4. The findings of specific combined exposuresand effects described in depth in this Appendix aresummarized in Table A.1.

beta activity on the skin. They modelled the immunosuppressive effects of whole-body gamma radiation in the sublethal tolethal range (111 Gy) on skin reactions produced by 50 Gy
from superficial beta radiation from 171Tm in male mice. For
gamma doses below 4 Gy, no interaction effects were detectable. For gamma doses in the range 48 Gy, the skin reaction
developed more slowly, but it was not much more severe. The
overall time for the resolution of the skin reaction, about 45
days, was also unaffected by high-dose whole-body irradiation. The authors ascribed the absence of any considerable
deviation from additivity in this system to the mismatch intime between maximal immunosuppression and localized
severe beta burns ranging from 2 to 10 days and 10 to 25
days, respectively. Although such beneficial mismatches intime are species-specific, these mechanisms may also be
important in humans. Deterministic combined effects in
Chernobyl power plant staff and emergency workers are
discussed in Annex J, “Exposures and effects of theChernobyl accident”.

A. RADIATION AND PHYSICAL AGENTS

4.
A strong antagonistic combined effect was found in anexperimental study of deterministic effects in rats. When the
animals received high external gamma (about 6 Gy) or beta(about 24 Gy surface dose) radiation, lethality was lower by afactor of 5 when the animals received a concomitant exposure
to the thyroid gland of 0.3 kBq g1 from 131I given orally[M13]. The protective influence of the combined treatment
was attributed to 131I-induced changes in the hormonal state in
the course of acute radiation sickness. In another study by thesame author with lower sublethal external doses of up to 3 Gy
and additional orally administered 131I, there was an increased
yield of mammary tumours in the combined treatment group
receiving low exposures from iodine (0.040.8 kBq g1). For
higher iodine exposures, however, the reverse was true [M30].
The combined effects observed seem to be deterministic andcan be attributed mainly to different organ doses rather thandifferent radiation qualities.

1. Combinations of different types
of ionizing radiation
2. Understanding how cellular damage produced by highlinear-energy-transfer (LET) radiation interacts with that
produced by low-LET radiation is important both in radiation
therapy and in evaluating risk. In view of the possible
radiotherapeutic applications, a wealth of data on the
combined action of neutrons, heavy ions, and gamma or
x rays was accumulated in the 1970s and early 1980s. This
information was reviewed in Annex L, “Biological effects ofradiation in combination with other physical, chemical andbiological agents”, in the UNSCEAR 1982 Report [U6].
Because the underlying damage mechanism is similar, theinteraction between different types of ionizing radiation is, ingeneral, of the isoadditive type in the case of cell killing.
Deviations from additivity are generally not very large andcan be explained in most cases by concomitant changes in
exposure rates and in exposures of critical target structures.
For example, survival of Chinese hamster V79 cells in vitro
after irradiation first with neon ions (LET = 180 keV µm1)
and then with x rays (225 kVp) was additive, as predictedfrom independent action. The system also showed no
dependence on the order of application [N5].
3. In extreme emergency situations, localized exposures tothe skin or other organs in the presence of elevated externalradiation fields is of considerable concern. Randall and
Coggle [R3] studied the deterministic effects of concomitantwhole-body irradiation and localized radiation trauma from

5.
Changes in the haematopoietic bone marrow, i.e. in thenumber of colony-forming units (CFU), of rats were observedby Brezani et al. [B1] after a single whole-body neutron doseof 2 Gy and combined single neutron (2 Gy) and continuous
gamma irradiation (6 Gy, daily dose rate of 0.57 Gy). Neutron
irradiation alone significantly reduced the number ofkaryocytes, including CFU-S in the bone marrow and induced
extensive cytogenetic damage. When followed by continuous
gamma irradiation, the primary damage from neutrons was
not enhanced, however CFU-S remained at a decreased level
for the whole time of irradiation. Recovery from damage
began only after termination of the continuous irradiation; its
course was similar to that after single neutron irradiation. A
long-lasting supra-additive influence of the combined

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

exposure to neutrons and gamma rays is neverthelessmanifested in later periods after irradiation by a reduction in
the total CFU-S number in the bone marrow.
6. Most studies ofstochastic effects from combined irradiations are undertaken in connection with cancer. For boneseeking radionuclides, a synergistic effect was found in micefor osteosarcomas after combined exposure toshort-lived 227Th(190 Bq g1, corresponding to about 10 Gy mean skeletalalpha dose) and to longer-lived 227Ac (1.9 Bq g1) boneseeking radionuclides. The beta emitter 227Ac produces protracted internal alpha exposures through ingrowth of the
decay product 227Th. At 700 days after intraperitoneal (ip)
injection of pure 227Th or 227Th contaminated with 1% 227Ac(combined exposure) in the form of citrate, an osteosarcoma
incidence higher than additive was found for the combined
exposure. With incidences of less than 1% for controls, 8% for227
Ac alone, and 36% for 227Th alone, the combined effect of
62% amounted to an interaction factor of 1.7 [L26]. In termsof the time for 50% tumour appearance, the interaction factor
was reduced to a barely significant 1.3. The authors speculated that the increased oncogenetic effectiveness of 227Th
contaminated with 1% 227Ac may be caused by the continuous
stimulation of cell proliferation or by the activation of retroviruses by protracted low-level alpha irradiation from 227Ac/
227
Th.
7. Bukhtoiarova and Spirina [B33] studied the combined
effect of external gamma irradiation and 239Pu on theincidence of osteosarcomas in inbred male rats. Osteosarcomas occurred more frequently and at earlier times anddisplayed a more pronounced multicentric pattern of growthand metastatic spreading than the malignancies induced byexposure to only one of the two agents. The differences
resulted from increased development of tumours anddecreased osteogenesis. A quantitative evaluation of the combined effect of the same radiation mix on biochemicalparameters of the rat immune system was undertaken by
Elkina and Lumpov [E5]. The combined effect of external
gamma radiation (137Cs, 14 Gy) and incorporated alpharadiation (239Pu nitrate, 9.393 kBq kg1 body mass) was
estimated by determining changes in nucleic acid metabolism
and the number of cells in rat thymus, spleen, and bonemarrow. The data obtained for the lower end of exposures
were consistent with an additive model. The same researchers
also studied aminotransferase and lactate dehydrogenase
activity in the blood of dogs exposed to the joint action of
external gamma and internal alpha radiation [E6]. After theeffect of external gamma radiation (0.252 Gy) and inhaled239
Pu submicron oxide containing 25% 241Am (approximately
710 kBq kg1) delivered separately and in combination,
activities of alanine-aspartate aminotransferase and lactate
dehydrogenase changed in an undulatory manner, tending toincrease at later times. The change was a function of type andlevel of radiation as well as time elapsed from the onset of
exposure. Even at the relatively high exposures used in theseexperiments, the combined effect of gamma and alpharadiation did not exceed the additive effect of the two factorsdelivered separately. In view of the deterministic nature of theendpoints studied, no inferences for controlled exposures areapparent.

219

8.
Several authors have shown that large radiation doses
influence biokinetics and hence exposure from incorporated
radionuclides. The influence of external gamma radiation on
239
Pu redistribution in pregnant and lactating rats was
described by Ovcharenko and Fomina [O1]. A quite high dose
range of acute external gamma radiation, from 0.5 to 4 Gy,
was investigated. Transplacental transfer of 239Pu to theembryo increased with dose to a maximum at 1 Gy and thendeclined. However, transfer of 239Pu via milk to newborn rats
was decreased by external gamma irradiation of lactating rats
with the dose of 0.5 Gy. The nature of the biologicalmechanisms responsible for the changes in biokinetics
remains elusive. There are no suggestions by the authors thatthese radiation effects on metabolism are stochastic in nature
and would extend to low doses and dose rates.
9.
Lundgren et al. [L19, L20] examined the carcinogenicity of a single, acute pernasal inhalation exposure of
3,201 male and female F344 rats to 239PuO2, followed one andtwo months later bywhole-bodyx-irradiation. Plutonium lungburdens were 56 or 170 Bq, and the x-ray exposure wasfractionated into two exposures totalling either 3.8 or 11.5 Gy.
Other groups of rats received control (sham) exposures. Minor
x-ray-dependent differences in 239Pu lung retention wereobserved; however, exposure to x rays significantly reduced
the median survival times in rats of both sexes [L19]. For agiven level of x-ray exposure (0, 3.8, or 11.5 Gy), the level of239
Pu exposure (0, 56, or 170 Bq) had no effect on mediansurvival time. A preliminary histological evaluation ofprimary lung tumours produced has been reported forapproximately two thirds of the rats in this study. The authors
noted an apparently antagonistic interaction between the two
agents in producing lung tumours; for example, crude tumour
incidences were 10.8% in rats receiving 11.5 Gy x-irradiation
alone, 9.2% in rats receiving a 170 Bq lung burden of 239PuO2
alone, but only 11.7% in rats receiving a combined exposure
at these levels [L20]. The authors cautioned, however, that asimple evaluation of the crude tumour incidence is insufficientbecause of the effect of exposure on lifespan. They further
state that analysis of this study is not yet complete.
10. An apparent synergism was described in an in vitro
study of the combined effect of alpha particles and x rays
on cell killing and micronucleus induction in rat lungepithelial cells (LEC) [B39]. The cells were grown on
Mylar films and exposed to both x rays and alpha particles,
separately or simultaneously. X rays and alpha particles
given separately caused dose-related increases in cell cycle
time, with alpha particles producing greater mitotic delay
than x rays. Damage from alpha particles and x rays given
simultaneously did not interact to further alter the cell
cycle. Cell survival data following exposure to x rays andalpha particles, combined or individually, were fitted bylinear-quadratic models. Survival curves following
exposure to alpha particles only, or to 1 Gy from alphaparticles plus graded x-ray doses, were adequately
described using only the linear (alpha) terms with valuesof the coefficients of 0.9±0.04 and 1.03±0.18 Gy1,
respectively. Survival following exposure to x rays only or
to 0.06 Gy from alpha particles combined with x rays was

220

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

best fitted using both alpha and beta terms (0.12±0.03)D +(0.007±0.002)D2, and (0.57±0.08)D + (0.3±0.02)D2,
respectively. The numbers of micronuclei in binucleatedcells produced by exposure to alpha particles or x rays
alone increased linearly with dose, with slopes of 0.48±
0.07 and 0.19±0.05 micronuclei per binucleated cell per Gy
for alpha particles and x rays, respectively. Simultaneous
exposure to graded levels of x rays and a constant alphadose of either 1.0 or 0.06 Gy increased micronuclei
frequency, with a slope of 0.74±0.05 or 0.58±0.04 micronuclei per binucleated cell and Gy, respectively. These
slopes are similar to that produced by alpha particles alone.
These studies demonstrated that both cell killing and theinduction of micronuclei were greater with combined
exposures than with separate exposures.
11. A refined model for the combined effects of mixtures ofionizing radiations was recently published by Lam [L4].
Assuming that ionizing radiation is a special group of toxic
agents whose general interaction can be calculated, the model
postulates the existence of a common intermediate lesion andthe relative action of lesions before, at, and after this common
stage. General quantitative dose-effect relationships of mixed
radiations can be derived from the dose-effect relationships ofthe components in the mixture. Again, only small deviations
from isoadditivity are predicted by this damage function,
which allows treating mixed irradiation as two different
increments of dose from the same radiation source.
12. A unifying concept to predict the expected combined
stochastic radiobiological effects of different ionizing
radiations was presented by Scott [S15]. Additive-damage
dose-effect models were developed for predicting theradiobiological effects of sequential and simultaneous
exposures. These additive-damage dose-effect models
assume that
(a)

(b)

each type of radiation in the combined exposure
produces initial damage, called critical damage, that
could lead to the radiobiological effect of interest; anddoses of different radiations that lead to the same levelof radiobiological effect (or risk) can be viewed as
producing the same amount of critical damage, whichis indistinguishable as far as the effects of subsequently
administered radiation are concerned.

The methodologies allow the use of known radiationspecific risk functions to derive risk functions for the
combined effects of different radiations, called global riskfunctions. For sequential exposures to different ionizing
radiations, the global risk functions derived depend on how
individual radiation doses are ordered. Global riskfunctions can also differ for sequential and simultaneous
exposures. The methodologies are used to account for somepreviouslyunexplained radiobiological effects of combined
exposures to high- and low-LET radiations. Since allradiation effects are traced to a common initial damage
mainly occurring in DNA, the model is basically additive.
13. At doses lower than those that induce deterministic
effects, no large deviations from additivity are found in theinteraction of different radiation qualities (see also Table A.1).
Although the mathematical modelling of mixed radiation
showing non-linear dose-response relationships with a single
radiation qualityyields apparent synergistic interactions when
the analysis of endpoints like survival is based on somecurrent definitions [Z2], these definitions are clearly
inappropriate for the approach used in this Annex. This pointis also made by Suzuki, who stressed the need for definitions
based on biological mechanisms [S3].
14. In summary, it can be stated that when dose rates andother possible confounders are taken into account, practicallyall the results from mixed radiation yielding more than thesum of the single agents can be explained by isoaddition, sothat general quantitative dose-response relationshipsfor mixed
radiations can be derived from the dose-response relationshipsof the components in the mixture [L4, L47]. There is no
indication that the influence of external radiation on the
biokinetics of radionuclides found at high doses is relevant at
occupational or environmental exposure levels.
2. Ultraviolet radiation
15. Ultraviolet (UV) radiation is recognized as animportant initiator and co-factor for human skin carcinogenesis. Genetic predisposition, i.e. skin type, age at
exposure, and duration of exposures are important determinants of risk for UV radiation-induced skin cancer.
Shore analysed 12 studies on the incidence of skin cancer
in irradiated populations with known skin doses [S29]. In
the absence of a proper control (skin exposed to ionizing
radiation but not to UV), it was concluded that at least for
combined exposures, there was no evidence of a dosethreshold for radiation-induced skin cancer. The data arecompatible with a linear dose-response relationship [S29].
The question whether relative risk or absolute risk models
are more appropriate remains open. Considerable variations in sensitivity to skin cancer induction among demographic and genetic subgroups may be mainly a reflectionof the large differences in UV exposures because oflifestyle, skin type, and tanning.
16. Combined exposure to UV and x rays leads tosynergistic interaction in killing mammalian cells [H53],
confirming previous studies in yeast [S87]. Only a smallinteraction was found for mutations at the hprt locus in
Chinese hamster cells [K56]. A recent study by Spitkovsky
et al. [S82] in human peripheral lymphocytes on theinteraction between x-ray doses of 5250 mGy and
20 J m2 of 254 nm UV light in DNA repair, measured byunscheduled DNA synthesis (UDS), indicated that the
repair of UV-induced damage was modulated by previous
x-ray exposures. For radiation alone, UDS was highest for2030 mGy and 150200 mGy and lowest at 100 mGy.
For combined exposures, i.e. ionizing radiation followed by
UV, UDS was highest in cells previously exposed to
100 mGy and lower than in UV-only controls for cells
previously exposed to 2030 or 150200 mGy. The
mechanism of this proposed adaptive response remains tobe elucidated.

Bacteria (E. Coli)
Rats

Gamma

UV radiation

Laser (633 nm)

Microwave field (200 µW cm-2)

Static field (58 mT)

Static field (10-400 mT)

Temperature

UltrasoundMineral fibres (intrapleurally)

Mineral fibres

Mineral fibres

Alkylating agents

Alpha, 239Pu

Low LETAlpha

X rays (5.5 Gy)

X raysGamma (3-9 Gy)

X raysX raysAlpha (radon,
6 000 WLM)

Alpha (radon)

Alpha (radon)

X rays (7.5 Gy)

Human

C3HT½ cells

Rats

Rats

C3HT½ cells

Transformation

Thoracic tumours

Lung carcinomas andmesotheliomas

Transformation

Cell killing, suppression of
proliferation

Survival

Growth and survival rate

Survival

Survival

Skin cancer

Osteosarcoma

Osteosarcoma

Cell killing and micronuclei
formation

Deterministic effect;change in hormone status

Deterministic effectPhysical agents

Endpoint studied

Supra-additive

Supra-additive

Supra-additive

None

Supra-additive

Subadditive

Supra-additive

Subadditive

Subadditive?

Supra-additive

Supra-additive

Supra-additive

Subadditive

Additive

Nature of effectSecondary nonlymphocytic leukaemia
after breast cancer therapy

Supra-additive

Chemical toxicants (genotoxic chemicals)

Human, mammals

Mice

Cell culture

HumansMale rats

Mice

Beta, 227Ac

Rat lung epithelial cells
in vitro

Rats

Alpha, 227Th

Gamma, whole body

Male mice

Study system

Gamma

I to thyroid

Gamma, whole body

Combining agent

Alpha

Beta,

131

Beta to skin

Radiation

Table A.1
Combined effects of ionizing radiation and other agents

Complex adaptive processes, important fortumour control

Field has to precede irradiation by 7-30 days forfull protection

Mechanism only found in bacteria

Meta-analysis of 12 epidemiological studies;
controls with ionizing radiation only were notavailable

Additive at low exposure

Comments

[C29]

[H11]

[B38]

[B38]

[H10]

[G6, M15,
Z3]

[S33]

[K16]

[G13, G16]

[V6]

[S29]

[E5]

[L26]

[B39]

[M13]

[R3]

Ref.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS
221

1,2-Diethylnitrosamine (DEN)

Paraquat(superoxide generating agent)

TPA

Sulfhydryl-carrying radioprotectors

alpha-Difloormethylornithine (DFMO)
antipromotor (1% in drinking water)

X raysX rays (3 Gy)

X raysX rays90

Sr/90Y (3 × 3 Gy
per week)
ICR mice

Male BALB/c and
C57Bl micesee Table A.3

C3HT½ cells

Survival, transformation

Stamen hair cell mutations

Reciprocal translocations

Skin tumour

Supra-additive

Supra-additive

Additive tosupra-additive

Supra-additive

Supra-additive

Supra-additive

Subadditive tosupra-additive

Supra-additive

Subadditive

Supra-additive
Supra-additive
Subadditive

Supra-additive

Supra-additive

Nature of effectSkin and bone tumours

Survival, tumour induction

Antagonism

Antagonism

Synergism

Chemical toxicants (non-genotoxic chemicals)

Mouse germ cells

Bleomycin (60 mg kg-1)

Gamma (1, 9 Gy)

Tradescantia

ICR female mice

4-Nitroquinoline 1-oxide

Sr/90Y(27 Gy, skin surface)

Liver carcinomas and foci

Mice

90

Liver foci

Rats

Diethylnitrosamine (DEN)(100 mg kg-1)

Thymic lymphomas

BDF1 mice

Neutrons (0.125-0.5 Gy)

Lymphomas

C57Bl/6N mice

Diethylnitrosamine (DEN)(100 mg kg-1)

Brain tumours

Rats

Gamma (0.75-3 Gy)

Gastrointestinal tumours
T-cell lymphomas
Neural tumours

Rats
Mice
Rats

Methyl, ethyl, butylnitrosourea(10 mg kg-1)

X rays (0.25-3 Gy)

Colon carcinogenesis

Rats

DMH, 1,2-dimethylhydrazine(0.15 g kg-1)

X rays (9 Gy)

Mutations in germ cells

Endpoint studied

Mice

Study system

Cyclophosphamide

Combining agent

X raysRadiation

Table A.1 (continued)

Tumour emergence delayed

Many systems and endpoints studied
with similar results

TPA not present in natural environment

Additive for sister chromatid exchangeInteraction of single-strand lesions, coveredby isoaddition

No tumours from single-agent exposure despite
acute effects

Effect mainly of increased foci appearance

Large sex differences, different histochemically characterized foci show
divergent response

Deterministic effect suggested by enhanced risk
in generating lympho-haemopoiesis
Killing of stem cells? (Radiation suppresses
schwannoma induction by ENU and also
spontaneous squamous-cell carcinomas)
Probably due to radiation-induced alkyltransferase
Effect traced to cellular kinetics and clonal
expansion
Lower radiation doses enhanced, higher doses
delayed leukaemogenesis

Interference of cyclophosphamide with DNA
repairComments[O12][M6, M7]

[G5]

[L13]

[D6]

[H39]

[M8]

[P26]

[S20]

[S21]

[S13]

[M32]
[S22]
[H6, K15]

[S27]

[E7]

Ref.

222
ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

Bromo-2'-deoxyuridine (BrdUrd)(3.2 mg per rat)

Nicorandil, inhibitor of free radical
production

Calyculin A, protein phosphataseinhibitor (2.5-10 mM)

Active smoking a

Active smoking b

Active smoking a

X rays (1.5 Gy)

X rays (4-10 Gy)

X rays (2-8 Gy)

Alpha (222Rn)

Alpha (222Rn)

Alpha (222Rn)

Mainstream smoke b

Mainstream smoke b

Diesel fumes b

Mainstream smoke a

Mainstream smoke a

Alpha (222Rn)

Alpha (222Rn)

Alpha (222Rn)

Alpha (239Pu)

Alpha (239Pu)

Active smoking

Active smoking b

Arsenic

X rays
(>9 Gy to target)

X raysAlpha (222Rn)b

Active smoking

Alpha ( Rn)

a

CCI4

Alpha (PuO2)

222

CCI4

Combining agent

Neutrons (1.7-3.3 Gy)

Radiation

Table A.1 (continued)

Humans (miners)

Humans (breast
cancer patients)

Humans (Hodgkin’s
disease patients)

F344 rats

Mice

Sprague-Dawley rats

Beagle dogs

Rats

Humans(thorotrast patients)

Humans (home)

Humans (miners)

Humans (miners)

BHK21 cells

Chinese hamster
V79 cells

Rats

Rats and hamsters

C57Bl6 mice

Study system

Lung cancer

MetalsLung cancer

Lung cancer

Lung lesions, lung cancer

Lung dose, histopathology

Lung cancer

Lung cancer

Lung cancer

Lug cancer

Lung cancer

Lung cancer

Lung cancer

TobaccoCell killing

Survival

Total tumour yield, latency

Modification of biokinetics

Liver carcinomas

Endpoint studied

None

Subadditive tosupra-additive

Supra-additive

Supra-additive

Supra-additive

None

Subadditive

Supra-additive

None

Supra-additive

Additive tosupra-additive

Supra-additive

Supra-additive

Subadditive

Supra-additive

Supra-additive

Nature of effect

Adjustment for arsenic exposure reduced theradon risk estimate (ERR WLM-1)

Possible bias in positive study

Clinical problem

Lung clearance reduced

Lung clearance reduced

Diesel fumes alone had no effectReduced dose to critical cells by thickeningof bronchial mucus?

Interaction only for radon

Proper lung dosimetry lacking, therefore little
statistical power

Little statistical power

More-than-multiplicative only for smoking
preceding radon

Confounders: other air pollutants; transfer tomodern workplaces and indoor exposure

Disruption of protein kinase mediated signal transduction?

Potential clinical problem

Results not yet known

No effect with chloroform

Comments[L18, T5]

[I9, I10,
N4]

[V7]

[F17]

[T2]

[M61]

[C21]

[C9]

[I5]

[P11]

[T18]

[C1, L18]

[N21]

[N20]

[A15]

[B16]

[B16]

Ref.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS
223

Mice

Arabidopsis thaliana

Cadmium (1 mg kg-1)

Lead (16-320 mg kg-1)

Gamma

Caffeine

Caffeine, theobromine, theophylline

N-methylformamide (0-170 mM)

Restricted food intake a

NaCl (1% in diet), ethanol (10%)

Tetrahydrocannabinol b

Vitamin A, E, selenium,
3-aminobenzamide a

Oestradiol-17 beta b

X raysX raysX raysX raysX rays (10 Gy)

Gamma rays (1.5-4 Gy)

Gamma rays (4 Gy)

Gamma, neutronsbCaffeine (1-7 mM)

X rays (0.24, 0.94 Gy)

Cs (-0.012 Gy)

137

-

Cs
( 2 500 Bq kg-)

Rats

Supra-additive

Supra-additive
Subadditive

Subadditive

Supra-additive
Supra-additive

Supra-additive

Subadditive tosupra-additive

Subadditive

Supra-additive
Supra-additive

Supra-additive
Supra-additive

Supra-additive
None

Nature of effectTransformation

Tumour incidence

Intestinal metaplasia

Tumour incidence

Subadditive

Supra-additive

Supra-additive

Subadditive

Mammary tumours

Questionable

Biological agents (hormones, viruses, other)

C3H10T½ cells

Rats

CD(SD):Crj rats

Animals

Survival

Apoptosis
Apoptosis

Oxygen radical
concentrations

Micronuclei induction
Embryonal development

Mitogens and cytotoxicants

Nitrogen fixation, dentrification, CO2 flux

Mutations

DNA damage in peripheral
lymphocytes

Cell-cycle delay
Chromosome aberrations

Lung dose
Lung tumours

Lung dose
Lung tumours

Endpoint studied

Antioxidants, vitamins and other dietary factors

Human colon tumourline

T lymphoma cells
TKG cells

In vitro chemistryPre-implantation
mouse embryos

Soil microbes

CHO cells

Beryllium (0.2, 1 mM)

X rays (1, 2 Gy)

Zinc (10-100 mg kg -1)
cadmium (0.5-16 mg kg-1)

F344 rats

Beryllium (50-450 µg lung burden)Alpha(60-170 Bq 239Pu)

134, 137

Rats

Study system

Beryllium (1-91 µg lung burden)

Combining agent

Alpha(150-6 700 Bq 239Pu)

Radiation

Table A.1 (continued)

High hormone levels applied

Epidemiological studies indicate importanceof dietary form

Breast adenocarcinoma increased by a factor 5

No effect on incidence of gastric tumours

Probably via reduced growth stimuli

Effect on alpha term, i.e. stronger
at lower exposures

Apparent threshold below 1 mM

Catalase activity reduced

Cadmium exposure has to precede radiation
by several hours

Multiplicative interaction probably limited
to S and G2 cells

Lung clearance reduced

Lung clearance reduced

Comments[B32][B24, H11]

[M24]

[W29]

[C26]

[L15]

[Z4]

[K29]

[M11]

[E19, E20]

[K42]

[P27]

[B37]

[F12]

[S42]

Ref.

224
ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

ab

Colon cancer

C57Bl mice

Mammals
Athymic nude andeuthymic mice

BALB/c mice

Testosterone and DES dimethylestradiol (0.2-2.5 mg implant) b

Retrovirus T1223/B a

Microbial substances a

Urethane a

Dimethylhydrazine-induced
inflammation of bowel a

X rays (2 × 10 Gy)

X raysX raysGamma rays (1-4 Gy)

X raysRelative for low doses.
Uncertain whether relative for low doses.

Radiation enhancement ofurethane-induced lungtumours

Normal and gonadectomized CD(SD);Cjr rats

Castration ± testosterone b

Beta rays (1.5 MBq 131I)
Male rates

Rats

DES pellet (release 1 µg d-1) a

Gamma rays (2.6 Gy)

Survival

Leukaemogenesis

Intestinal metaplasia

Thyroid neoplasms

Mammary tumours

Mammary tumours

Rats

Diethylstilbestrol (DES)(12.5 mg kg-1) a

Mammary tumours

Endpoint studied

Neutrons (0.064 Gy)

Rats

Study system

Prolactin a

Combining agent

Neutrons

Radiation

Table A.1 (continued)

Additive

Additive

Subadditive

Supra-additive

Subadditive
Supra-additive

Supra-additive

Supra-additive

Supra-additive

Supra-additive

Nature of effectImmunosurveillance by T cells not importantin this system

Stimulation of immune system?

Recombinant viral DNA found in the genomeof every tumour

DES in females
Testosterone in females; DES in males

Irradiation in late pregnancy or duringlate lactation clearly more effective

Increased time span between radiation andhormone did not reduce interaction

Comments

[W2]

[K41]

[A12]

[A25]

[W28]

[H12]

[I11, S40]

[S24]

[Y6]

Ref.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS
225

226

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

17. In the early study of molecular genetics, a wealth of data
were accumulated on interactions between UV and ionizing
radiation in bacterial systems. For a review, see Annex L,“Biological effects of radiation in combination with otherphysical, chemical, and biological agents”, in the UNSCEAR
1982 Report [U6]. Recently, laser applications have become
important in industrial settings. A sparing effect from visible
light on irradiated bacteria was reported by Voskanian et al.
[V6]. The study measured the combined effect of laser(helium-neon laser, 633 nm) and alpha radiation on thesurvival of Escherichia coli K-12 cells of different genotypes.
Pre- and post-irradiation exposures to laser radiation
diminished the damaging effect of alpha particles. The
increase in survival was more pronounced for post-irradiation
exposure. There is a well-known molecular basis for enhanced
DNA repair and hence for survival: photoreactivation withvisible light after UV irradiation. The protective mechanism
involved in the repair of damage from alpha irradiation,
especially the one involved in pre-irradiation exposure to laser
light, remains to be elucidated. However, at this stage there
are no such mechanisms known in mammalian cells. Despitelarge human populations with considerable combined
exposures to ionizing and UV radiation to parts of the skin, no
indications of a critical interaction are apparent.
3. Low- and high-frequency electromagnetic
radiation
18. The photon energies of all frequencies of electromagnetic radiation below infrared are clearly too low to
produce direct chemical damage to DNA. However, there
is a large body of published data suggesting the presence ofeffects at exposure levels below those from critical thermal
effects, i.e. local heating by several degrees Celsius. (Heatstress is discussed in Section A.4.) The epigenetic influencesof heat stress could only act on later stages of cancer
development. Whether so-called athermic levels of highfrequency non-ionizing radiation may interfere with cellsignalling, levels of cellular calcium, or systemic melatoninremains disputed on the level of the single agent.
19. Tyndall undertook an investigation [T3] to ascertain
the combined effects of magnetic resonance imaging fields
and x-irradiation on the developing eye in mice from thestrain C57Bl/6J. Dams in groups were subjected toabsorbed doses of 50, 150, and 300 mGy. Other dams were
exposed to T2 spin-echo magnetic resonance imaging
fields under clinically realistic conditions following
exposure to 300 mGy from x-irradiation. It was found thatthe 300 mGy dose had significant teratogenic effects on theeye of C57BL/6J mice. Groups exposed to both types ofradiation fields demonstrated malformation levels similar
to those in animals irradiated only with 300 mGy fromionizing radiation. The results confirmed the teratogenic
effects of low-level x rays but gave no evidence for anenhancement of the teratogenicity of x-irradiation on eye
malformations in the mouse system tested.
20. Somewhat unexpected results of combined effect of
microwave exposures of non-thermal intensityand ionizing

radiation were reported in rats and chicken embryos by
Grigor’ev et al. [G13, G16]. Rats were pre-exposed toelectromagnetic radiation of power flux density (PFD)
200 µW cm2 30 minutes daily for 8 days, followed the next
day by single whole-body gamma irradiation at 5.5 Gy. Preexposure to microwave radiation reduced the mortality rate of
the test animals by33% compared with the controls. Immunobiological examinations revealed a significant increase in thestimulation index in mitogen (phytohemagglutinin, PHA)
induced lymphocytes. The imprinting of chicks was disrupted
when they were irradiated in early embryogenesis for 5
minutes with microwaves (PFD = 40 µW cm2) and then withgamma rays at a dose of 0.36 Gy.
21. The same group also described changes in humoral
immunity and in autoimmune processes under the combined
action of microwave, infrasonic, and gamma irradiation [G13,G16]. The exposure regimens for rats and rabbits were
9.3 GHz and 0.1 GHz (200 and 1,530 µW cm2, respectively),
infrasound (8 Hz, 115 db), and gamma radiation (cumulative
dose of 5.5 Gy). It was shown that pre-irradiation withmicrowaves increased the resistance of the animal to gamma
radiation, but microwaves combined with infrasoundenhanced the biological effect of gamma radiation. Since nohypotheses on possible mechanisms are suggested, noinferences applicable to controlled human environments canbe drawn at this stage from the extremelyhigh exposure levelsin this study.
22. A very strong radioprotective effect of static magnetic
fields of 10, 120, and 350 mT on the survival of mice (CBA× C57Bl/6) after acute 60Co irradiation with a dose of 9 Gy
was described by Schein [S33]. The adaptive effect of an
exposure of 6 hours in a static field increased with time andwas strongest in animals irradiated 30 days later. The weakest
field, 10 mT, led to a survival of up to 60% of the animals,
whereas controls had survival rates of only 0%4%. The
mechanisms behind this antagonism are speculated to beunspecific stress-induced stimulation of endocrine systems bymagnetic fields, an increase in surviving stem cells after
ionizing radiation, or a faster proliferation and differentiationof bone marrow stem cells in adapted animals.
23. Growth and survival rates of cultured cells (FM3A)
were investigated in a static gradient magnetic field witha strength of 58 mT at the center and a mean gradient of
0.6 T m1 [K16]. The magnetic field alone reduced thegrowth rate by 5% and survival by 20%. The combined
effect of 60Co irradiation followed by exposure to themagnetic field showed synergism.
24. Magnetic fields have been shown under certain
reaction conditions to perturb the rates at which radical
pairs recombine. An example is catalase-catalysed decomposition of H2O2, which is increased by 20% in an
extremely high magnetic field of 0.8 T [M35]. In theory,
this could lead to changes in the kinetics of free-radical
production and recombination [S60]. To measure theinteraction potential of this indirect genotoxic effect ofmagnetic fields with ionizing radiation, the exposures

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

would have to be simultaneous and not sequential, as
described in the preceding paragraphs.
25. In assessing the association between exposure toelectromagnetic fields and cancer, Koifman [K17] defined
the elements necessary for quantitative analysis. Obtaining
more accurate measurements of exposure to electromagnetic fields is a key to understanding any possible
association. In certain circumstances, strong electromagnetic fields may stimulate growth and hence fulfill thecharacteristics of a cancer-promoter in biomechanistic
models of carcinogenesis. This leads to the hypothesis thatelectromagnetic fields do not act alone to affect health, as
is assumed in many epidemiological studies, but only
where their action is combined with that of other initiator
agents.
26. In summary, no straightforward inferences fromexperimental results to exposures in occupational settings
are possible at this stage for the combination of electromagnetic and ionizing radiation. From the standpoint of
mechanistic considerations, there is little evidence forpotentially harmful interactions between the two radiation
modes for controlled exposure levels in the workplace or in
the clinic.
4. Temperature
27. Heat kills mammalian cells in a predictable andstochastic way [D3]. Heat stress at the cell and tissue levelmay disrupt energy metabolism (local depletion of oxygen
and ATP) as a result of the enhanced reactivity of mostenzymes, the production of heat shock proteins, and finallydenaturation and cell death. Critical changes leading to aloss of proliferative capacity involve cell membrane
blebbing, probablyowing to detachment of the cytoskeleton
from the plasma membrane [R23]. A slow mode of cellkilling by hyperthermia in CHO cells involves the formation of multi-nucleated cells from damage to centrioles[D3]. Above 42.5°C, cell-survival curves for Chinese
hamster ovary cells in culture where the abscissa is theduration of heat treatment are similar to the curves for
x rays. At 42°C and below, the survival curves tend to
flatten out with time as tolerance to the elevatedtemperature develops. The cell-cycle dependence ofsensitivity to heat contrasts with that of x rays, with lateS-phase cells being the most sensitive to hyperthermia
treatment. Cells at low pH or deficient in nutrients also
show elevated heat sensitivity. Temperature is therefore an
important modifier of radiation sensitivity in many therapies
to control tumour growth. In general, hyperthermia increases
the relative susceptibility of tumour cells to radiationcompared with healthy tissue. Very hot or very cold
ambient temperatures are rarely encountered in the modern
workplace and the temperatures that do prevail generally
do not change the body core temperature. No correlation
with elevated radiation exposure is apparent in such
workplace settings. The same is true for recreationalsettings and even for hot spas with elevated radon levels.
Therefore the combined action of high and low tempera-

227

tures remains in the realm of clinical research, and thefollowing paragraphs give only some cursory remarks onrecent in vitro work.
28. At the mechanistic level, it is important to note that thelarge effects found in hyperthermia treatments cannot be
attributed solely to changes in blood flow and concomitantchanges in local oxygen pressure alone. The disruption ofenergy metabolism due to considerably accelerated biochemical reactions and a decrease in molecular stability areimportant far below the threshold of protein denaturation.
Dauncey and Buttle [D2] found a tendency towards elevatedplasma concentrations of growth hormone and prolactin in
14-week-old pigs acclimated to 35° or 10°C, respectively. In
mammalian cell culture (L5178Y), protease inhibitors such as
phenylmethylsulfonyl fluoride were shown to potentiate
hyperthermic cell killing [Z13]. It is suggested that proteaseinhibitors sensitize by inhibiting the proteases that are needed
to degrade denatured proteins induced by heat. In response toheat, cells and tissue produce proteins of mainly 70 and 90
kilodaltons. These proteins are called heat-shock proteins,
although many other agents such as arsenite and ethanol also
induce them. Their appearance coincides with the development of thermotolerance, an important effect that can influence the slope of the survival curve by a factor of up to 10.
The development of thermotolerance and the production ofheat-shock proteins occur during heating at temperatures up
to 42°C (CHO cells) but are delayed by several hours for heat
treatment with higher temperatures [H36].
29. Skin is the only tissue whose temperature might differ
considerably from the core temperature. Therefore, Zölzer et
al. [Z3] studied the influence of radiation and/or hyperthermia on the proliferation of human melanoma cells in
vitro. DNA synthesis and content were both determined with
two-parameter flow cytometry. In controls, most of the
S-phase cells showed incorporation of BrUdR. The fraction ofquiescent S-phase cells increased after irradiation (up to 8 Gy
from x rays) and/or hyperthermia (up to 6 hours at 42°C or up
to 2 hours at 43°C). There was a clear dose dependence forradiation and hyperthermia alone or in combination. In
general, the combined effect seemed to be additive.
30. Combination effects of radiation and hyperthermia were
found, however, in several other in vitro cell systems.
Matsumoto et al. [M15] treated cultured human retinal
pigment epithelial cells by radiation, hyperthermia, or a
combination of the two. The effect on cell proliferation was
evaluated by counting the cell number and measuring theuptake of bromodeoxyuridine. x-irradiation with a dose of
1 Gy or 3 Gy was not effective in suppressing proliferation of
the retinal pigment epithelial cells. Similarly, heat treatment
at 42°C for 30 minutes did not suppress proliferation.
However, combining hyperthermia at 42°C for 30 minuteswith 3 Gy irradiation suppressed cellular growth of the retinal
pigment epithelial cells to 36% of the control, as estimated bycell counting, and to 48% by the bromodeoxyuridine uptake
assay. The effect of radiation combined with heat on threehuman prostatic carcinoma cell lines was investigated by
Kaver et al. [K6]. Cells were exposed to different radiation

228

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

doses followed by heat treatment at 43°C for 1 hour. Heat
treatment given 10 minutes after radiation significantly
reduced the survival rate of all the cell lines studied. The
combined effect of radiation and heat produced greater
cytotoxicity than predicted from the additive effects of the two
individual treatment modalities alone. Impairment of DNA
repair with elevated temperature is considered an importantmechanism [W36].
31. Growth, cell proliferation, and morphological alterations in vivo in mammary carcinomas of C57 mice exposed tox rays and hyperthermia were followed by George et al. [G6].
Radiation doses of 10, 20, or 30 Gy from x rays or heating to
43°C for 30 minutes preceded or not by exposure to 10 Gy
were studied. Tumour growth, cell proliferation kinetics,
induction of micronuclei, and morphological changes innecrosis and vascular density were simultaneously determined. These showed very complex adaptive responses.
Treatment with radiation and/or hyperthermia produced only
a delay in tumour growth of between 1 and 3.8 days. However, the effects of the treatments became more apparent when
the amounts of muscle and necrosis were deducted from theoriginally measured tumour volume. Radiation-induced G2
block of the cells was observed 12 hours after radiation alone.
After combined treatment, however, the G2 block was delayed
beyond 12 hours. Whereas the amount of necrosis wasmarkedly enhanced five days after treatment with 10 Gy plus
heat, as well as after 30 Gy, no changes in the density of small
blood vessels could be observed during this period. These
results clearly demonstrate that the apparent changes intumour volume after x rays and hyperthermia do not truly
reflect the response of the constituent cells and that there aremany other factors, for instance cell proliferation andmorphological alterations, that influence the combined effectsof radiation and hyperthermia.
32. Heat shock before, during, or immediately after
exposure to ionizing radiation can increase cell killing in
a supra-additive manner [B70]. The heat-shock treatment
was shown to inactivate the Ku auto-antigen binding toDNA, and this binding capacity of Ku was directly related
to the hyperthermic radiosensitizing effect. The Ku autoantigen is the regulatory subunit of the DNA-dependent
protein kinase and is directly involved in DNA doublestrand break repair and V(D)J recombination.
33. In general, it can be said that because of the hightemperatures and exposures needed to produce enhanced cellkilling in poorly oxygenated tissue, combined effects fromhyperthermia and ionizing radiation are not relevant outside
the realm of tumour therapy. Temperature in combination
with ionizing radiation can act synergisticallyon cell survival,
cell proliferation, and cytogenetic damage. However,temperatures higher than those found in the human body areneeded to cause these effects.
5. Ultrasound
34. Possible effects from ultrasound exposures alone or in
combination with ionizing radiation are of some concern
because ultrasound is so widely used in diagnostic procedures. Above a threshold level, ultrasound by itself may
induce cavitation, leading to mechanical damage to cellular
structures and to microlesions. Kuwabara et al. [K20]studied the effects of ionizing radiation and ultrasound at
exposure levels typical for diagnostic purposes on the
induction of chromosomal aberrations and sister chromatid
exchanges in peripheral lymphocytes. No statisticallysignificant increases in the frequencies of dicentric andring chromosomes or sister chromatid exchanges werediscovered after ultrasound exposure alone at the diagnostic level (Table A.2). Nor could elevated frequenciesof these phenomena be found following exposure toultrasound before or after ionizing radiation, comparedwith the frequencies found after the same dose of ionizing
radiation alone. However, simultaneous exposure to ultrasound and ionizing radiation seemed to induce a slightenhancement of sister chromatid exchanges, although nosignificant changes were noted in the yields of dicentric
and ring chromosomes.

Table A.2
Effects of combined exposures to ionizing radiation and ultrasound in peripheral human lymphocytes[K20]
Exposure
Dicentrics and rings

Sister chromatid exchanges

Radiation

UltrasoundNone (control)

None (control)

3 Gy

None
40 min (immediately following)
80 min (immediately following)
30 min (simultaneous)

0.61±0.08

0.52±0.07

7.92±0.54
6.31±0.53
7.00±0.47
9.80±0.91

None
30 min (simultaneous)

1.12±0.11
1.10±0.11

9.96±0.50

4 Gy

35. Continuous-wave ultrasound and neoplastic transformation was assayed in vitro by Harrison and BalcerKubiczek [H10] in C3H10T½ cells in suspension. An

6.64±0.40

initiation-promotion protocol for neoplastic transformationinduced by continuous-wave ultrasound was used. Cells
were insonated at 1.8 MHz for 40 minutes. Two ultrasonicANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

229

intensities were used: 1.3 and 2.6 W cm2 spatial average.
The first intensity was found to be non-cytotoxic; thesecond was above the threshold level for cavitation andresulted in immediate lysis of 20% of the cells (cavitationinduced cell killing), followed by the clonogenic survivalof 64% of the remaining cells. Ultrasound was delivered
alone or in combination with x rays (2 Gy, 240 kVp given
before ultrasound) and/or TPA (0.1 µg ml1 after irradiation). Under all treatment conditions, ultrasound had noeffect on transformation at the 95% confidence level. The
effects of high-energy shock waves, i.e. therapeutic levelsof ultrasound generated by a lithotripter in combination
with 137Cs gamma rays were shown to act additively orslightlysupra-additivelyin colony-forming assays and cellcycle analysis [F29]. Both pellets of single cells andmulticellular spheroids of the bladder cancer cell line RT4gave similar results.

mutagens but quite powerful inducers of human mesotheliomas and bronchial cancers. Fibre dimensions, fibre
durability, and surface characteristics are important properties
affecting their carcinogenicity. In the case of asbestos, there isclear evidence for the induction of chromosomal aberrations
and aneuploidy [B13]. A possible mechanism of asbestos cell
toxicity is phagocytosis and accumulation of the fibres in theperinuclear region of cells. During mitosis, the fibres would
then interfere with chromosome segregation, and chromosomal abnormalities would result. In addition, mechanicalirritation and cell killing may lead to growth stimulation andtranscellular epigenetic promotion. The production of active
oxygen species on fibre surfaces was proposed as a directly
acting genotoxic mechanism; however, the relatively long
diffusion length from the site of radical production outside thenucleus to the target structures argues against the importanceof this pathway.

36. In conclusion, it can be said that the ultrasoundintensities used for diagnostic purposes and ionizing radiation
did not interact to cause cytogenetic damage in treated cells.
However, sister chromatid exchanges were slightly increased
in one study. In vitro transformation rates caused by ionizing
radiation were not changed by ultrasound.

40. Recent reviews of mortality and cancer morbidity inasbestos worker cohorts with large cumulative exposures
showed an ERR for pleural mesothelioma of about 1 foreach fibre-year ml1 of air [A3]. For lung cancer, an ERR
from 0.0009 to 0.08 per fibre-year ml1 has been found[N9], which, in absolute terms, is considerably higher thanthe mesothelioma risk. The ratio of the number of mesotheliomas to the excess number of cases of lung cancer
ranges from 0.06 to 0.78.

6. Dust, asbestos, and other mineral fibres
37. The combination of radiation exposure and exposure todusts and fibres is quite common in important industrial
environments such as mining, metallurgical industries, andpower plants. Some dusts and fibres are pathogenic or
carcinogenic by themselves. Both experimental results frommammals and epidemiological evidence are available [B9,
B13, C22, K13, P1, P5]. In cases where the main biologicaleffect results from soluble toxicants that dissolve from thesurface of dust particles to interact with biological structures,the interaction is basically between radiation and a chemical,
which is dealt with in Section B of this Appendix.
38. Silica is often considered to be a co-carcinogen throughthe route of silicosis. Harlan and Costello [H9] studied 9,912
metal miners (369 silicotics and 9,543 non-silicotics) toinvestigate the association between silicosis and lung cancer
mortality. When lung cancer mortality in silicotics and nonsilicotics was compared, the age-adjusted rate ratio was 1.56
(95% CI: 0.912.68). Further adjustment for smoking yieldeda rate ratio of 1.96 (95% CI: 0.983.67), and the value for
employment in mines with low levels of radon was 2.59 (95%
CI: 1.444.68). The statistical power of the study was too
weak toquantifysingle contributions and interactions between
metal, radon, silica, and smoking. For high dust loads andconcomitant exposures to gamma radiation and radon inearlier times, there is indication for an increased lung cancer
risk (standardized mortality ratio = 2.5 with 20 years of
employment and hired before 1960) in the phosphate industry[B74].
39. The molecular mode of action of mineral fibres is quitedistinct from radiation and genotoxic chemicals interacting
directly with nuclear DNA. They are relatively ineffective as

41. Few epidemiological data exist describing potential
interactions between mineral fibres and radiation. In acase-control analysis of deaths from lung cancer among
persons employed at the Portsmouth Naval Shipyard at
Kittery, Maine, in the United States, elevated odds ratios
for exposures to ionizing radiation, asbestos, and welding
by-products were found in a first crude assessment. Further
analysis of data on radiation exposure, controlling for
exposures to asbestos and welding, found no evidence fora risk related to radiation exposure. The low cumulative
radiation doses and the absence of data on cigarettesmoking and socioeconomic status precluded an assessment of possible interactions among the three toxic agents[R1].
42. The synergistic effects of the combined exposures toasbestos and smoking in the causation of human lungcancer was one of the first examples of a supra-additive
interaction of importance for protection in the workplace[S19]. In most studies, very high risk ratios were observedin asbestos-exposed subjects who were heavy smokers. Theinteraction observed in most cases conforms more closely
to a multiplicative model than an additive one. Brown et al.
[B7] were able to show in organ cultures derived fromFischer F344 rats that the ability to metabolize
benzo(a)pyrene was significantly reduced after in vivo
exposure to crocidolite, thus suggesting possible
mechanisms leading to a departure from linearity. Work by
Fasske [F2] showed that after the combined instillation of
1 mg chrysotile and 0.5 mg benzo(a)pyrene, lung tumours
arose much earlier than after the instillation of only one of
the carcinogens.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

43. Regarding animal experimentation, Bignon et al.
[B38] inoculated radon-exposed Sprague-Dawley rats
intrapleurally with asbestos fibres, glass fibres, or quartz.
In rats given mineral materials, bronchopulmonary carcinomas and mixed carcinomas were observed, as well as
typical mesotheliomas and combined pulmonary pleuraltumours, whereas in rats inhaling radon alone, only
bronchopulmonary carcinomas occurred. A clear cocarcinogenic effect of the insult from the minerals wasestablished for malignant thoracic tumours. Significant
differences in survival time were found for exposures todifferent types of dust, depending on the additional tumour
types induced. The same group also studied whether
similar co-carcinogenic effects would take place overlonger distances, i.e. from subcutaneous injection of chrysotile fibres. Neither mesotheliomas nor evidence of cocarcinogenic effects were found in the animals treated withboth radon and asbestos fibres [M19]. Three groups ofanimals were used: 109 rats that inhaled radon only (dose= 1,600 working-level months [WLM]); 109 rats given a
subcutaneous injection in the sacrococcygeal region of
20 mg of chrysotile fibres after inhalation of radon
resulting in the same dose; and 105 rats injected with fibres
only. As already stated, no mesotheliomas occurred in anyof the three groups. The incidence of lung cancer was 55%
in the second group, 49% in the first, and 1% in the thirdgroup. Statistical analysis using the Pike model showedthat the carcinogenic insult was slightly higher in thesecond group than in the first group. Electron microscopy
analysis of fibre translocation from the injection site
showed that less than 1% of injected fibres migrated to theregional lymph nodes and only about 0.01% to the lungs.
After injection, the mean length of the fibres recovered inlung parenchyma increased with time, suggesting thatshort fibres are cleared by pulmonary macrophages,
whereas long fibres remain trapped in the alveolar walls.
Kushneva [K49] studied pathological processes in thelungs of white rats exposed intratrachealy to 50 mg offinely dispersed quartz dust and to 3 hours of 3 108 Bq m3
radon. Supra-additivity is clearly implied but only
described qualitatively.
44. To assess the possible co-carcinogenic effects ofmineral dust in radon-prone mines, five groups of 30
Sprague-Dawley rats received minerals typically found inmetal mines (nemalite; biotite, present in many granites;
iron pyrite; chlorite) by intratracheal instillations one
month after the end of a 1,000 WLM radon exposure. No
or only slight co-carcinogenic effects were found [M62]. In
earlier work with the same experimental system toinvestigate the effect of intrapleural injection of asbestos
fibres (chrysotile), glass fibres, and quartz on the yield ofradon-induced thoracic tumours, a clear promoting effectwas noted [B38].
45. Densely ionizing alpha particles, similar to thoseemitted by radon progeny, are highly effective in inducing
transformations in cell cultures such as CH310T½ cells.
The yield of foci from combined alpha/asbestos exposure
is clearly greater than would be predicted from the sum of

the effects found with single-agent exposures. Figure A.I
shows a clearly supra-additive interaction with asbestos
fibres [H11].
0.08
TRANSFORMANTS PER SURVIVING CELLS (%)

230

0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
Asbestos fibres

Alphaparticles

Expected
Observed Spontaneous
frequency
Asbestos fibres andalpha particles

Figure A.I. In vitro transformation of C3H10T½ cells
exposed to asbestos fibres and alpha particles alone
and in combination [H11].

46. In an experimental study, Donham et al. [D10]studied possible combined effects of asbestos ingestion andlocalized x-irradiation of the colon in rats based on the
hypothesis that the mucous produced by goblet cells that
normally coats the normal bowel surface protects against
tissue penetration by ingested asbestos. X-ray treatment
results in localized damage to the colonic mucosa andtheoretically disrupts the normal mucous coating, allowing
increased tissue penetration by the fibres. To study this,segments of the colons of laboratory rats were exposed to
x-irradiation. The animals were then divided into threegroups, which were fed a diet containing 10% chrysotile
asbestos, a diet containing 10% non-nutritive cellulose
fibre, or a standard laboratory diet. Autopsies and histopathology were performed on all animals that diedspontaneously and those that were killed at 350 days.
Various types of inflammatory and degenerative lesions
were commonly seen, but there was little difference infrequency between the diet groups. Five adenocarcinomas
and two sarcomas were seen in the fibre groups (threetumours in the asbestos group and four tumours in thecellulose group), but no tumours were seen in animals on
the standard diet. There was no significant difference intumour rates between the asbestos and cellulose groups,
nor was there a significant difference between the combined fibre groups and the standard diet group. Ingested
asbestos did not increase the risk of tumour development
and does not, therefore, seem to be co-carcinogenic or topromote tumours by disrupting the mucous coating.
47. In summary, it can be stated that mineral dust andfibres such as asbestos generallyact through non-genotoxic
mechanisms. These include mechanical irritation and cellkilling. However, chromosomal aberrations, especiallyaneuploidy, can be induced by interfering with the spindle
apparatus of mitotic cells. At exposure levels found inANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

workplaces until the early 1940s, there was a clearly supraadditive interaction between asbestos and tobacco smoke
exposure in the causation of lung cancers, with a concomitant
shift in the cancer spectrum from mesotheliomas to bronchopulmonary carcinomas. A similar supra-additive interaction
and shift in the cancer spectrum was observed in animals
exposed to both asbestos and radon. The much lower occupational exposures experienced today considerably decrease therisk for potential detrimental interactions between dust/fibres
and radiation. However, in view of the proven interaction
effects in humans, any stochastic and/or genotoxic effects ofthese agents merit further consideration.
7. Space flight
48. In space flight, which involves an extreme situationof controlled exposures, a multitude of stressors act in
combination on astronauts, the most important being
microgravity. Its biological and medical role has been
extensively reviewed [M71]. Microgravity effects may
occur at all levels of biological organization, and inprinciple can also lead to modifications of radiation action.
From an experimental point of view there are no clear-cut
results at the organ and tissue level. With simple organisms,
a synergistic action of microgravity and radiation has beenreported for teratogenic effects [B80]. Antipov et al. [A14]analysed structural and functional changes in the central
nervous system of experimental animals exposed to theisolated and combined effects of space flights. Theyevaluated the significance of ionizing and non-ionizing
radiation, hyperoxia, hypoxia, acceleration, vibration, andcombined effects of some of these factors for anatomic andphysiological changes in the rat brain. Neuronal functions
were found to be sensitive to ionizing radiation and hypoxia,
but these synapses were shown to be highly resistant toshort-term hyperoxia and electromagnetic radiation [A13].
Along with radiation, the investigated stressors had
additive, synergistic, and antagonistic effects on the central
nervous system. However, as significant effects and deviations from the sum of effects from exposure to isolated
stressors were always linked to high exposures andexposure rates, they have little relevance for exposure
situations on the ground.
49. In radiobiological experiments in space, a more-thanadditive interaction between microgravity and radiation
was reported in several cases (reviewed in [H47]). Insect
embryos in particular appear to be susceptible. Conflicting
results were reported for cellular systems. In human
lymphocytes that were exposed to 32P-irradiation in space,chromosomal aberrations were significantly increased
compared with ground controls [B18]. However, thefollow-up experiment by the same authors did not show
this interaction [B19]. More recently, experiments on theinteraction of space microgravity and DNA repair were
performed by Hornek et al. [H14]. Microgravity had nomeasurable effect on strand rejoining of x-ray-induced
DNA strand breaks in Escherichia coli (120 Gy) and in
human fibroblasts (5 and 10 Gy) or on the induction of
SOS reponse in E. coli (300 Gy). In yeast no microgravity

231

related effects on the repair of DNA double-strand breaks
were found both for cells irradiated previously on ground[P31] or during flight using a 63Ni beta source [P32].
Therefore, repair of radiation-induced DNA damage seems
not to be disturbed by microgravity, and other mechanisms
must be involved in the reported interaction between
radiation and space gravity.
50. At similarly high exposures, Vasin and Semenova[V3] showed synergistic effects for combined stress fromradiation and vibration or normobaric hyperoxia. A study
was made of the combined effect of normobaric hyperoxia
and vibration on the sensitivity of hybrid mice (CBA ×
C57Bl)F1 and F2(CBWA) to gamma radiation. Both single
and protracted (for five days, daily) vibration before
irradiation aggravated acute radiation sickness. Hyperoxia
also enhanced the development of the intestinal form ofradiation sickness. The combined effect of the two additional factors aggravated the intestinal syndrome of acuteradiation sickness. These deterministic effects have no
direct implication for present-day controlled exposure
situations. Nevertheless, the changes of many parametersthat are normally stable in experimental work on earth
make well-designed studies in space potentially importantin addressing the combined effects of physical agents.

B. RADIATION AND CHEMICALTOXICANTS
51. A multitude of natural and man-made chemicals withcancer-initiating and -promoting potential are present in
the human environment and may interact with radiation.
Classification based on their mode of action is often
difficult, but at least a crude separation can be made into
substances that mainly act by damaging DNA directly(genotoxic substances) and non-genotoxic substances[C50]. The former group includes chemicallyactive species(activation-independent chemicals) or species dependent
on biotransformation and their active metabolites(activation-dependent chemicals). The mode of action iseither direct, by forming covalent links with DNA, or
indirect, via radical attack of DNA. The latter group
comprises chemicals ranging from nonspecific irritants andcytotoxins to natural hormones and growth factors andtheir analogues that interact with the regulatory systems ofcells and organs. At this point, chemicals that protect
against ionizing radiation should also be mentioned. Many
endogenous and exogenous sulfhydryl-carrying molecules
as well as other radical-scavenging agents considerably
reduce the primary damage and hence the clinical effects
caused by radiation [M6, M7]. A wealth of experimental
data is available to describe the action of single chemical
agents, but the literature on interactions between thesesubstances and other agents is far more sketchy. It is
important to note that recent efforts to quantify tissue dosesof chemical toxicants and their metabolites showed thedecisive importance of interactions in activation anddeactivation/excretion processes. For example, an assessment of the toxicity of benzene and its metabolites was

232

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

shown to depend crucially on the presence of othertoxicants such as toluene, and this effect extended toconcentrations found in human exposures [M60].
1. Genotoxic chemicals
52. The large group of genotoxic chemicals may befurther subdivided on the basis of their need to be activatedby metabolism. Most chemicals require metabolic activation through the generation of highlyreactive electrophiles,
which form DNA adducts by binding covalently to nucleic
acids. The metabolism of any individual chemical can bevery complex, because the chemical can be the substrate ofseveral metabolizing enzymes. Genotoxic chemicals canalso be subdivided based on whether the reactive compound acts directly by covalent binding to DNA orindirectly by the generation of free radicals. In the latter
case, effects similar to those of radiation can be envisaged.
(a) Activation-independent alkylating agents
53. Modern cancer therapy involves many combined
treatments using radiation and genotoxic drugs. Although
exposures are well known and strong interactions exist,
this human experience is of limited direct importance forrisk assessment at low doses, because with therapy, cellkilling is the main endpoint envisaged. Therefore thissubject is considered separately in Section D of thisAppendix. The occurrence of second primary tumours inhealthy tissue adjacent to treated tumours is of great direct
relevance.
54. Morishita et al. [M32] examined the effects of x rays
on N-methyl-N-nitrosourea (MNU)-induced multi-organ
carcinogenesis in both sexes of ACI rats. Rats were treatedwith MNU (25 or 50 mg kg1) at 6 weeks of age and/orwith x rays (3 Gy) at 10 weeks of age. The incidence ofadenocarcinomas in the small and large intestines of malerats treated with 50 mg kg1 MNU and x-irradiation (small
intestine, 48%; large intestine, 32%) was significantly
higher than the sum of the incidences resulting from50 mg kg1 MNU alone (small intestine, 17%; large
intestine, 8%) and with radiation only(small intestine, 0%;large intestine, 0%) and also higher than the frequency of
adenocarcinomas in the large intestine of males treatedwith 25 mg kg1 MNU alone (0%). Strongly synergistic
effects in these high-exposure studies were restricted to thegastrointestinal system. When MNU or 1,2-dimethylhydrazine (DMH) treatment was started two months after
x-irradiation, no induction of gastric tumours was observedwith MNU [W3], and only a low incidence was observedwith DMH [A7]. Surprisingly, an inverse relationshipbetween incidences of gastric tumours and intestinal
metaplasias was apparent. These findings again indicate
the importance of the order and timing of the exposures in
the induction of combined effects. It comes as a further
surprise that the presence of intestinal metaplasia, longconsidered a basis for further malignant growth, does notexert a positive influence on the induction of gastricneoplasia by MNU in the rat.

55. Seidel [S22] studied the effects of radiation on
chemicallyinduced T-cell lymphomas (thymomas) in BDF1
mice. N-methyl-N-nitrosourea or butylnitrosourea (BNU)
were the main inducers, and x rays in various doseschedules were applied. The radiation was seen to shorten
the latency period between induction and lymphoma
emergence in protocols of 12 exposures of 0.25 Gy. Thiseffect was most pronounced compared with chemicallyinduced non-irradiated controls with a prolonged median
induction time as a result of a dose reduction of thechemical (median induction time 2736 weeks instead of
1618 weeks under optimal conditions using 50 mg kg1 of
MNU). Irradiation 25 weeks before administrating
40 mg kg1 of MNU also enhanced leukaemogenesis.
Again, mice with regenerating lymphohaemopoiesis after
lethal irradiation and bone marrow transplantation weremore sensitive to both chemicals than were the controls.
Combined effects from radiation and N-ethyl-Nnitrosourea (ENU) on neural tumours in Wistar rats werereported by Hasgekar et al. [H6]. The animals received
2 Gy whole-body irradiation, followed immediately by
10 mg kg1 of ENU on the day of birth. Of 33 rats given
ENU alone, 14 developed 22 tumours of the nervous
system, of which 15 (68%) were gliomas and 7 (32%) were
schwannomas. Of 34 rats given both irradiation and ENU,
12 were found to harbour 15 neural tumours, of which 14(93%) were gliomas and 1 (7.1%) was a schwannoma. The
pretreatment with irradiation seems to have resulted inselective suppression of schwannoma induction. Whether
this antagonistic relationship is a result of overkill orwhether it may be relevant for lower radiation doses
remains to be elucidated.
56. The combined effects of radiation and BNU on
murine T-cell leukaemogenesis was studied by Seidel andBischof [S20] in BDF1 mice. The animals were exposed to
BNU (0.02% in drinking water) for 12 weeks, and they
died of thymic lymphomas with median latency periods of
1220 weeks. Groups of mice received weekly radiation
doses of 0.061.0 Gy in addition to BNU. Lower doses (12× 0.25 Gy) enhanced leukaemogenesis, high doses (12 ×0.75 Gy) delayed it, and intermediate doses (12 × 0.50 Gy)
had no effect. Doses lower than 12 × 0.25 Gy had marginalenhancing effects. After a dose of 12 × 1.0 Gy, the micedied earlier than after treatment with BNU alone, and aswith the dose of 12 × 0.75 Gy, some extrathymic lymphomas were observed. The numbers of CFU-S in the femur
and the spleen showed a dose-dependent depression, in
addition to the decrease from BNU alone. In lymphocyte
stimulation assays with Con A and LPS and also in themixed lymphocyte reaction, a reduced proliferation was
found, again dependent on the radiation dose. Thus, there
was an inverse correlation between leukaemogenesis andthe degree of stem-cell reduction or depression of these
immune parameters.
57. Stammberger et al. [S13] analysed the activity ofO6-alkylguanine-DNA alkyltransferase (AT) in the fetal
brain and liver and made long-term observations of Wistar
rats that were treated in utero either with x-irradiation

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS(1 or 2 Gy), with ENU (50 mg kg1), or with both in combination. They hoped to reveal any relationship between
the O6-alkylguanine repair capabilityand tumour incidence
in the organs of the offspring. The AT activity in the brain
was affected to the same extent in the fetuses as in thedams. There was a 61% decrease in AT activity in fetuses
24 hours after ENU treatment. This correlated with asignificant increase in the incidence of brain tumours in
the treated offspring (44%) compared with control animals.
The inductive effects of x-irradiation on AT activity (131%for 1 Gy and 202% for 2 Gy) corresponded with a reduction in the incidence of tumours after the combined treatment (27% and 8.3% tumour incidence, 103% and 158%
AT activity). Comparing biochemical and morphologicalresults suggests that this antagonistic effect may be theresult of the AT induction by x rays.
58. Yokoro et al. [Y6] found that whole-body irradiation
facilitates chemically initiated T-cell lymphomagenesis inmice. This was attributed to the amplification of the cell
population susceptible to a chemical carcinogen in thetarget tissues, bone marrow, and thymus during therecovery phase after irradiation. Split administration of
ENU showed different effects in the different phases ofcarcinogenesis leading to T-cell lymphomas. Once more
the authors emphasized that after a cell has been initiatedby a genotoxic agent, its fate is determined by the presenceof promoters and inhibitors and that modifiers of target
cells play a crucial role in the induction yield of tumours.
The possibility of synergistic effects in carcinogenesis due
to changes in cellular kinetics brought about by combined
treatment with radiation and ENU was studied by Seyama
et al. [S21]. Lymphomas in female C57Bl/6N mice wereused as a model system. A single intragastric administrationof 5 mg (about 200 mg kg1 body weight) of ENU was only
slightlylymphomagenic, inducing thymic lymphomas in 20%
of mice; the incidence was elevated to 92% if the ENU
treatment was preceded (five days earlier) by 4 Gy fromwhole-body x-irradiation, which alone is seldom lymphomagenic. A high yield of lymphoma (84%93%) was also
obtained when 5 mg (about 200 mg kg1) of ENU wasdelivered in two split doses four days apart of 4 mg and
1 mg (160 and 40 mg kg1), indicating that cellularkinetics or clonal expansion, but not two agent-specificdifferent initiation events in the combined treatment, is at
the root of this apparent synergism. Drastic injury to boththe thymus and bone marrow caused by either 4 Gy wholebody x-irradiation or the first dose of ENU (4 mg, or about
160 mg kg1) was followed by a vigorous regeneration
within a few days. The maximum induction rate oflymphoma was obtained when the subsequent dose of ENU(1 mg, or 40 mg kg1) was given at the peak of DNA
synthesis in the bone marrow and thymus following thefirst treatment. The data indicate that the principal effectof irradiation or the first dose of ENU was to provide asusceptible cell population, and that a high yield of
lymphomas was brought about by the action of thesubsequent dose of ENU on a larger number of potentiallyradiation-modified target cells engaged in heightened DNA
synthesis.

233

59. A clear antagonistic effect of ENUand x-irradiation wasobserved by Knowles [K14, K15] for neurogenic tumours inneonatal rats. After neonatal injection of rats with 10 mg kg1of ENU, whole-body x-irradiation with 1.25 Gy caused a
reduction in induced neurogenic tumours, which was greatest
when radiation was given 1 day after ENU and progressively
decreased with irradiation at 5 and 30 days. Although xirradiation did not affect the range of histological appearances in the tumours, malignant schwannomas, particularly
those of the trigeminal nerve, were significantly reduced by
1.25 Gy given after ENU (10 mg kg1). The mean latency forclinical signs of tumour appearance was not affected byradiation. Another important finding in this study also points
to the importance of the size of stem cell pools ininteractions: a significant reduction in the high spontaneous
incidence of squamous-cell carcinomas of the mouth in theinbred strain used after 1.25 Gy from x-irradiation. The
reduction was greater after irradiation at 5 days of age than at
30 days. A large study on the incidence rates of neural,
pituitary, and mammary tumours in Sprague-Dawley rats
treated with x-irradiation and ENU during the earlypost-natal
period was undertaken by Mandybur et al. [M2]. These lateeffects of early post-natal treatment with ENU, preceded by xirradiation to the head, were studied in 226 neonatal CD rats.
The animals were divided into six groups, each receiving oneof the following treatments: x-irradiation with 5 Gy to thehead on the third post-natal day; ip injection with 30 mg kg1
ENU on the fourth post-natal day; ip injection with 30
mg kg1 ENU on the seventh post-natal day; a combination of
x-irradiation with 5 Gy to the head on the third post-natal day,followed by ip 30 mg kg1 ENU on the fourth post-natal day;a combination of x-irradiation of 5 Gy to the head on the third
post-natal day, followed by ip 30 mg kg1 ENU on the seventh
post-natal day; and untreated controls. The results indicatedthat (a) x-irradiation to the head alone significantly extended
the lifespan of females compared with that of control females
and did not affect the survival of males; (b) x-irradiation did
not influence the latency period or mortality from neurogenic
tumours when ENU was given 1 or 3 days afterwards; (c)ENU itself was a factor in shortening latency periods for
mammary tumours; (d) x-irradiation alone did not increase
the incidence of mammarytumours and revealed noprotective
effect on the ENU-induced mammary carcinogenesis; (e) xirradiation increased the prevalence ofpituitarytumours in thefemales; (f) no enhancement of pituitarytumours by ENU was
observed; and (g) there was a statistically significant
association of pituitary and mammary tumours in females.
Again, these widely divergent findings speak against thepossibility of simple concepts for the interaction of different
genotoxic agents.
60. Post-natal development and cancer patterns in NMRI
mice after combined treatment with ENU and x-irradiation
on different days of the fetal period were studied by Wiggenhauser and Schmahl [W10]. When mice were irradiatedto 1 Gy on day 14, 15, or 16 of gestation, this did not resultin an increased tumour frequency in the offspring until 12months. Mice treated with ENU (45 mg kg1) on day 15 ofgestation developed a significantly increased tumour
frequency in the lungs and liver and in the ovaries. After

234

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

combined treatment in the sequence x rays plus ENU withan interval of 4 hours, a significantly increased incidenceof animals with tumours was observed in the offspringtreated on gestation day 14 or 16. Moreover, the treatment
on day 16 exhibited the highest tumour frequency per
examined animal (5.7) of all treatment groups. Although
the result was due to a relatively uniform increase of all
tumour types, the frequency of liver tumours was mostmarked. In the reverse sequence (ENU plus x rays), thetotal tumour outcome was not significantly altered compared with the effects of ENU alone. However, detailed
analysis also showed a significant augmentation of theliver tumour frequency with treatment on day 15.
(b) Metabolism-dependent alkylating agents
61. Maisin et al. [M8] studied the effects of x rays alone
or combined with the initiator diethylnitrosamine (DEN)on liver cancer induction in infant C57Bl/Cnb mice. The
number of induced liver foci and carcinomas was found todepend essentially on the dose of DEN. X rays did not
produce any combined effect on the induction of foci or
carcinomas when given seven days before or after administration of DEN [M34]. Using the same system for
exposures to DEN and neutrons (average energy =3.1 MeV), it was shown that even high-LET irradiation(0.1250.5 Gy) initiated only small numbers of nodular
lesions, whereas DEN alone increased liver nodules
significantly and proportional to dose (0.32.5 µg). A
supra-additive interaction between the two initiating agentswas found mainly in the increased rate of foci appearance
after 1.25 µg of DEN and 0.125 Gy of neutrons, both given
seven days before or after DEN exposure [M33]. Peraino etal. [P26] studied three altered hepatocyte foci (elevatedgamma-glutamyl transpeptidase [GG+] and/or ironexclusion [Fe]) in Sprague-Dawley rats exposed to DEN(0.15 µmol g1) and/or gamma rays (0.75, 1.5, and 3 Gy)
shortly after birth. The exposure was followed by a phenobarbital (0.05%) diet to promote focus expression. Radiation
alone was a weak hepatocarcinogen. A strong synergism
was seen at the lower radiation doses for the induction of[GG+] foci but not for other focus phenotypes. A
qualitatively different type of genetic damage for DEN(point mutations) and for radiation (rearrangements) is
postulated from the result. Large sex differences in theyield of DEN-induced [GG+/Fe] foci by a factor of up to
10 are additional indicators of the complexity of thissystem.
62. The potential for pulmonary carcinogenic interactions
between 239PuO2 and the tobacco-specific nitrosamine 4-(Nmethyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a
genotoxic lung carcinogen, was studied in 740 male rats
[L17]. The animals received 239PuO2 by inhalation to result inlung burdens of 0 or 470 Bq. The NNK was administered bymultiple ip injection at doses of 0, 0.3, 1.0, or 50 mg kg1. The
highest dose of NNK markedly reduced the median lifespanof the rats, whereas in the other treatment groups survival was
minimally reduced in comparison with the controls. Resultson carcinogenicity are not yet available from this study.

63. An apparent synergism between low-LET ionizing
radiation and the carcinogen 1,2-dimethylhydrazine (DMH)in the induction of colonic tumours in rats has been
described by Sharp and Crouse [S27]. They evaluated theinteraction of radiation (9 Gy to the abdomen only) andDMH (150 mg kg1) with respect to colon carcinogenesis
in male Fischer 344 rats. Radiation was administered 3.5
days before the DMH. At eight months post-treatment, theincidence of DMH-induced colon tumours was doubled byprior radiation exposure. When the protocol of radiation
plus DMH was repeated three times at monthly intervals,
a 15-fold increase in tumour incidence (from 5% to 74%)
was observed at six months post-treatment. This finding
demonstrated an apparent synergy between radiation andthe chemical carcinogen. Throughout the study, theappearance of carcinomas was associated with pre-existingcolonic lymphoid nodules. The reproducibility of tumour
induction as well as the range of tumour incidence generated by treatment variations in this system appeared to besensitive enough to allow the examination of combined
effects of much lower doses of radiation and/or chemical
carcinogens. The model could be used to evaluate therelationship between existing lymphoid aggregates, whichalter local epithelial cell kinetics and are associated withfenestrations in the basement membrane. The quantification of the development of colon cancer in congruent
sites may assist in defining dose-response curves for combined agents and may also provide a system for evaluating
the mechanisms underlying their interactions. When DMH
treatment was started two months after x-irradiation, onlya slight increase in gastric tumour incidence was recorded[A7]. These tumours occurred on top of a background ofradiation-induced gastrointestinal metaplasia.
64. Ehling and Neuhäuser-Klaus [E7] studied theinduction of specific-locus and dominant-lethal mutations
by combined cyclophosphamide (see also Section D.1.a foruses in combined modalities in tumour therapy) and radiation treatment in male mice. Unlike radiation, this widelyused antineoplastic agent, used alone, induced recessive
mutations in spermatozoa and spermatids but not inspermatocytes and spermatogonia. Pretreatment (with60 mg kg1) 24 hours before radiation, however, enhanced
the frequencyof specific-locus mutations in spermatogonia.
The mutational spectrum among seven loci remained thesame as in animals treated only with radiation. The
synergistic interaction was mechanistically explained by
the interference of cyclophosphamide, a strong inhibitor of
DNA and RNA synthesis, with repair of radiation-induced
damage.
65. The effect of radiation on chemical hepatocarcinogenesis has also been examined in male ACI/N rats [M26].
The number of neoplastic nodules or hepatocellular carcinomas in rats given N 2-fluorenylacetamide (FAA) (0.02%
in diet for 16 weeks) followed by x-irradiation (3 Gy) was
significantly greater than in rats given FAA alone (p <0.001). In addition, the incidence of hepatocellular carcinomas in rats given the combined treatment was also higher
than in rats given FAA alone (p < 0.003). No liver lesions

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

were found in animals receiving only an x-ray dose of
3 Gy. The authors suggested that these highly supraadditive results indicate that ionizing radiation acts as apromoter in this model.
66. An inhibition of urethane(ethyl carbamate)-induced
pulmonaryadenomas byinhaled 239Pu in random-bred maleA2G mice was reported as far back as 1973 by Brightwelland Heppleston [B66, B67]. This early study of combined
exposure to alpha radiation and a genotoxic chemicalcomprised four groups, each of 32 animals, receiving
plutonium inhalation followed by urethane (PU), plutonium followed by saline (PS), mock inhalation followed byurethane (MU), and mock inhalation followed by saline(MS). Exposures consisted of initial lung burdens of
925 Bq 239Pu and ip urethane injections of 1 mg g1 bodyweight two weeks later. Eight weeks after the injections,
PS-treated animals showed no increase in pulmonary
tumours over control animals (MS), whereas practically all
animals in the PU and MU groups had multiple tumours.
The number of tumours per animal 8, 16, and 24 weeks
after urethane treatment was clearly lower in the PU group,
which had 4.2, 11.4, and 13 as compared with 8, 24.4, and
38 in the MU group. An earlier hypothesis, that thisfinding is the result of alpha irradiation counteracting
immuno-suppression by urethane, is rejected on the basisof ultrastructural evidence. Severe morphological changes
in mouse type-II cells in the vicinity of alpha particles
indicate that functional impairment of the initiated cells is
the main cause of the effect. The authors said, however,that this apparent antagonism needs to be viewed withcaution; it remains to be determined, they concluded, if
much smaller local plutonium doses would augment
urethane tumorigenesis.
67. The transgenerational combined effects of x rays
(2.2 Gy) and urethane were studied by Nomura [N23, N24]in three different mice strains (ICR, LZ, and N5). Urethane
treatment of F1 offspring of either irradiated males or females
yielded an 18% incidence of tumour nodule clusters in thelung compared with only 2.8% in offspring of non-irradiatedcontrols. Tumour clusters were defined as having 12 or morenodules. The transgenerational effect of radiation alone
resulted in lung tumours (at least one tumour nodule) in 7.5%of the animals, whereas the value in unexposed controls was
4.7%.
68. The interaction of gamma rays with urethane in lungtumorigenesis in mice in relation to the immune status has
been studied by Kobayashi et al. [K41]. Male athymic nude
mice (nu/nu) and their female heterozygous litter mates(nu/+) were treated with 14 Gy of 137Cs gamma rays and
0.5 mg g1 of urethane. Gamma-ray exposure alone caused
relatively few lung tumours (in up to 10% of animals);urethane alone caused tumours in 70%80%. The combined
effect was supra-additive. There was a tendency towards
higher yields in nu/+ mice, suggesting that impaired
immunosurveillance from T-cell deficiency does not increaselung tumorigenesis in this system. Since relativelyradiationresistant macrophages and natural killer cells had higher

235

activities in nu/nu mice, the authors concluded that the
influence of immunological status on tumorigenesis remainedunresolved.
69. A strong synergism was found by Hoshino andTanooka [H39] for skin tumours in beta-irradiated ICR
mice painted later with 4-nitroquinoline 1-oxide (4NQO);
27 Gy of 90Sr/90Y radiation or 20 applications of 5 mg ml1
4NQO in benzene to the skin alone did not produce any
skin tumours in groups of 50 mice. Radiation followed by
4NQO painting with an interval of 11408 days between
the two treatments resulted in an incidence of malignant
skin tumours (squamous-cell carcinomas and papillomas)
of up to 17%. There was no significant decrease of thesynergistic effect with increasing interval, the greatest
effect being seen with an interval of 234 days.
70. A notable finding indicating the considerable uncertainties and misinterpreting the results of experimental
animal studies was described by Little et al. [L55], who
studied the potential synergistic interactions between 210Po(185 Bq, resulting in a lifetime lung dose of about 3 Gy)and benzo[a]pyrene (0.3 mg) in the induction of lungcancer in Syrian golden hamsters. It was shown that simultaneous administration by intratracheal instillation led toadditive effects. A significant apparent synergism wasfound when benzo(a)pyrene was given 4 months after the210
Po. Most of this effect could be ascribed, however, to apotentiating effect of the seemingly innocuous 0.9% NaClinstillation solution alone.
71. The effects of repeated low exposures at high dose
rates such as used in some diagnostic radiologic proceduresat the time of the study were published by Lurie and Cutler
in 1979 [L56]. The induction of lingual tumours by 7,12dimethylbenz[a]anthracene (DMBA) and radiation to thehead and neck was studied in Syrian golden hamsters.
Treatment schedules were topical application of 0.5%
DMBA in acetone on the lateral middle third of the tongue
three times a week for 15 consecutive weeks, about 200
mGy radiation exposures (x rays with 100 kV peak) of thehead and neck once a week for 15 consecutive weeks, or
concurrent radiation and DMBA treatments for 15 consecutive weeks. Histopathology was performed 35 weeks
after the start of the treatment. Animals receiving radiation
alone had no detectable changes. The combined treatment
led to an excess of lingual papillomas compared withanimals receiving only DMBA (35% versus 15%). In addition, an excess of non-lingual oral tumours (lip, gingiva,
and floor of the mouth) was found in the animals receiving
the combined treatment compared with the DMBA-treatedanimals. Whether this radiation enhancement of DMBAinduced tumorigenesis has implications for the lower
combined exposures found for cigarette-smoke-derived
carcinogens in the bucal cavity of humans and dental
x rays, remains to be elucidated.
72. Studies on chromosome aberrations from the combined effect of gamma rays and the mutagen thiotepa on
unstimulated human leukocytes showed no significant

236

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

difference from the sum of their separately induced effects.
The sequence of treatment and the interval between them(up to 4 hours) did not affect the frequency of chromosome
aberrations [B21].
73. Leenhouts et al. [L13] investigated the combined effectof 1,2-dibromoethane (DBE) and x rays on the induction ofsomatic mutations in the stamen hair cells of tradescantia
KU 9. At low radiation doses, a synergistic interaction was
found between the two agents for both DBE exposure followedby acute x rays and chronic simultaneous exposures. The
synergism was considered to result from an interaction of
single-strand lesions in the DNA. It was concluded that thistype of interaction would not be too important for radiologicalprotection. However, it could be of significance in evaluating
the effects of chemicals at low exposure rates.
(c) Free-radical-generating chemicals
74. Superoxide (O2) generating agents such as the
dipyridilium compound paraquat might also interact
directly with the fixation or repair of radiation-induced
damage. Geard et al. [G5] investigated the combined
effects of paraquat and radiation on mouse C3H10T½ cells.
Effects on oncogenic transformation, chromosome alteration, cytokinetics, or cellular survival were the endpoints
measured. Paraquat alone is a cytotoxic agent and is alsoa weak radiosensitizer. Treatment with 0.1 mM for 24hours results in about 30% cell survival and enhances thecell-killing effects of 137Cs gamma rays by a factor of about
1.2. The drug appears to function lethally by initiatinginterphase cell death and also by slowing cell cycling. In
combination with radiation (3 Gy), paraquat acted either
additively (sister chromatid exchanges) or with a greaterthan-additive effect (cell survival and oncogenic transformation).
75. De Luca et al. [D6] studied the induction of reciprocaltranslocations in mouse germ cells (BALB/c) by bleomycin
alone or combined with radiation (see also Section D.3 forbleomycin used in combined modalities in tumour therapy).
The dose-response relationships after treatments with doses of20, 40, and 60 mg kg1 of bleomycin as well as the combined
effect of bleomycin and gamma rays were studied. A positive,
significant correlation between the dose of bleomycin and thefrequency of translocations was found. Both potentiation andadditivity were found when the yields of translocations
induced after combined treatments, separated by a lapse of 24hours, were compared with the sum of translocation
frequencies induced after the corresponding single treatments.
Potentiation occurred in the treatments with 1 Gy plus 9 Gy
and 60 mg kg1 of bleomycin plus 9 Gy, while additivity
occurred in the treatments with 60 mg kg1 of bleomycin plus
1 Gy and 1 Gy plus 60 mg kg1 of bleomycin. In mice
irradiated with 1 Gy plus 9 Gy and mice treated with 60
mg kg1 of bleomycin plus 9 Gy, similar translocation yields
were found. The potentiating effect of bleomycin was found tobe similar to that obtained with non-radiomimetic compounds
such as triethylenemelamine, cyclophosphamide, and adriamycin. The high doses involved and the erratic changes fromsynergistic to additive relationships preclude extending inferences from these experiments beyond cancer therapy tooccupational or non-occupational settings.
76. In summary, there are many examples of strong deviations from hetero- and isoadditivity in the interactions
between genotoxic chemicals and ionizing radiation (Table
A.1). Owing to generally high exposures to both agents understudy, deterministic effects were shown or suspected to be thecause of strong deviations from additivity in many studies.
Thus, the several cases of synergism found seem to be mostly
the result of modifications of the biokinetics and the metabolism of the chemical rather than of agent-specific genotoxicity at different stages of the pathological processes.
Similar considerations hold for antagonistic effects, wheredepletion of stem cells and inhibition of cellular growth may
be a factor in the high dose range. Additional risks, beyond
the level predicted from isoaddition, from the combined effectsof ionizing radiation and genotoxic chemicals at low exposures levels are, accordingly, not specifically demonstrated
by the many epidemiological and experimental studies
reviewed in this Section.
2. Non-genotoxic chemicals
77. Many chemicals in the human environment or their
metabolites do not specifically attack DNA but influencecell proliferation and cell differentiation on an epigenetic
level. Specific mitogens may interfere with regulatory
mechanisms and cell-cell signaling, but many substanceswith a high chemical reactivity act as unspecific irritants
or toxicants via membranes or proteins. Toxin-induced cell
death will induce proliferation in neighbouring cells,
which may enhance the progression of premalignant cells.
Substances acting in a non-specific manner, for example
lipophilic solvents, quite often show highly non-linear
dose-response relationships with apparent thresholds.
Other agents may interfere with the critical cellular
processes involved in repairing damage to cellularconstituents such as DNA. The assessment of possiblesynergistic effects at the exposure levels relevant to thisAnnex is very difficult, because of the high exposures usedin experimental systems and the apparent threshold levels.
78. The tumour promoter 12-O-tetradecanoylphorbol-13acetate (TPA) has the potential to enhance the yield ofradiation-induced tumours. This has been well documented
in vitro and in animal systems. The combined effects ofpaternal x-irradiation and TPA on skin tumours in two
generations of descendants of male mice was studied by
Vorobtsova et al. [V5]. Progeny of outbred SHR male micenon-irradiated or exposed to a single dose of whole-body xirradiation (4.2 Gy) were skin-painted twice a week for 24
consecutive weeks from the age of four months onwards
with acetone or with TPA in acetone (6.15 µg ml1). The
incidence and number of skin papillomas were monitored
between week 2 and week 20 after the last application of
the promoter (TPA). Exposure to acetone was neverfollowed by skin tumour development in the progeny ofeither irradiated or non-irradiated males. Two weeks after

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

TPA treatment, the incidence of skin tumours in theprogeny of non-irradiated mice was 21% in males and 37%
in females, and 20 weeks later it was 12% in males and
15% in females. The skin tumour incidence in the progenyof the irradiated male mice 2 and 20 weeks after the last
painting was clearly elevated: 75% and 68% in males and
50% and 43% in females, respectively. Some of the F1
offspring of irradiated male mice were mated before thestart of TPA treatment, and F2 progeny were exposed toacetone or TPA as F1. The incidence of skin papilloma 2
weeks after the last TPA painting was 58% in males and
40% in females, whereas at 20 weeks after the last
exposure to the promoter it was 53% and 36%,
respectively. In the progeny of irradiated male mice there
were more animals with multiple (>4) skin papillomasthan in the progeny of non-irradiated mice. The incidenceof other than skin tumours in offspring was also clearly
increased in TPA-treated progeny from irradiated malemice. The authors suggested that irradiation of males
before mating increases the susceptibility of progeny in atleast two generations to promoters of carcinogenesis as aresult of persisting genomic instability. On the other hand,
Brandner et al. [B30] found no influence of ip-administered TPA on the incidence of radiation lymphomas in
C57Bl/6 mice. Female C57Bl/6 mice, given four xirradiations each with 1.7 Gy, developed lethal lymphomas
in more than 90% of animals 270 days after irradiation.
Intraperitoneal application of TPA, 30 ng g1 twice weekly
for 240 days, had no influence on survival of the animals
or on incidence of the malignant lymphomas. However, theincidence in radiation-only treated animals was already sohigh that this test was highly insensitive to the promoting
effects of TPA.
79. Jaffe et al. [J2] studied the effect of proliferation andpromotion time on radiation-initiated tumour incidence in
Sencar mice. In this system, a single subcarcinogenic doseof ionizing radiation followed by 60 weeks of TPA
treatment led to the formation of squamous-cell carcinomas. Even TPA pretreatment before irradiation seemed toresult in an overall increase in total tumour incidence,
including both epidermal and non-epidermal tumours [J1].
Based on these findings, the effect of the proliferative stateof the skin before irradiation and the promotion duration
after irradiation on tumour incidence was further investigated in CD-1 mice. To examine the influence of the proliferative state of the skin, a 17 nmol TPA solution wasapplied to one half of the mice 24 hours before irradiation.
The skin was irradiated using 4 MeV x rays at a dose rateof 0.31 Gy min1. Animals received a single dose of x raysof 0.5 or 11.3 Gy, followed by twice weekly applications of
TPA (8 nmol). The animals were then promoted for either
10 or 60 weeks. All animals promoted with TPA for thesame duration had a similar incidence of papillomas
regardless of radiation or TPA pretreatment. Increasing thepromotion duration did not significantly alter the incidenceof squamous-cell carcinomas at either initiation dose. At
the lower initiation dose, only animals that were promoted
for 60 weeks developed squamous-cell carcinomas. TPA
pretreatment at the higher dose resulted in a slight decrease237

in tumour incidence; however, this was not statisticallysignificant. The incidence of basal-cell carcinomas wasradiation-dose-dependent and appeared to be independentof TPA promotion. Again, as in many other cases, nocommon pattern emerged for the different tumour types.
80. The interaction between ionizing radiation and TPA has
been studied using a three stage model of initiation, promotion, and progression. Ionizing radiation is well established as
an initiator, whereas its potential for promotion and progression is less well known. Therefore, Jaffe and Bowden [J1]performed a three-stage experiment using ionizing radiationin the third stage of mouse skin carcinogenesis. CD-1 mice
were initiated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), followed by biweekly promotion with TPA. After
20 weeks of promotion, the animals were treated with either
acetone, TPA (twice a week for two weeks), or eight fractionsof 1 MeV electrons (1 Gy per fraction over a period of 10days). The conversion ofpapillomas to squamous-cell carcinomas was 80% for animals treated with ionizing radiationcompared with 25% for tumour-bearing animals treated with
TPA. Ionizing radiation increased the number of cumulative
carcinomas per group. The absence of an increase in thenumber of cumulative papillomas per group due to late
exposure to ionizing radiation suggests that the dose andfractionation protocol used in this study enhanced theprogression of pre-existing papillomas.
81. The tumour-initiating and -promoting effects ofionizing radiation in mouse skin was also studied with
TPA by Ootsuyama and Tanooka [O2]. Neither single
24 Gy 90Sr/90Y beta irradiation followed by repetitive
treatment with TPA nor single pretreatment with 7,12dimethylbenz-(alpha)-anthracene (DMBA), followed byrepetitive 4.7 Gy beta irradiation, produced tumours above
the level of significance within a period of 210 days, while
a positive control, DMBA + TPA, yielded a high incidenceof papilloma in a shorter period. In this system, DMBA
seemed to exert an action antagonistic to beta particles in
the induction of malignant tumours. It was concluded thatthe tumour-enhancing activity of repetitive radiation isqualitatively different from the promoting activity of TPA.
82. Nomura et al. [N6] were able to show that in utero
irradiation at early stages of embryogenesis, which was not
visibly carcinogenic by itself in a tester strain of mice (PT ×
HT F1), followed by post-natal application of TPA, led to ahigh incidence of skin tumours. Radiation doses in thissystem were 0.3 and 1.0 Gy of 180 kVp x rays, respectively,
at about 10.5 days after fertilization. Two dose rates, 0.54 and0.0043 Gy min1, were used. The incidence of both embryonicmutations, determined as spots of different coat color, andtumours increased with in utero doses. Low-dose-rateirradiation led to a large (about 80%) reduction in tumour
incidence.
83. TPA also causes enhanced transformation ofirradiated mouse 10T½ cells (Figure A.II). For the loss-ofcontact inhibition, two genetic steps and modulation by
epigenetically acting substances were proposed by Little

TRANSFORMANTS PER SURVIVING CELLS (x 10 -3)

238

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

10
9
8
7
6
x rays plus TPA

54
3

x rays
2
1
0
0

1

2

3

4
5
DOSE (Gy)

6

7

8

9

Figure A.II. In vitro transformation of C3H10T½ cells
exposed to x rays (50 kV) with and without postirradiation incubation in TPA (0.1 µg ml1) [H3].

[L24]. TPA promotes following exposure to x rays or tofission-spectrum neutrons without any effect on cell
survival [H3]. However, treatment of unirradiated cellswith 0.1 µg ml1 of TPA resulted in a small increase intransformation frequency above background (i.e. from
1.1 105 to 1.0 104). Thus, besides being a promoter, TPA
seems to be also a weak initiator. The enhancement factorof TPA for radiation-induced transformation was greater
after low doses than high doses of either radiation. In
addition, TPA caused the RBE of neutrons as compared tox rays to increase with increasing dose. For x-ray doses
from zero to approximately 1.2 Gy, TPA raised transformations to frequencies approximately equal to those due
to neutrons alone. Analysis of TPA enhancement in thecontext of the combined effect of two inducing agents, TPA
plus radiation, indicates that with either x rays or neutrons,
TPA acts synergistically. The main mechanism of action of
TPA is suggested by the finding that the dependence oftransformation frequency on the density of viable cells is
also altered by the tumour promoter. In contrast to theconstant frequency of transformants per surviving (orviable) cell, which was observed after a fixed dose of x raysor neutrons for a range of cell inocula, the increase in thefrequency of transformation caused by TPA and radiation
was dependent on cell inocula. The frequency of transformation from combined treatment decreased with increasing
size of the inoculum, from approximately 20 to 6,000
viable cells per 90-mm Petri dish, a result that the authorsinterpreted as an interference with cell-to-cell communication by TPA plus the fading of initiation events causedby radiation.
84. DNA base analogues are another group of substanceswith the potential to modify the effects of radiation andother genotoxic agents (see also Section D.2). 5-Bromo-2'deoxyuridine (BrUdR) is an analogue for thymidine andwidely used in tumour diagnosis, cytogenetics, and flow
cytometry. Important examples of epigenetic and (indirect)
genetic effects are the inhibition of differentiation in
cultured myoblasts and photosensitivity of patients,

respectively. Anisimov and Osipova [A15] investigatedcarcinogenesis induced by combined neonatal exposure to
BrUdR and subsequent whole-body x-irradiation of rats.
Outbred LIO rats at 1, 3, 7, and 21 days of post-natal life
were exposed to subcutaneous injections of 3.2 mg of
BrUdR per animal and/or at the age of 3 months to single
whole-body x-irradiation at a dose of 1.5 Gy. In males,
treatment with BrUdR alone decreased the latency of all
tumours and increased the incidence of malignant tumours
and the number of tumours per rat compared with controls.
Combined exposure to BrUdR and x-irradiation increased
total and malignant tumour yield and multiplicity over thatin all other groups. More testicular Leydigomas, tumoursof prostata, kidney, and adrenal cortex, and leukaemia
were seen in male rats exposed to BrUdR plus x rays,
compared with male rats treated with BrUdR or xirradiation alone. In female rats, treatment with BrUdR
alone decreased the latency for the total number of tumours
and increased their incidence and number per rat, incomparison with controls. Combined exposure of females
to BrUdR and x rays did not increase total tumour
incidence in comparison with females that had only been
irradiated; however, it shortened tumour latency. The
incidence and multiplicity of malignant tumours andincidences of pituitary adenomas, mammary adenocarcinomas, and uterine polyps were significantly increased,
whereas the latency of kidney tumours was decreased infemales exposed to BrUdR plus x rays, compared with allother groups. The data from this experimental model
provide, together with other studies, evidence that perturbation of DNA induced by the nucleoside analogue BrUdR
contributes substantially to the spontaneous developmentof tumours and enhances the sensitivity of target cells tocarcinogenesis induced by x-irradiation as well as bychemicals or hormones.
85. Information on the effects of the interaction of thoriumand phenobarbital, an anticonvulsive drug inducing liverdetoxification functions and showing promoting activity, may
be available from earlier epileptic patients. Thorium exposure(thorotrast) resulting from angiographic procedures correlatedwith the use of anticonvulsive drugs. Olsen et al. [O15, O16]
found considerably increased risks for liver cancer, but sincethorotrast exposure was considered a confounder in bothstudies, no definitive quantitative information on combined
effects from thorium and phenobarbital was given.
86. The potentially important interaction of phenobarbital,a widely used anticonvulsant and sedative, with x-irradiation
was studied by Kitagawa et al. [K18]. Male newborn WistarMs rats received whole-body x-irradiation of 0.5, 1, and 4 Gy
at 8 or 22 days. After weaning they were fed either a basal dietor a diet containing 0.05% phenobarbital. The x rays induced
numerous adenosine-triphosphatase-deficient islands appearing in the liver by week 22 of age. However, no hepatic
tumours were observed by 22 months after radiation, even in
phenobarbital-treated animals.
87. Supra-additivity was also found for a combination of
fast-neutron irradiation and subcutaneouslyapplied carbon

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

tetrachloride in male and female C57Bl6 mice. The
animals received a single whole-body dose of 1.7 or 3.3 Gy
from fast neutrons, followed nine weeks later by a single
subcutaneous injection of carbon tetrachloride. Carbon
tetrachloride markedly increased the incidence of radiation-induced liver carcinomas, whereas chloroform, which
was also tested in this system, did not influence theincidence of radiation-induced tumours [B16].
88. The potential for carbon tetrachloride to modify the
biokinetics of an inhaled, soluble form of plutonium is also
being examined in both F344 rats and Syrian hamsters[B16]. Groups of animals were exposed to carbon tetrachloride in whole-body chambers at concentrations of 0, 5,
20, or 100 ppm for 6 hours per day, 5 days per week, for a
total of 16 weeks. After 4 weeks of exposure, approximately one half of the animals were exposed by a single
pernasal inhalation exposure to 239Pu nitrate. Serial sacrifices of groups of animals were conducted at 4 hours and2, 4, 6, or 13 weeks after plutonium exposure for the quantification of 239Pu in lung, liver, kidney, and bone (femur)
and for the evaluation of histologic changes in various
tissues. Results describing possible carbon tetrachloride
effects on plutonium disposition are not yet available fromthis study. Another subgroup of rats and hamsters was
exposed to a radioactively labelled insoluble tracer particle.
Tracer particle clearance was analysed for 13 weeks following exposure, and no significant clearance differences wereobserved between carbon-tetrachloride-treated and control
groups.
89. Since ionizing radiation and tumour-promoting agentsincrease the level of ornithine decarboxylase (ODC) involvedin polyamine biosynthesis, the effect of alpha-difluoromethylornithine (DFMO), an inhibitor of ODC, on tumour yieldfrom beta radiation was tested in female ICR mice [O12]. The
chronic radiation exposure consisted of three times 3 Gy90
Sr/90Y surface dose per week to the back. DFMO was added
to the drinking water in a final concentration of 1%. It
significantly delayed the time of tumour emergence from 245
days with radiation exposure only to 330 days in animals also
given DFMO. The antagonistic effect of DFMO was also
observed for bone tumours.
90. Monchaux et al. [M61] addressed the importantquestion of possible synergistic contributions from diesel
fumes present in mine air to radon-induced lung tumours.
Three groups of 50 male Sprague-Dawley rats were exposed
to radon (1,000 WLM) and/or diesel exhaust (300 hours;
2225 ppm CO and 45 mg m3 diesel particles), with thediesel exposure succeeding the radon exposure by one month.
Contrary to the strong synergistic effect of cigarette smokefound in this system (discussed under tobacco), exhausts had
only a slight, non-significant effect on the risk for thoracic
tumours from radon. Diesel exhausts alone were not carcinogenic.
91. Since phosphorylation and dephosphorylation ofproteins play an important role in cellular metabolism,
Nakamura and Antoku [N21] studied the effect of239

calyculin A (CL-A), a specific inhibitor of protein
phosphatase 1 and 2A isolated from the marine sponge
Discodermia calyx, on x-ray-induced cell killing in
cultured mammalian cells (BHK21). At concentrations
above 2.5 nM, CL-A enhanced the radiation effectconsiderably. As also shown in another cell culture systemwith the inhibition of protein kinases [H40], agents thatinterfere with protein-kinase-mediated signal transduction
after radiation exposure may enhance damage andrepresent a new class of radiosensitizers.
92. Many non-genotoxic agents clearly produce strong
synergistic effects with ionizing radiation. The combined
effects of this class of agent are summarized in Table A.1.
Table A.3 lists more detailed effects of TPA, probably thebest-studied modifier of genotoxic agents, on several
endpoints. These studies are of great importance for the
elucidation of mechanisms affecting expression of risk. At
this stage, however, no functional analogues of potent
experimental enhancers of radiation risk, such as TPA or
DNA bases, are known to exist in critical concentrations in
the human environment.
3. Tobacco
93. The important interaction of tobacco smoke andradiation was introduced in the main text of this Annex.
Epidemiological studies of uranium miners have allowed
the risks and interaction coefficients to be quantified, atleast for higher radiation doses. The complex compositionof tobacco smoke makes the interaction not simply a binary
combination, however. Some 4,000 individual chemicalcomponents of cigarette smoke have been identified, and anumber of additional unidentified components surely exist(for example, extremelyreactive, short-lived compounds or
those present in very low concentrations) [G1]. Identified
compounds in smoke include several known carcinogens of
the polycyclic aromatic hydrocarbon and nitrosamine
classes.
94. The studies reviewed below refer to mainstream
smoke, sidestream smoke, or environmental tobaccosmoke. Mainstream smoke is defined as the smoke
originating from the butt end of a cigarette; it is generated
during the active puffing process. Sidestream smoke is thesmoke released at the burning tip of a cigarette, whether
the cigarette is being puffed or simply smoldering. Lastly,
environmental tobacco smoke is a mixture of sidestream
smoke and exhaled mainstream smoke. This term mostaccurately describes the smoke that would be found within
an enclosed space with a smoker present. Tobacco smoke
contains relatively small amounts of DNA-reactive
carcinogens, such as nitrosamines, polycyclic aromatichydrocarbons, and pyrolysis products, such as carbolines.
Hence enhancing and promotional factors, e.g. catechols,
other phenols, and terpenes, are an important component.
Probably because it reduces pressure from the action ofpromoters, discontinuation of smoking progressively
reduces the risk of cancer development with time since
withdrawal [W1].

240

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

Table A.3
TPA as a modulator of transformation and cancer yield from ionizing radiation
Endpoint

Experimental system

Interaction

Proposed mechanism

Outcome

Ref.

Transformations
in surviving cells

10T½ cell culture

x rays, TPA

Initiation, promotion

Higher linear yield
Loss of threshold(see Figure V)[H3]

Transformations
in surviving cells

10T½ cell culture

x rays/neutrons, TPA

Initiation, promotion

Enhancement factor
greater
at lower exposures
RBE of neutrons
enhanced
at higher doses[H3]

Transformations
in surviving cells

10T½ cell culture

Radiation, TPA

Two genetic steps,
epigenetic modulation

Genetic effect fadingwith culture time
TPA interferes withcell-cellinteraction[L24]

Squamous-cellcarcinoma

CD-1 mice

Beta radiation, TPA;
MNNG, TPA, beta
radiation

Initiation, promotion,
progression

High papilloma yieldwith
TPA only
Progression tocarcinoma
by radiation[J1]

Skin papilloma

Mice

Beta radiation, TPA;
DMBA, beta radiation

Initiation, promotion

Promotion by repetitive
irradiation different
from TPA[O2]Skin papilloma

SHR mice

Radiation (4.2 Gy)
to father
TPA to offspring
F1 and F2

Genetic modification,
promotion

Skin tumours elevatedin
TPA-treated offspring
Weaker effect in female
offspring[V5](a) Epidemiological studies
95. In the last few years, joint analyses of original data
sets [C1, L18] and meta-analyses of published results [T14]
have yielded detailed assessments of risk patterns fromcombined exposure to high-LET alpha radiation fromradon and its short-lived decay products and tobaccosmoke, and have allowed investigators to test risk models.
The most comprehensive and complete analysis of radoninduced health risks was published by Lubin et al. [L18].
The review contains a joint analysis of original data from11 studies of male underground miners; 2,736 lung cancer
deaths among 67,746 miners were observed in 1,151,315
person-years. A linear relationship was found for the ERR
of lung cancer with the cumulative exposure to radon
progeny, estimated in working level months (WLM). Thiscoefficient (ERR/WLM) was stronglyinfluenced byvarious
factors. Contrary to the low-LET experience from Hiroshima and Nagasaki, ERR/WLM decreased significantlywith attained age and time after cessation of exposure toradon progeny. A stronger decline of risk with time since
exposure than in survivors of the atomic bombings was
also found. A considerably higher lung cancer risk was
initially found for exposures received at low rates ascompared with high rates. Depletion of stem cells at risk inhigh dose rate exposures was implied. However, theepidemiological database was said to be too weak to project

this indication of an inverse dose-rate effect to nonoccupational settings, i.e. to typical indoor radon exposuresand exposure rates [L18]. Also, a recent reassessment of
the Beaverlodge cohort, which earlier on gave the strongest
indication of such an effect, no longer does so. Revised
exposure estimates of this study of miners with relatively
low exposures now bring the modifying effects of risk withtime since exposure and age at risk in line with those fromother studies [H46]. The highly significant decrease in
ERR with time since exposure may be explained withmicrodosimetric considerations. In the case of high-LET
alpha radiation from radon progeny, the minimal local
dose from one single alpha track averaged over a cell
nucleus is already in the range of several hundred milligray, whereas one electron track yields a dose to the nucleus
in the range of only 13 mGy. This means that even at thelowest possible nuclear dose from alpha exposure, stem cells
that are hit carry a multitude of DNA lesions, which may
considerably impair long-term cell survival and maintenanceof proliferative capacity [B25, B27].
96. In the joint analysis by Lubin et al. [L18], data on
smoking were available for 6 of the 11 cohorts, but assessments were limited by incomplete data on lifetime tobacco
consumption patterns and sometimes exotic tobacco use, suchas in water pipes in the Chinese study. Most studies for whichsmoking data could be analysed were generally not informa-ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

tive enough to allow deciding between an additive or a multiplicative joint relationship for radon progeny and smoking.
The Chinese cohort seemed to suggest an association moreconsistent with additivity, while the Coloradocohort suggested
a relationship more consistent with a multiplicative interaction. For all studies combined, the joint relationship of
smoking and radon progeny exposures with lung cancer was
stable over the different age groups and deviated quite clearly
from either a purely additive or a multiplicative relationship.
The most recent analyses of the BEIR VI Committee [C46],
which were based on an update of these data, suggest that thejoint effect is statistically closer to a multiplicative than an
additive interaction. To further characterize the association,
more detailed data on tobacco use would be needed. Age at
onset of smoking, amount and duration of smoking, and typeof tobacco were recognized as important determinants of risk.
Such a refined analysis of smoking patterns is possible only inthe prospective part of ongoing studies and is subject,furthermore, to potential bias in the affected individuals owing
to the rapidly decreasing public acceptance of smoking. In
general, the single-exposure subcohorts of lifetime nonsmokers are very small in all studies. The statistical power ofthe conclusions on the interaction between radon and tobaccosmoke is correspondingly small. Applying the two-mutation
clonal expansion model of carcinogenesis of Moolgavkar et al.
to data from the Colorado plateau miners shows no interaction
between radon and tobacco smoke in any of the three steps[M39], but the predicted lung cancer incidence caused byradon and smoking remains more than additive and less thanmultiplicative, an indication of isoadditivity.
97. Microdosimetric considerations are also important in
extrapolating the inverse dose-rate effect found foroncogenic endpoints caused by alpha radiation in generaland for lung cancer in miners [L36]. Brenner [B40]
postulated that protraction enhancement is a mechanism
limited to cells receiving multiple hits over a human
lifespan. Since a typical domestic exposure to radon
progeny of 14 WLM yields a very small probability ofmultiple traversals in a cell nucleus (<1% for the mosthighly exposed stem cells in the tracheobronchial
epithelium), dose-rate effects are probably of no relevance,and lung cancer risk per unit exposure will not increase
further at low radon levels.
98. Two recent analyses by Yao et al. [Y7] and Thomas etal. ([T18] with erratum) on the radon-smoking interaction
showed a considerable influence of timing of exposures. The
former study found a higher lung cancer risk for exposure toradon progeny and tobacco use occurring together ascompared to radon exposure preceding tobacco use. The
second study on Colorado uranium miners found asignificantly more-than-multiplicative effect for smoking
followed by radon, whereas radon exposure followed by
tobacco use produced an essentially additive effect. Thesefindings are in conflict with earlier notions based on
experimental results in rats, whereby radon is an initiator andtobacco smoke, a promoter [G20]. However the relevance of
this animal system is questionable, because tobacco smoke
alone does not produce lung tumours in this system.

241

99. Despite the remaining uncertainties, it is quite clear thatthe joint effect of radon progeny exposure and smoking isgreater than the sum of each individual effect. The combined
analysis [L18] shows that a linear exposure-response estimate
for radon and lung cancer is compatible with the data andgives a relative risk that is about three times higher in nonsmokers than in smokers. Assuming a 10-fold difference inthe tobacco-caused lung cancer risk between smokers andnon-smokers, this means that the lung cancer risk for smokers
expressed in absolute terms is higher by a factor of about 3.
Such a supra-additive effect, if also demonstrated to hold forpresent occupational and non-occupational exposure settings,
would be of great importance for the regulation of smoking
and radon progeny in the human environment. Until now,
little quantitative evidence has come from indoor radonstudies. The few case-control studies published are inconclusive [A28, P11]. Only one larger study [P11] was indicative of an indoor radon risk and its modification by tobaccothat is comparable to what is predicted from miner studies. It
remains doubtful whether the results from the many casecontrol studies under way will in the near future allow
narrowing of the uncertainties that surround indoor radon risk
and possible interactions with smoking. Based on inconclusive results from 1,000 computer-simulated large case-control
studies assuming an ERR of 0.015 WLM1, Lubin et al. [L33]questioned the assumption that epidemiological studies, evenwhen pooled in meta-analyses, will produce reliable estimatesof risk from residential radon exposure. Errors in exposure
assessment, migration, and confounding bysmoking are at theroot of this pessimistic assessment. At least for the second
confounder, studies in Europe based on much longer mean
residence times may offer better statistical power. Several
large indoor case-control studies under way will narrow
uncertainties in the next few years. First results from theUnited Kingdom [D33] and Germany [W35] are indicative ofa lung cancer risk in the range of ICRP projections. However,confidence intervals are relatively large and include zero risk
in most analyses.
100. Because of the limitation of the indoor radon studies,risk estimates based on miner data remain the main basis
for predicting lung cancer from indoor radon exposure. A
best linear estimate of the risk coefficients found in the
joint analysis of Lubin et al. [L18, L35] for the indoor
environment indicates that in the United States, some
10%12%, or 10,000 cases, of the lung cancer deaths
among smokers and 28%31%, or 5,000 cases, of the lungcancer deaths among never-smokers are caused by radon
progeny. About half of these 15,000 lung cancer deaths
traceable to radon would then be the result of overadditivity, i.e. synergistic interactions between radon andtobacco. Based on the same risk model, Steindorf et al.
[S47] predicted that about 7% of all lung cancer deaths in
the western part of Germany are due to residential radon.
This corresponds to 2,000 deaths per year, 1,600 in males
and 400 in females. The attributable risk estimate was
4%7% for smokers and 14%22% for non-smokers. The
most recent central estimates for the proportion of radonattributable lung cancer deaths in the United States in 1995
was recently provided by the BEIR VI Committee [C46] in242

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

1998, based on an updated data set of the miners studies
reported by Lubin et al. [L18]. The Committee applied a
sub-multiplicative relation to model the joint effect of
tobacco smoking and radon. Depending on two different
models (exposure-age-concentration model or exposureage-duration model) about 14% or 9% of all lung cancer
deaths among ever-smokers and 27% or 19% among
never-smokers were estimated to be attributable to radon.
Because of the many differences between mines and homes
and the additional carcinogens such as arsenic, dust anddiesel exhaust in mine air, these figures should beinterpreted with caution. A population-based case-control
study of incident lung cancers among women in Missouri
who where lifetime non-smokers or long-term ex-smokers
yielded a very low and non-significant estimate of theattributable lung cancer risk from radon in non-smokers[A4].
101. It has been questioned whether toxicants other thanjoint exposures to radon progeny and cigarette smoke
contribute considerably to the high lung cancer risk foundin miners [I5]. Heavy exposures to mine dust containing
silicates, diesel exhausts, and fumes from explosives may
add to or combine with the two main lung carcinogens,radon and cigarette smoke. Patients who received thorotrast continuously exhale the very short-lived 220Rn derived
from 232Th deposits in the body and therefore provide amodel for lung carcinogenesis by radon without concomitant dust exposure. Ishikawa et al. [I5] studied thelung cancer incidence in a Japanese thorotrast cohort andfound 11 lung cancer cases in 359 thorotrast autopsy cases.
The analysis revealed that while the proportion of smallcell lung cancer considered to be related to alpha radiation
was significantly increased, the overall lung cancer incidence was not significantly higher than in controls, in spiteof the high levels of 220Rn in the patients' breath. The
authors took this as an indication that the risk for radoninduced lung cancer is not as high as expected from risk
coefficients deduced from miner studies. To substantiate
this hypothesis, the build-up of 220Rn decay products in thelung air space before exhalation and the resulting exposure
to critical stem cells would have to be quantified.
102. Owing to the generally good linear correlation
between radon progeny exposure and lung cancer in the
major miner studies, few additional carcinogens in mine
dust were considered in depth. Toxic metals are, however,of special concern. Results from the Chinese [X1],Canadian (Ontario), [K21] and Czech [T41] cohorts
showed arsenic to be an important additional risk factor forlung cancer. Adjustment for arsenic exposure reduced theradon risk estimate in these cohorts considerably. Even in
the most recent joint analysis by Lubin et al. [L18], othermine exposures were difficult to interpret, since theinformation was quite limited and of poor quality. In mostcases these concomitant exposures to suspectedcarcinogens or promoters are typically highly correlatedwith radon progeny exposures in a given study andtherefore difficult to assess independently (see also
following Section B.4).

103. The mechanism of interaction between DNA lesions
caused by radon progeny and those caused by chemical
toxicants contained in tobacco smoke is not known. Thereis clear evidence that the prevalence of mutations in criticalgenes is dependent on the type of insult. The most common
known gene mutations in lung cancer cells are found in thetumour-suppressor gene p53, which is thought to be crucial
in the initiation of this and many other types of cancer.
Several groups analysed the molecular changes in theconserved regions of the p53 gene in lung cancer tissue andreported differences between non-smokers (survivors of theatomic bombings and unirradiated controls), Japanesesmokers, and uranium miners with high radon exposures[T12, T13] (see also Annex F, “DNA repair and mutagenesis”). The non-smokers from Hiroshima showed
mainly transition mutations (all G:C to A:T) but no G:C to
T:A transversions. By contrast, the changes in 77 Japanesesmokers showed a predominance of G:C to T:Atransversions in which the guanine residues occur in the
non-transcribed DNA [T12]. In 16 of 52 lung cancers ofminers, a specific transversion AGG to ATG at codon 249
was reported [T13]. The prevalence of 31% for thismutation in miners was compared with only 1 reported
case in 241 published p53 mutations from lung cancers in
the general population (mainly smokers). Such a marker
might help to define a causal relationship, but even in thefirst study, only a minor fraction of the p53 genes fromlung cancer tissue of miners, all of whom had a uniquegenotoxic exposure, showed the specific change. However,later studies were not able to confirm the initial finding[B73, L53]. As was pointed out, a multitude of different
primary lesions can lead to the same cellular and clinicalendpoints, in this case a non-functional repressor protein
and lung cancer, respectively, and highly specific molecular markers of single agents in all affected individuals
are not to be expected.
104. A difficult matter of some concern for the protectionof the public is the combined exposure to indoor radon
progeny and environmental tobacco smoke. The presenceof environmental tobacco smoke in homes has been
implicated in the causation of lung cancer. In the absenceof direct epidemiological information, the clearly higherthan-additive combined effects of smoking and radon
progeny in mine air may lead to the application of amultiplicative model for risk assessment. While of interest
in its own right, environmental tobacco smoke also
influences the risk imposed by radon and its decay products
through its strong influence on aerosol characteristics. Theinteraction between radon progeny and environmental
tobacco smoke alters the exposure, intake, uptake,biokinetics, dosimetry, and radiobiology of those progeny.
Crawford-Brown [C10] developed model predictions of thevarious influences of environmental tobacco smoke onthese factors in the population of the United States andprovided estimates of the resulting change in the dose fromaverage levels of radon progeny. It was predicted thatenvironmental tobacco smoke produces a very small, nonmeasurable increase in the risk of radiation-induced
tracheobronchial cancer in homes with initially very high

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS
80
70

Neither
Breast cancer radiation therapy
Smoking

60
RELATIVE RISK

particle concentrations for both active and never-smokers
but that it significantly lowers the dose in homes withinitiallylower particle concentrations for both groups whengeneration 4 of the tracheobronchial tree is considered thetarget site. For generation 16, the presence ofenvironmental tobacco smoke generally increases the lungdose from radon progeny, although the increase should beunmeasurable at high initial particle concentrations.
Although the author shows that the dose-modifying effects
of environmental tobacco smoke are negligible, the main
problem, a potential synergism between environmental
tobacco smoke and radon progeny, was not assessed.

243

Both

50
40
30
20
10

105. A smaller but still considerable cohort may be at risk
from the combined effects of low-LET radiation and tobaccosmoke, namely cigarette-smoking women who underwent
breast cancer radiation therapy. Ionizing radiation has alreadybeen shown to be a lung carcinogen after breast cancer
radiation therapy. Neugut et al. [N4] used a case-control study
to explore whether cigarette smoking and breast cancer
radiation therapy have a multiplicative effect on the risk ofsubsequent lung cancer. Case and control women werepersons registered with primary breast cancer in theConnecticut Tumour Registry who developed a second
malignancy between 1986 and 1989. Cases, i.e. thosediagnosed with a subsequent primary lung cancer, werecompared with controls diagnosed with a subsequent nonsmoking, non-radiation-related second malignancy, and ageadjusted odds ratios were calculated with logistic regression.
No effects from radiation therapy were observed within 10
years of initial primary breast cancer. Among both smokers
and non-smokers diagnosed with second primary cancers
more than 10 years after an initial primary breast cancer,
radiation therapy was associated with a threefold increased
risk of lung cancer. A multiplicative effect was observed, withwomen exposed to both cigarette smoking and breast cancer
radiation therapy having a relative risk of 32.7 (95% CI:
6.9154) (Figure A.III). Further evidence for a direct causal
relationship was the observation that the carcinogenic effectof radiation was seen only for the ipsilateral lung and not forthe contralateral lung in both smokers and non-smokers. The
authors concluded that breast cancer radiation therapy, asdelivered before 1980, increased the risk of lung cancer after
10 years in non-smokers, and a multiplicative effect wasobserved in smokers. The significance of the findings is,however, strongly reduced by the fact that the study also
indicates a large difference in the incidence of ipsilateral andcontralateral lung tumours for smokers who had no radiation
therapy(Figure A.III), resulting in concerns about unidentified
bias [I10]. A similar case-control investigation was based on
61 lung cancer cases from the Connecticut Tumour Registry
who had received radiation therapy for the treatment of breast
cancer [I9]. The authors of this study found no indication ofa strong positive association between smoking and radiotherapy in the 27 cases where information on cigarette use
was available. Therefore, it is not possible at this stage todecide whether current treatment practices involving much
lower radiation doses to the lung may need to be reassessed inview of the detriment (late stochastic effects) for young breast
cancer patients who smoke.

0
Both lungs

Ipsilateral lungContralateral lung

Figure A.III. Age-adjusted relative risk of lung cancer
for separate or combined exposures to radiation andcigarette smoke [N4].

106. Long-term survivors of Hodgkin's disease display anincreased lung cancer risk. Van Leeuwen et al. [V7] conducted a case-control study with 30 lung cancer cases from acohort of 1,939 patients treated for Hodgkin's disease from1966 through 1986 in the Netherlands to investigate theeffects of radiation, chemotherapy, and smoking. Comparing
patients who had a radiation dose of more than 9 Gy to thearea where malignant growth developed with those who had
less than 1 Gy, the relative risk was 9.6 (95% CI: 0.9898, p
for trend = 0.01). Patients smoking more than 10 pack-years(number of years with more than 1 pack per day) after
diagnosis had a sixfold higher risk than patients with less than
1 pack-year. A multiplicative interaction was observed
between the lung cancer risk from smoking and fromincreasing levels of radiation. On the other hand, no such
trend was found with the drugs mechlorethamine or procarbazine, either in relation to the number of cycles ofchemotherapy or to cumulative dose. It was suggested that
Hodgkin's disease patients should be dissuaded from smoking
after radiotherapy [V7].
(b) Animal studies
107. Although there are no well-suited animal model
systems in which to examine potential carcinogenic interactions between environmental tobacco smoke andradiation, the issue of interactions between exposure to
mainstream cigarette smoke and either radon or 239PuO2
has been examined. Relationships between increased risk
for lung cancer in animals and exposure to radon and/orradon progeny [G11] or to 239PuO2 [C2] have recently beenreviewed.
108. Studies conducted in France involved the whole-body
exposure of rats to diluted mainstream cigarette smoke
administered either before or after exposure to radon [C9].
Rats received high-level exposures to smoke for ten 15minute periods four times weekly for one year. Smoke
exposures given before the exposures to radon did not
influence radon-induced tumour incidence, but smoke

244

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

exposures given after radon exposure increased the tumour
incidence by a factor of 23 over rats receiving radon
alone. These data indicated that cigarette smoke may haveacted to promote radon-induced carcinogenesis, as
reviewed in the UNSCEAR 1982 Report [U6].
109. In contrast, studies conducted on dogs exposed to thesmoke from 10 cigarettes per day for 45 years combinedwith radon suggested that the incidence of lung tumours
was less than that in dogs receiving radon alone [C21].
Lung tumours were produced in 8 of 20 dogs receivingradon alone, whereas tumours developed in only 2 of 20
dogs receiving both agents. The investigators speculated
that increased mucus flow may have led to a reduced
radiation dose to target cells in the smoke-exposed dogs;
however, the small number of animals made interpretationof these results difficult.
110. Thus, despite the directly relevant epidemiological
data on smoking and albeit high exposure to radon
progeny, a significant problem remains, for example, forextrapolations to low exposures, in that the epidemiologicaland animal data related to lung cancer are in agreement for
rats [C9] but in disagreement for dogs [C21]. Archer [A21]tried to explain this disagreement by advancing ahypothesis based on an additive interaction of the two
agents at the level of initiation and on temporal differencesof cancer expression. The hypothesis is that among cigarettesmokers a given radiation exposure induces a finite number oflung cancers that have shorter latency periods as a result of
the cancer-promoting activity of smoke.
111. In a study with hamsters exposed to 210Po,
benzo(a)pyrene was used as a substitute for tobacco smoke[L24]. As compared with animals exposed only to ionizing
radiation (lung cancers incident in about 3% of theanimals) or only to benzo(a)pyrene (no incident cases inover 280 treated animals), animals receiving benzo(a)pyrene instillations after exposure to ionizing radiation
were at a much higher risk (about 50%) of developing alung tumour. It is noteworthy that the instillation of saline
after radiation exposure also induced lung tumours inabout 30% of the animals.
112. Douriez et al. [D30] investigated the role ofcytochrome P-450 1A1 (CYP1A1) inducers on radoninduced lung cancers in rats. CYP1A1 is the member ofthe cytochrome P-450 gene family producing the mostmutagenic activation products from polycyclic hydrocarbons. All three inducers tested (methylcholanthrene,
5,6-benzoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin)
increased the incidence of epidermoid carcinoma to 100%,
independent of whether the inducer itself was converted toa powerful carcinogen or not. Depletion of retinoid acid in
CYP1A1-stimulated rats is implicated as a further stepleading to increased susceptibility to lung cancer. Since
tobacco smoke is a powerful inducer of CYP1A1, thismechanism could account for the supra-additive effects inradiation-exposed smokers.

113. Preliminarystudies on an interaction between 239PuO2
and cigarette smoke were reported by Talbot et al. [T2].
The experiments were designed to show whether exposure
to cigarette smoke for 12 months enhances the incidence oflung tumours in mice that had previously inhaled 239PuO2.
The main difference found was a reduced growth rate inboth smoke- and sham-exposed mice relative to that ofcage controls. After 3 months of treatment, histopathology
and morphometry of lung sections found only slightsmoke-induced changes. On a per-unit-area basis, thesechanges included a reduced proportion of alveolar space
and an increased number of pulmonary alveolar
macrophages that were larger than those from shamexposed or control mice and had an increased proportionof binucleated cells. All mice in a second study were
initially exposed to 239PuO2, then subsequently divided into
three treatment groups as above. Cigarette smoke exposure
was shown to increase lung weight and inhibit clearance of239
Pu from the lung. The authors pointed out a dosimetricproblem: the group receiving 239PuO2 and subsequently
tobacco smoke would receive a higher radiation dose to thelung than those receiving 239PuO2 alone. Although this
aspect is important for elucidating the mechanisms bywhich synergism or antagonism occur, for radiation
protection, an apparent combined effect traced to amodification of exposure/dose conversion factors by one
agent would still be considered synergism or antagonism.
114. A cigarette-smoke-induced reduction in the lungclearance of inhaled 239PuO2 was also observed in a study
in rats [F15, F28]. Animals were first exposed by a wholebody inhalation mode to diluted mainstream cigarettesmoke at a concentration of 100 or 250 mg m3 of total
particulate matter for six hours per day, five days per week.
Control rats received filtered air alone. After three months,
all groups of rats received a single pernasal exposure toradioactively labelled insoluble tracer particles; then therats were returned to their respective cigarette smoke orfiltered air exposure. External whole-body counting of the
tracer was continued for six months, and substantial
smoke-induced clearance inhibition was found. Lifetime
radiation doses were 3.8 Gy, 4.4 Gy, or 6.7 Gy for thecontrol, 100 and 250 mg m3 total particulate mattergroups, respectively [F28]. The results for the highest levelof cigarette smoke exposure suggested that the radiationdose increased by a factor between 1.6 and 1.7 by thiseffect, compared with the group of rats receiving filtered
air alone. It should be noted that cigarette smoking has
been shown to reduce the lung clearance of relatively
insoluble particles in humans as well as in animals [C5].
115. The study described above is part of a carcinogenicity
experiment in which 2,170 male and female F344 rats
received exposures to cigarette smoke and/or 239PuO2 [F17].
Groups of animals were exposed for up to 30 months tofiltered air or to low or high concentrations of cigarettesmoke. For each of these groups, approximately one half ofthe rats also received a single pernasal inhalation exposure
to 239PuO2 that resulted in an initial lung burden of
approximately 400 Bq. Cigarette smoke exposure did notANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

markedly influence survival, but it did result in decreased
weight gain and a variety of lung lesions such as alveolar
macrophage hyperplasia, interstitial fibrosis, chronic-active
inflammation, hyperplasia of the alveolar epithelium, andbronchial mucous-cell hyperplasia. A preliminary evaluation of lung cancer in females indicated that crude lungtumour incidences were approximately 7% in rats exposed
to high concentrations of smoke, 20% in rats exposed to239
PuO2, and 74% in groups receiving both agents (FigureA.IV). Thus, the interaction was clearly synergistic. This
study illustrates the manner in which a dose from one agent
can be markedly affected by exposure to a second agent,leading to a clear synergism in carcinogenic response. Less
certain, however, is the extent to which the interaction
resulted strictly from the impaired clearance (and associatedincreased radiation dose) in the combined exposure groups
rather than a more fundamental interaction between theradiation and cigarette smoke constituents at the molecular or
cellular level. Another mechanism by which synergism could
occur might relate to the localized radiation dose rather thanthe dose to the whole organ. For example, the synergistic
interaction between smoking and radiation in this example
could result from the alpha radiation dose delivered at the siteof smoke-induced lung lesions, where the processes of cell
hyperplasia, fibrosis, and activated phagocytes were alreadyoccurring.
80
70

No plutonium
Plutonium

INCIDENCE (%)

60
50
40
30
20
10
0

(0%)
No smokeLow smokeHigh smoke

Figure A.IV. Incidence of lung tumours in female rats
exposed to plutonium dioxide in combination withvarying levels of cigarette smoke [F17].

116. In an early study by Cowdry et al. [C49], the carcinogenicity of 90Sr beta irradiation to the skin of Swiss mice
applied twice weekly and 3 tar paintings per week to
distributed skin areas was studied. Surface doses wereabout 2 Gy per fraction and 200 Gy totally. Skin tumour
incidences after 30 months were 12.3% for radiation alone,
42.9% for tar paint alone, and 61.3% for the combined
treatment. It was concluded that there is no synergism
between the two carcinogens in the study system. It isnoteworthy that a monthly skin irradiation of several Gy
surface dose did not produce any skin tumours, whereas in
the control group only painted with acetone (the solvent for
cigarette tar) an incidence of 6.8% was seen after
30 months.

245

117. In view of the many active substances contained incigarette smoke, possible interactions are very numerous.
It is outside the scope of this Annex to cover this fully, but
the many reported interactions of caffeine with cigarettesmoke components may merit mention, especially because
caffeine at higher concentrations also modifies the effectsof ionizing radiation. Rothwell [R13] found an inhibitionof cigarette-smoke-induced carcinogenesis in mouse skinby caffeine. Other recent reports showed an inhibition of
tobacco-specific nitrosamine-induced lung tumorigenesis
in A/J mice by polyphenols extracted from green tea andcruciferous vegetables [C24, X2]. It is believed that these
dietary compounds act as antioxidants (see also Section B.6).
(c) Cellular studies
118. To examine the interaction between radiation exposureand smoking, Piao and Hei [P8] studied the toxicity andoncogenic transforming incidence ofalpha-particle irradiation
with and without concurrent exposure to cigarette smoke
condensate on C3H10T½ cells in vitro. In this system,
additive modes of interaction between cigarette smoke
condensate and ionizing radiation were observed for the
oncogenic transforming potential of both gamma rays andalpha particles. In a recent study made possible by thedevelopment of charged-particle microbeams, it was shownthat even for radon alone, induction of transformation in
C3H10T½ cells in vitro from exactly one alpha particle wassignificantly lower than for a Poisson-distributed mean of one
alpha particle through a cell nucleus [M66]. This implies thatcells traversed by multiple alpha particles contribute most of
the risk, and that a linear extrapolation from high exposures
may overestimate the transforming potential of high-LET
radiation in low-level exposures. If generally applicable, such
results would speak against the potential of combined effects
at low exposures to surpass values expected from linear doseeffect relationships and additivity.
119. The combined genotoxic effect of cigarette smoke
condensate and gamma radiation was also studied in a simple
eukaryotic organism [S8]. The induction of gene conversion
in diploid yeast (Saccharomyces cerevisiae) strains wasinvestigated following exposure tocigarette smoke condensate
and gamma radiation. Cells exposed to a combination ofcigarette smoke condensate and low-LET radiation showed an
additive response irrespective of the order of treatments. The
system also showed large differences in sensitivity dependingon growth status, with log-phase cells being 23 times moresensitive than stationary cells. The relevance of these findings
is limited by the fact that critical toxicants in tobacco smoke
require activation by biotransformation, a mechanism that is
highly species- and tissue-specific.
(d) Summary
120. In summing up the many results from the wellstudied interaction between tobacco smoking and highlevels of radon exposure, it can be stated that thisimportant combined exposure leads to clearly overadditiveeffects for lung cancer in humans. Some of the more

246

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

important findings are summarized in Table A.1. The quite
different dose-effect relationships for the two agents,
apparently linear for radon and clearly non-linear for
tobacco smoke, speaks against iso-addition and for a true
synergism. However, large uncertainties remain withregard to quantifying the health effects of these important
agents at prevailing levels of combined exposures in thepresent-dayworkplace and in non-occupational settings. Inview of the complexities involved in the toxicologicalassessment of tobacco smoke, which is itself a combinationof genotoxic and non-genotoxic agents, it is not possible atthis time to extend inferences from mechanistic considerations to low combined exposures. Several large casecontrol studies under way involving non-occupational
exposures may help to solve this enigma by creating better
estimates and reducing the uncertainties surrounding
synergistic effects from smoking and radon.
4. Metals
121. Toxic metals are important trace pollutants in thehuman environment. They interact in many ways with cell
constituents and may produce oxidative gene damage or
influence enzyme activity at low concentrations, e.g. bycompeting with essential metal ions [H38]. Carcinogenictransition metals are capable ofcausing promutagenic damage
such as DNA base modifications, DNA-protein cross-links,
and strand breaks [K7]. The underlying mechanism seems to
involve active oxygen and other radicals arising from metalcatalysed redox reactions. Cadmium, nickel, cobalt, lead, andarsenic may also disturb DNA repair processes [H48]. Leadneurotoxicity, an example of an important non-genotoxic
metal effect, is a result of intracellular regulatory dysfunction
caused by this heavy metal. Lead activates calmodulindependent phosphodiesterase, calmodulin-sensitive potassiumchannels, andcalmodulin-independent protein kinase C(PKC)[G31]. The latter effect alreadyoccurs at picomolar concentrations and indicates second messenger metabolism as apotential sensitive site for the disruptive action of lead.
Epidemiologically proven metal lung carcinogens are arsenic,
cadmium, chromium, nickel, and antimony [M65]. In thecritical field of underground mining, possible metal effects
have to be assessed together with high-LET radiation fromradon. Arsenic in particular has been shown to be a major risk
factor in combined exposures to mineral dust, radon, metals,
and diesel fumes [K48, T5]. The risk-enhancing effects of iron
dust seems to be limited to very high dust concentrations,leading to changes in lung function [B74]. An elevatedstomach cancer risk in Ontario gold miners was statisticallyassociated with chronium exposures but not with arsenic,
mineral fibres, or diesel emissions [K50].
122. Multiple exposures to radon, arsenic, and tobaccosmoke were common in several uranium mines (see also
Section B.3). An assessment of 107 living tin miners who
had lung cancer and an equal number of age-matched
controls from tin miners without lung cancer provided noevidence for synergism between radon and arsenic or
between arsenic and smoking [T5]. That there is no
obvious synergism between this heavy metal and radon

progeny exposure is implied by the fact that the risk oflung cancer among workers exposed to arsenic (and radon)in mining only is slightly less than for miners whose exposure
came from smelting operations. In a study on gold miners
with quite low radon exposures, linear regressions indicatedthat exposure to 1 WLM of radon decay products increases
lung cancer mortality rates by 1.2%, a finding comparable toother studies, and that each year of employment in a poorly
ventilated mine (before 1946) with an arsenic content of thehost rock of 1% is associated with a 31% increase in lungcancer mortalityrates [K21, K51]. Adding an interaction term
to allow for a deviation from additivity for the combined effectof arsenic and radon decay products did not improve the fit.
Noteworthy is the fact that the duration of the arsenic
exposure seems to be more important than its intensity [T5].
123. The induction of radical-scavenging metallothionein byhigher concentrations of heavy metals may confer protection
against ionizing radiation. Single ip injection of cadmium(1 mg kg1) two hours before radiation exposure increased
the yield of DNA lesions in peripheral blood lymphocytesof mice, but cadmium injection 2448 hours in advance ofionizing radiation reduced DNA damage in lymphocytes incomparison with untreated animals [P27]. In this study theprotective effect was due to reduced levels of initial DNA
damage per unit dose of radiation as well as accelerated
DNA repair measured in a single-cell gel assay.
124. Beryllium is another metal that has been examinedfor potential interactions with radiation. Although notconsidered to be a genotoxic metal [A10], beryllium is aknown animal carcinogen and has recently been classified
as a demonstrated human carcinogen [W9]. The potential
for carcinogenic interactions between inhaled beryllium
oxide and 239PuO2 was examined in rats [S42]. The agentswere administered alone or in combination at initial lungburdens of 191 µg beryllium and 0.156.7 kBq 239Pu.
Beryllium oxide exposure induced few tumours and did notmarkedly influence Pu-induced lung tumorigenicity,
despite the fact that beryllium oxide exposure decreased theclearance of 239Pu from the lung and thus served to increase
the total radiation dose to the lung.
125. Another ongoing study investigates the potential
carcinogenic interactions between inhaled beryllium metal
and 239PuO2 in some 5,456 male and female F344 rats [F1].
Preliminaryresults from the studydemonstrate that inhaled
beryllium metal is a potent rat lung carcinogen; over 90%
of rats that survived at least 12 months after inhaling an
initial lung burden of 450 µg beryllium developed benign
and/or malignant lung tumours [F12]. At lower lungburdens of approximately 50 µg beryllium, some 65% of
exposed rats (39 of 60 rats) developed at least one
malignant lung tumour. When this level of beryllium was
combined with a lung burden of 60 and 170 Bq 239PuO2,
which alone caused crude malignant lung tumour
incidences of 8% (6 of 60 rats) and 7% (2 of 27 rats),
respectively, crude tumour incidences ranged from 57%(16 of 28 rats) to 90% (54 of 60 rats) [F16, F28]. Thus,
indications of a more-than-additive response were observed.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

As was the case for the cigarette smoke or beryllium oxide
exposures described above, inhalation of beryllium metal
was found to markedly decrease the clearance of 239PuO2
from the lung [F1], serving to increase the radiation dosein groups receiving combined exposures and to leave inquestion the role of beryllium-induced increased radiationdose vs. a more fundamental interaction between the two
agents at the molecular or cellular level. In addition, theinvestigators reported that exposure to beryllium markedly
reduced the median lifespan of exposed animals, and they
noted that a complete analysis of the combined carcinogenic effects of the two agents would require an analysis
more sophisticated than an examination of crude tumour
incidence [F15]. Specifically, the authors noted that theage-specific tumour incidence for the two agents alone andin combination should be analysed, and it was noted that
this analysis is under way.
126. The potential for beryllium and x radiation
administered alone and in combination to affect cell-cycle
kinetics, cell killing, and induction of chromosomal
aberrations was examined in mammalian cell culture(Chinese hamster ovary cells, Figure A.V) [B37]. Beryllium
was administered in a soluble form (BeSO4) at 0.2 or 1 mM
concentrations, and x rays at levels of 1 or 2 Gy. It was foundthat exposure to beryllium significantly inhibited the capacityof the cells to repair DNA damage induced by x rays. The
combined exposures were characterized by a multiplicativemodel when total chromosomal aberrations were examined
hours after exposure. Both agents caused an accumulation ofcells in the G2/M stage of the cell cycle, and an analysis using
varying times between exposures suggested that themultiplicative interaction observed may have been limited tocells in the S and G2 stages of the cell cycle.
0.8

0 mM beryllium
0.2 mM beryllium

0.7

1.0 mM beryllium

RELATIVE RISK

0.6
0.5
0.4
0.3
0.2
0.1
0
0

1

2

0
1
DOSE (Gy)

2

0

1

2

Figure A.V. Induction of chromosome aberrations in
Chinese hamster ovary cells from exposures to x rays
and beryllium [B37].

127. Micronucleus formation in mouse bone marrowpolychromatocytes as a measure of the modulation of themutagenic action of gamma rays by chromium and leadsalts was used by Vitvitskii et al. [V12]. Chromium (VI)247

ions enhanced radiation effects in acute and chronic experiments. Acute exposures of lead (II) ions below 15 mg kg1
body weight had an antagonistic effect, i.e. they decreased thenumber of gamma-ray-induced micronuclei, whereas higher
doses increased it. Chronic combined action of lead (III) ionsand gamma rays resulted in a lower yield of micronuclei. For
an extrapolation to environmental exposures and humans, anelucidation of the underlying mechanisms, i.e. heavy metal
influence on cell kinetics and/or on DNA damage and repair,
will be necessary.
128. The combined effect of 134,137Cs and lead (Pb2+) at thesoil concentrations found in highly contaminated habitats
in the Russian Federation on the mutation rate in the plant
Arabidopsis thaliana (L:) Heynh has been investigated[K42]. At concentrations of 2202,500 Bq kg1 and 16320
mg kg1, respectively, both antagonistic and synergistic effects
were seen. The radiation-induced mutation rate wassignificantly reduced in the presence of 16 mg kg1 Pb2+,
whereas higher lead concentrations increased the rate inplants grown in soil with up to 1,000 Bq kg1 radiocaesium.
At the highest radiation level and 32320 mg kg1 Pb2+, anapparent decrease in the mutation rate was linked to alarge number of sterile seeds. In an ecological study, thecombined effect of zinc or cadmium and external radiation
on microbial activity in soil was determined by measuringnitrogen fixation, dentrification, and CO2 flux [E20]. At
metal concentrations in soil of 10100 mg kg1 for Zn2+
and 0.516 mg kg1 for Cd2+, small radiation doses ranging
from 3.6 to 12 mGy led to a supra-additive effect in theinhibition of microbial activity in soddy-podzolic soil. It
was further shown that the enzyme level of invertase
increased in combined exposures, whereas catalase anddehydrogenase activities were lower [E19].
129. A sensitive assay in spring barley (Hordeum
vulgare L.) leaf meristem to record effects from ionizing
radiation and/or heavy metals was developed by Gerask’in
et al. [G34]. The radiation-induced frequency of cells withaberrant chromosomes in the intercalary meristem allows
doses to be registered in the range of a few tens of
milligray [G35]. Irradiations were performed at the shootstage and involved doses of 40, 80, and 200 mGy at a dose
rate of 2 Gy h1. Lead (II) and cadmium (II) were applied
as nitrates in two concentrations of 40 and 200, and 3 and20 mg kg1 of soil, respectively. The authors claimed thatin this system, radiation and heavy metals alone exhibit
clearly non-linear relationships, i.e. supralinearity, with ahigher slope for aberrations at lower doses than at higher
doses. Combined exposures show an antagonism for low
doses of ionizing radiation (40 mGy) and for all leadconcentrations. At doses of 80 and 200 mGy, a slightly
supra-additive effect is reported. For cadmium, supraadditivity is found at low metal concentrations of 4 mg kg1
for 80 and 200 mGy but not for 40 mGy. At high metal
concentration, less-than-additive effects were found. Although these findings may be important for environmental
assessments and potentially extendable to mechanistic
studies, no direct inferences to humans are warranted at
this stage.

248

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

130. Metals and ionizing radiation have been shown to
produce combined effects in many biological systems(Table A.1). Because metals cause many biological effects
with no or very low thresholds, possible interactions would
potentially extend to very low exposures. In the case of
relatively unspecific damage to DNA, such as oxidative
attack, iso-addition would be predicted. As an example of asynergistic effect at high exposure levels, a thresholdphenomenon, decreased lung clearance of internal
radionuclide content by high metal concentrations, was foundto be the cause of the combined effect. No supra-additiveeffects are seen in the albeit weak database on combined
occupational exposure to radon and arsenic. The relative
importance of different damage-inducing mechanisms ofmetals for combined exposures in human remains to beelucidated.
5. Mitogens and cytotoxicants131. Although mitogenic and cytotoxic compounds are
generally non-genotoxic agents and could have been included
in Section B.2, theyare considered here separately, principally
because of their ability to stimulate cell proliferation. The
combination of mitogens or differentiation-inducing agentswith radiation has some potential as a cancer therapeuticstrategy. Experiments in this area employ high doses, but
studies intended to elucidate the mechanisms of interaction
may still be relevant outside the clinic. Leith and Bliven [L15]
investigated the x-ray responses of a human colon tumour cellline after exposure to the differentiation-inducing agent
N-methylformamide (NMF). A human colon tumour line was
exposed for three passages to varying concentrations(0170 mM) of NMF and the change in sensitivityto ionizing
radiation was examined in vitro. The linear-quadratic
formalism of survival with two constants (alpha and beta) wasused to characterize the single graded dose-survival curves. As
the NMF concentration increased, the alpha parameterincreased and the beta parameter decreased, yielding a
concentration-dependent radiosensitization that was mostmarked in the low-dose region of the survival curve. Upon
removal of NMF, the original radioresistance was regained
within two or three cell culture doubling times.
132. Müller et al. [M11] studied the formation of
micronuclei in preimplantation mouse embryos in vitro
after combined treatment with x rays and caffeine. The
exposures to caffeine were 0.1 or 2 mM and to x rays, 0.2
or 0.9 Gy. X rays as well as caffeine induced micronuclei.
The dose-effect curve after irradiation was linear for thedose range measured (03.8 Gy). Caffeine only induced
micronuclei at concentrations higher than 1 mM; between
1 mM and 7 mM, however, there was a linear increase in
the number of micronuclei. A considerable enhancement of
the number of radiation-induced micronuclei was observed
when irradiation of the embryos was followed by treatment
with caffeine. The sum of the single effects was clearlyexceeded by the combination effects. An earlier study inthe same laboratory [M12] was on the effects of a combination of x rays (0.2, 0.9, or 1.9 Gy) and caffeine (0.1, 1, or
2 mM) on the formation of blastocysts (96 hours post-conception), hatching of blastocysts (144 hours postconception), and on the cell numbers of embryos at
different times (48, 56, 96, and 144 hours post-conception).
The embryos were irradiated in the G2 phase of the two-cellstage (28 or 32 hours post-conception), either 1 hour afteror immediately before application of caffeine. Caffeine waspresent during the whole incubation period (until 144
hours post-conception). Specific conditions under which
caffeine markedly enhanced the radiation risk, i.e. underwhich the combination effect exceeded the sum of thesingle effects, were described. This was the case, inparticular, for embryonal development, for which the risk
was almost doubled, whereas the enhancement of risk was
smaller for the proliferation of cells. The amount ofcaffeine necessary for supra-additivity, however, is so high(at least 1 mM caffeine for rather long times) that it isclearly above the range achievable in vivo by consumptionof caffeine-containing beverages. At physiological levels,
caffeine also displays antioxidant properties and inhibits
carcinogenesis induced in rats and mice by various known
carcinogens. Examples are the inhibition of smokecondensate-induced carcinogenesis in mouse skin [R13]and gastric tumour promotion by NaCl in rats [N10].
Based on these and other findings, Devasagayam et al.
[D1] suggest that at lower concentrations, the potency of
the antioxidant action of caffeine far outweighs thedeleterious effects, if any, from its inhibition of DNA
repair.
133. Besides the interference of caffeine with repair processes
as a consequence of its effect on cell-cycle blocks at highconcentrations, this ubiquitous substance also scavenges
oxygen species induced by radiation and genotoxic chemicals[K27, K28]. The chemical basis of this effect was shown to be
the removal of free electrons and hydroxyl radicals by
caffeine. The reaction rate constants for these two reactions
were shown to be about 1.5 1010 M1 s1 and 6.9 109 M1 s1,respectively [K27]. The former value is high enough tocompete with oxygen for the scavenging of free electrons andtherefore may reduce oxidative damage involving superoxideanion (O2), hydroperoxyl radical (HO2), and hydrogen
peroxide (H2O2). This mode of action is backed by recent
findings in barley seeds that caffeine affords protection only athigh oxygen concentrations but potentiates radiation damage(albeit less damage) at low oxygen pressures [K29].
134. Both a reduction of the radiation-induced G2/M phasearrest and the antioxidant effect of caffeine may indirectly
influence apoptosis and modulate survival and expansionof cells with a modified genome. In different systems, anenhancement of the degree of DNA fragmentation bycaffeine, theobromine, theophylline, and 2-aminopurine
was found in murine T-lymphoma cells [P10], whereas in
TKG cells, 2 mM caffeine eliminated the degradation of
DNA entirely [Z4]. At this stage, it is doubtful whether
these findings have any meaning for risk assessments at
controlled exposure levels.
135. Caffeine, which may potentiate radiation damage at
higher concentrations owing to its release of protective cell-

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

cycle blocks, seems also to influence the clastogenic effects ofradiation and other genotoxic agents (see also Section B.5).
Several studies found an inhibition of oxic radiation damage[K25, K26]. Stoilov et al. [S43] found both potentiation andprotection against radiation-induced chromosomal damage inhuman lymphocytes. Temperature and concentration wereshown to be decisive for the direction of the effect.
136. Kalmykova et al. [K3] evaluated the effectiveness ofjoint exposure to 239Pu and tributyl phosphate on the
induction of leukopenia in Wistar rats. It was shown in thissystem that the additive effect of the two agents delivered
simultaneously was exceeded only at high doses, i.e. acutelevels. With levels ranging from subacute effective tominimum effective, the effect of the combined treatment
was less than projected from additivity.
137. Cattanach and Rasberry[C27] reviewed the literature on
the genetic effects of combined treatments with cytotoxic
chemicals and x rays. Some pretreatments clearly enhanced
the yield of genetic damage. With spermatogonial cells,
chemicals that kill cells can substantially modify the genetic
response to subsequent radiation exposure over several days or
weeks. Both enhancement and reduction in the genetic yield
were found, and the modifications also depended on the typeof genetic damage scored, with specific-locus-mutation
response differing from that for translocations. Selective
killing of rapidly dividing cells in the areas most heavily
damaged by radiation was a suggested explanation [C28]. In
general, such interactions based on perturbations of cell
kinetics should be of little relevance for lower exposure levels.
138. Cyanate (KOCN)-induced modification of the effect of
gamma radiation and benzo(a)pyrene was studied by
Serebryanyi et al. [S80] in cultured CHO-AT3-2 cells.
Sensitizing effect was found for radiation and benzo(a)pyrene
effects such as cell viability, micronuclei induction, andmutations in the thymidinekinase and Na+/K+-ATPase loci.
The authors suggested that repair inhibition and/or changes in
the cell chromatin structure produced byKOCN is responsible
for these sensitizing effects. The proposed mechanisms as wellas the concentration and dose ranges used in the experimentpreclude direct transfer to occupational or environmental
levels.
139. In summary, many studies assessing deviations fromadditivity in combined exposures between mitogens/cytotoxicants and ionizing radiation are found in the literature(Table A.1). In most cases, the high exposure levels applied
and the biological endpoints studied do not allow the transfer
of results to humans. However, any endogenous or dietarylevels of agents influencing stem-cell population size or
kinetics will have the potential to modulate response toradiation.
6. Antioxidants, vitamins, and otherdietary factors
140. The genetic effects of combined treatments of radioprotecting agents and x rays were reviewed by Cattanach and249

Rasberry [C27]. Chemicals such as cysteamine, mexamine,and glutathione given in advance of radiation were not always
protective but gave contradictory results, with significant
protection of specific germ-cell stages being restricted todifferent dose ranges. This might be attributable to thedifferent radiation sensitivities and cell-cycle kinetics of thegerm-cell stages tested. Some pretreatments clearly enhanced
the yield of genetic damage.
141. Dietary caloric intake and type of food are importantvariables affecting the rate of spontaneous DNA damage,as was discovered recently in humans [D11, S37, S38].
These findings are supported by similar findings of reduced
oxidative damage to mitochondrial and nuclear DNA in
food-restricted rats and mice [C12]. It is known fromexperiments with rats that caloric restriction of food iscorrelated with a lower incidence of cancer, an increased
lifespan, and less free-radical damage to lipids, proteins,and DNA [W4, Y3, Y5, Y14]. Dietary fat is associatedwith increased breast cancer risk. In a study involving 21
women at high risk, the level of the oxidized thymine(5-hydroxymethyluracil) per 104 thymine was 9.3±1.9 in
the nucleated peripheral blood cells of women consuming
57 g of dietary fat per day compared with 3±0.6 for womenconsuming 32 g per day [D11, F3].
142. Diet can also modify the effectiveness of chemical
carcinogens, sometimes by a large factor. Some of theunderlying mechanisms have been identified. Rats with adeficiency of riboflavin in their diet become highly sensitive
to liver tumour formation when treated with 4-dimethylaminoazobenzene, because reduced levels of a flavin adenine
dinucleotide-dependent azo dye reductase increase theeffective dosage of the carcinogen [C25]. On the other hand,a protein-free diet prevents liver toxicity of dimethylnitrosamine in rats, and a fat-restricted diet decreases tumour
induction in mammary glands of rats. Silverman et al. [S32]studied the effect of dietary fat on mammary cancer inductionin Sprague-Dawleyrats given 3.5 Gywhole-bodyx-irradiation
at 50 days of age. Rats on a high-fat diet (20% lard) from 30
days of age had more tumours than rats on a low-fat diet (5%lard) and a higher multiplicity of carcinomas per rat. Rats on
the low-fat diet exhibited longer median tumour latency
periods than did those on the high-fat diet. Spontaneous breast
cancer incidence in humans is also influenced bythe level andtype of fat intake. Potential mechanisms in dietary-fatdependent mammarytumorigenesis were reviewed byWelsch[W5]. Yoshida et al. [Y14] reported that caloric restriction
significantly reduced the incidence of x-ray-induced myeloid
leukemia in C3H mice. Again, in this system, caloric
restriction either before or after irradiation also significantly
prolonged the lifespan of the animals.
143. In some instances, the degree of tumour formation
depends on the amount of food provided during the promoting phase and not on the nutritional status at time of
exposure. Polyunsaturated oils are potent promoters,
probably also for humans [W14]. It is now generally
accepted that restricted food intake, particularly during
development phases, reduces the incidence of neoplasms

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

and increases longevity. An epigenetic effect, namely ageneral decrease in cell duplication rates, especially in
endocrine-sensitive organs, is at the root of this finding[C26].
144. Several vitamins and many food constituents display
radical scavenging activities and antioxidant properties. Thereis considerable scientific and economic interest in the stillunresolved question whether diets enriched in vitamins,
antioxidants, carotinoids, and selenium reduce the risk ofcancer [W13]. Vitamin A and retinoic acid derivatives are
considered important micronutrients involved in themodulation of cancer risk in humans. Vitamin A seems toaffect the incidence of lung cancer in smokers and tobacco
chewers positively. Hence, clinical trials in Finland and theUnited States randomized the use among smokers of artificial
beta-carotene (precursor of vitamin A) and, in the Finnish
study, the use of artificial alpha-tocopherol (vitamin E) [H7,O14]. Surprisingly, these two studies found significant
increases in lung cancer risk related to beta-carotene use.
Whether this finding is due to the dietary form of theprovitamin remains to be elucidated. Human cervix andbladder cancer are somewhat more frequent in individuals
with low vitamin intake [S45]. These beneficial effects arethought to arise from differentiation of epithelial tissues andfrom improved cell-cell communication. Vitamins E and K
are benzo- and naphthoquinones and therefore potential
antioxidants. Reduction of tumour induction by the former inanimal systems was shown only at levels much higher thanare found in the human organism.
145. Selenium also reduces tumour risk in animal systems.
Its salts are indicated as a co-factor for glutathione
peroxidase. Vitamins C, E, and K, the latter two in thelipid phase and its boundary, prevent the formation of
nitrosamines and nitrosamides and seem to be important in
the protection of the gastro-intestinal linings, the liver, andthe respiratory tract [M31]. Although any molecule withantioxidant and radical scavenger activity is also a potential
radioprotector, the extreme speed of the interaction of reactive
species formed by radiation with DNA would require highconcentrations to make a difference. For combined effects, theavailable information indicates that micronutrients areimportant. The sizeable influence of vitamin A, vitamin E,
selenium, and 3-aminobenzamide as radioprotectors in the
C3H10T½ transformation assay is shown in Figure A.VI
[H11].
146. Borek et al. [B24] studied the anticarcinogenic action ofselenium and vitamin E. The single and combined effects ofthese chemicals were examined on cell transformations
induced in C3H10T½ cells by x rays and benzo(a)pyrene andon the levels of cellular scavenging and peroxide destruction.
Incubation of C3H10T½ cells with 2.5 µM Na2SeO3(selenium) or with 7 µM alpha-tocopherol succinate(vitamin E) 24 hours prior to exposure to x rays or thechemical carcinogens resulted in an inhibition oftransformation by each of the antioxidants with an additiveinhibitory action when the two nutrients were combined.
Cellular pretreatment with selenium resulted in increased

levels of cellular glutathione peroxidase, catalase, and nonprotein thiols (glutathione) and in an enhanced destruction ofperoxide. Cells pretreated with vitamin E did not show thesebiochemical effects, and the combined pretreatment withvitamin E and selenium did not augment the effect ofselenium on these parameters. These results support thenotion that free-radical-mediated events play a role inradiation and chemically induced transformation. They
indicate that selenium and vitamin E act alone and in additive
fashion as radioprotecting and chemopreventing agents. The
results further suggest that selenium confers protection in partby inducing or activating cellular free-radical scavenging
systems and by enhancing peroxide breakdown, whilevitamin E appears to confer its protection through another,complementary mechanism.
TRANSFORMATION PER SURVIVING CELLS (x 10-4 )

250

16
14
12
10
8
6
4
2
0
Gamma rays
only

TPA

Gamma rays plus
Vitamin A Selenium Vitamin E Benzamide

Figure A.VI. Effect of chemical and dietary factors on
response of C3H10T½ cells in combined exposureswith gamma rays giving a dose of 4 Gy [H11].

147. The importance of sulfhydryl groups as an antagonist
of or protector against radiation-induced radical attack on
DNA is well known from molecular and in vivo studies[M4, M6, M7, V4]. In view of the fast and localized actionof ionizing radiation, these substances have to be smallenough to reach the target to be protective and to bepresent there in considerable concentrations for noticeable
effects. Even for small water-soluble substances displaying
sulfhydryl groups, such as cysteamines, this is difficult toachieve in humans. Therefore the use of sulfhydryl
radioprotectors is limited by their toxicity and the shortperiod during which they are active [M4]. At
environmental levels, no protective effect is to beenvisaged. However, for lipophilic substances such asvitamins A, E, and K or for coenzymes with high-affinity
binding to active centres, local concentrations in specificcompartments might become high enough for a protective
effect, even with low dietary intakes. In addition to these
directly acting protectors, immunomodulators such as
endotoxins and bacterial or yeast polysaccharides are
known to protect against the deleterious effects of radiation[M4]. Their mode of action, stimulation of the reticuloendothelial system, is probably irrelevant for stochastic
effects.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

148. Ethanol consumed in alcoholic beverages is known toincrease the incidence of several cancers of the oral cavity
and the oesophagus, especially in combination with active
cigarette smoking [I8]. No data from human studies on
ethanol and ionizing radiation are available, but theirritating effect of higher concentrations of ethanol makes
this form a potential tumour promoter. Mechanistic studies
also suggest that ethanol modifies the biochemical activation in the oral cavity and the oesophagus of some tobaccospecific carcinogens [S44], a mechanism of no direct relevance to radiation. Acetaldehyde is a toxic reactive metabolite of ethanol in tissue where biotransformation occurs.
Very important in view of population health are behaviouralchanges and a tendency to malnutrition in alcohol addicts,
which may increase their susceptibility to toxicants in theenvironment or at the workplace. The many direct andindirect effects of alcohol consumption on radiation-inducedchanges at the cellular, organ, and behavioral levels werediscussed in depth in a review by Ushakov et al. [U18]assessing experiences in human populations affected bythe
Chernobyl accident.

251

1% NaCl diet were much higher than other combinedtreatment groups. The incidence of atypical hyperplasia
was less than 6% in all treatment groups, and no promoting effect on gastric tumorigenesis was observed. These
results demonstrated that the occurrence of intestinal metaplasia induced by x rays can be significantly modified bybasic and common food constituents, but this is not
associated with any influence on gastric neoplasia.

149. Iodine as a constituent of thyroxine, the hormone of
the thyroid gland, is often deficient in inland areas, where
geological factors and the absence of seafood produce a diet
low in iodine. Since its fission yield, relative volatility, andhalf-life make 131I one of the critical fission products that
may be present in environmental exposures, the potential
increase of thyroid dose per unit uptake in humans withiodine-deficient diets is a major concern in radiation
protection. It is still not known whether the higher
stimulation of the gland in iodine deficiency by endogenous
hormones will also alter the radiosensitivity of the stem
cells and the risk coefficient for thyroid carcinoma. The
wealth of data, mainly from therapeutic procedures innuclear medicine, showing little to no carcinogenic
potential for 131I, even at high exposures [U2], is somehow
contradicted by recent results showing large increases inthyroid carcinoma in children affected by the Chernobyl
accident. Initial measurements of iodine in urine from
Belarus indicated that areas most heavily affected byiodine
deposition are also deficient in a dietary supply of iodine
and are endemic goitre areas.

151. A potentially important interaction was investigatedby Montour et al. [M24], who studied the modification ofradiation carcinogenesis by marihuana (tetrahydrocannabinol, delta(9)-tetrahydro-cannabinolic acid). Male,
female, and ovariectomized female Sprague-Dawley rats
were irradiated with doses of 1.5, 3, or 4 Gy, respectively,from 60Co gamma rays at between 40 and 50 days of age.
The animals were injected three times weekly with either
marihuana extract or with alcohol-emulphor carrier. Mean
survival time in males was significantly shorter in the 4 Gyplus marihuana group compared with the three othergroups, whose mean survival times did not differ.
Throughout the 546-day period in which the male rats
were observed, the total number of tumours other thanfibrosarcomas was significantlygreater following radiationand marihuana administration (22) than following
irradiation alone (6). Fifteen of the tumours originated inbreast or endocrine tissues. No differences were seen in theunirradiated groups. In the females, which were observedfor 635 days, the total number of breast tumours was
significantly higher in the combined treatment group (38)compared with the group treated with radiation alone (22).
This was entirely due to a marked difference in the adenocarcinoma incidence, which was 21 (radiation plus marihuana) compared with 4 (radiation alone). The number of
adenofibromas was similar in the two groups. In theunirradiated female groups, the breast adenocarcinoma
incidence was 8 in the marihuana group and 2 in thecontrol group. Ovariectomy resulted in a lower breast
tumour incidence in all groups. Non-breast tumours weremore frequent in the ovariectomized-irradiated groups.
Radiation plus marihuana produced more non-breast
tumours (25) than radiation alone (17) in the ovariectomized females.

150. The modifying influence of sodium chloride (NaCl),miso (Japanese soybean paste), and ethanol on thedevelopment of intestinal metaplasia after x-ray exposure
was examined by Watanabe et al. [W29] in CD(SD):Crj
rats. Intestinal metaplasia in the glandular stomach isconsidered a precursor lesion for differentiated gastricadenocarcinoma. Five-week-old rats were treated with two
doses of 10 Gy from x rays to the gastric region at a threeday interval. After exposure, the rats were given NaCl (1%or 10% in diet), miso (10% in diet), or ethanol (10% in
drinking water) for 12 months. The number of alkaline
phosphatase-positive foci of intestinal metaplasia in rats
given a 1% NaCl diet after x rays was significantly
elevated compared with that in rats given x rays alone or
x rays with a 10% NaCl diet. In the pyloric gland mucosae,
total numbers of metaplastic foci in rats given x rays and152. Dietary factors are proven modifiers of risk fromdiverse agents at levels found in human populations andprobably also influence the production and repair ofendogenously arising lesions. Absence or deficiency ofimportant coenzymes and nutrients on one side and highlevels of directly or indirectly acting mitogens on the otherinterfere with molecular, cellular, and tissue responses toionizing radiation. In view of the many mechanisms
involved, the full spectrum of interactions from antagonisms to synergisms must be expected (see also
Table A.1). A reduction in the radiation risk may occur insituations where growth stimuli are reduced owing tonutritional deficiency or where the number of stem cells at
risk are reduced. Synergisms are to be expected where
reduced levels of radical scavengers or coenzymes needed
for repair increase the yield of primary damage from

252

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

ionizing radiation or impair the speed and accuracy ofcellular responses to damage. In general, the health risks
not only from ionizing radiation but also those from mostother deleterious agents in the human environment will beaffected by deviations from an optimum diet.

C. RADIATION AND BIOLOGICAL AGENTS,
MISCELLANEOUS
1. Hormones
153. Many hormones are potent growth stimulators.
Considerable evidence is available for the modulation ofcancer risk by hormones. Animal experiments have shownthat increased levels of thyroid-stimulating hormone (TSH)
can enhance tumour growth and increase the risk of cancer[D31]. Thyroid stimulating hormone is increased duringpuberty and pregnancy as a result of increased levels of
female sex hormones [H37, P25]. There is epidemiologicalevidence suggesting that the development of thyroid cancer
after high-dose radiation exposure in females can bepotentiated bysubsequent child bearing. Marshall Islanders
who were exposed to radioactive fallout from a nuclear
weapons test in 1954 received high thyroid doses fromradioiodines. Women who later became pregnant were athigher risk of thyroid cancer than exposed women who
remained nulliparous [C43]. The numbers, however, were
small.
154. The same effect was found in a population-based
case-control study in Connecticut in the United States
involving 159 subjects with thyroid cancer and 285
controls [R11]; 12% of the cases but only 4% of thecontrols reported prior radiotherapy to the head and neck.
Among women, this risk appeared to be potentiated bysubsequent live births (RR = 2.7). The risk for ever parous
alone was, however, higher (1.6) than for priorradiotherapy (1.1). Another case-control study, carried out
in Washington in the United States, linked a 16.5-foldincreased risk of thyroid cancer to prior radiotherapy of thehead and neck among 282 females and 394 controls [M17].
Overall, 20.2% of the cases but only 1.5% of the controls
reported earlier radiotherapy (RR = 16.5; 95% CI:
8.133.5). In this study, pregnancy following radiotherapy
was associated with only a small additional risk (RR =1.3), which was far from statistically significant (95% CI:
0.115.7) [M9]. Combined with similar findings from
Sweden [W30], these studies suggest that TSH-mediatedtissue proliferation in adolescence and pregnancy may be
a risk factor in radiation-induced thyroid cancer.
155. The long-term use of tamoxifen, a synthetic antioestrogen that has been shown to reduce mortality frombreast cancer and the occurrence of contralateral breast
cancer, increases the risk for endometrial cancer. In a casecontrol study of woman treated for breast cancer, Sasco etal. [S26] showed that tamoxifen or radiation castration(which included high doses to the uterus as well as theovaries) considerably increased the risk for subsequent

endometrial cancer. The odds ratios for tamoxifen use formore than five years and radiotherapeutic castration were
3.5 and 7.7, respectively. Women who had undergone
combined treatment had an odds ratio of only 7.1. Since
the study was based on small numbers (43 cases and 177
controls), the power is not sufficient to postulate anantagonism, but there is enough evidence to reject anenhancement of risk between the two carcinogenic factors.
156. One well-studied interaction is that between radiationand the natural hormone oestradiol-17 beta (E2) in mammary carcinogenesis. In a publication by Broerse et al.
[B32], the combined effects of irradiation and E2 administration on the mammary gland in different rat strains were
investigated. Three rat strains, Sprague-Dawley, Wistar
WAG/Rij, and Brown Norway, with different susceptibilities to the induction of mammary cancer, were irradiated with x rays and mono-energetic neutrons; increased
hormone levels were obtained by subcutaneous implantation of pellets with E2. Mean plasma levels were 100300
pg ml1 plasma, while normal levels in these rat strains
were about 50 pg ml1. The latency period for the hormonetreated animals was shown to be considerably shorter thanfor animals with normal endocrinological levels. Administration of the hormone alone also appreciably increased
the proportion of rats with malignant tumours. At the highlevels of hormones applied in the study, there was little
indication that radiation and hormones produced any
supra-additive effect, but the single-agent effect levels inthis study might have been too high to properly assess thisother effect. The effect of hormone administration andirradiation on mammary tumorigenesis was the same forhormone administration one week prior to or 12 weeks
after irradiation. The RBE values for induction of mammary
carcinomas after irradiation with 0.5 MeV neutrons have amaximum value of 20 and are not strongly dependent on
hormone levels.
157. The carcinogenic and co-carcinogenic effects ofradiation on rat mammarycarcinogenesis and mouse T-cell
lymphomagenesis were studied by Yokoro et al. [Y6]. For
both experimental models, the study clearly showed theimportance of the promotion stage and of the physiologicalcondition of target cells at the time of initiation. In rat
mammary carcinogenesis, prolactin was shown to be a
powerful promoter regardless of the initiating agent. The
authors also suggested that an enhancer like prolactin
might be useful in detecting the carcinogenicity of small
doses of carcinogens; for example, a high RBE of 2.0 MeV
fission spectrum neutrons was demonstrated by theapplication of prolactin to radiation-initiated mammary
carcinogenesis in rats. Because cellular reactions are
somewhat different for different LET values, it remains tobe proven that the sensitizing effect of a hormone isindependent of radiation quality.
158. Shellabarger et al. [S24] investigated the influence of
the interval between neutron irradiation and diethylstilbestrol (DES) on mammary carcinogenesis in female ACI
rats. Both radiation and DES are carcinogens for theANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

mammary gland of ACI female rats and act in a synergistic
fashion, particularly with regard to the number ofmammary adenocarcinomas per rat when DES is given at
about the same time as radiation. DES, in the form of acompressed pellet containing a mixture of cholesterol and
DES, formulated to average 1.25 mg of DES per 100 gbody weight, was given to groups of approximately 28 rats
at 2 days before or 50, 100, or 200 days after 0.064 Gy
from 0.43 MeV neutron irradiation. Every time DES wasgiven to irradiated rats, it was also given to non-irradiatedrats. When the total number of mammaryadenocarcinomas
375 days after administration of DES was analysed as apercentage of 24 sites per rat at risk, DES and radiation
always produced a response that was larger than the sumof the responses of DES alone and radiation alone. The
supra-additive interaction between radiation and DES did
not decline as the time interval between irradiation and
DES was lengthened, which suggests that neutron-initiatedmammary carcinogenesis is not subject to repair, since
DES promotion continues to be effective for long times.
159. Irradiation of pregnant Wistar rats at days 7, 14, and
20 of pregnancy, followed by DES treatment after nursing
for one year, showed a strong correlation of mammary
gland tumours with the hormonal status of the gland
during radiation exposure [I11]. Irradiation alone (2.6 Gy)
resulted in a 23% incidence of mammary gland tumours.
The additional implantation of a DES pellet (releasing
about 1 mg d1) increased this value to 35% and 93% forradiation exposure at days 14 and 20, respectively. The
data suggest that the initiation of tumorigenesis by gamma
rays is critically dependent on the developmental status of
the gland at exposure. Since no group with DES exposure
only was included, no direct assessment of the combined
effects is possible from this study. When the radiation
exposure was delayed to day 21 of lactation or day 5 postweaning, combined treatment with gamma rays and DES
resulted in an incidence of mammary gland tumours of
94% and 73%, respectively. Since the value from combined
treatment in virgin animals was only 24%, it is suggested
that the differentiation state of the radiation-exposed tissue
is more relevant than the hormonal and proliferative stateof the cell populations at risk [S40]. Rats with weaning
experience receiving only a gamma dose at day 21 oflactation or DES had tumour incidences of 35% and 27%.
When compared with the combined effect of 94%, asynergistic effect has to be postulated.
160. The effect of age and oestrogen treatment on radiation-induced mammary tumours in rats was analysed by
Bartstra et al. [B71, B72]. The excess normalized risk of
mammary carcinoma was 0.9 for 1 Gy and 2.2 for 2 Gy in
the age groups 8, 12, 16, 22, and 36 weeks, with nosignificant differences between the age groups. However,
irradiation at 64 weeks yielded fewer carcinomas than in
the controls, the excess normalized risk being 0.7 and 0.3
for 1 and 2 Gy, respectively. After oestradiol-17 beta2
treatment, the excess normalized risk for carcinomas was
7.7 for both 1 and 2 Gy in the age groups 8, 10, 12, and 15
weeks, with no significant differences between the age

253groups. However, in the age groups 22, 36, and 64 weeks,
the excess normalized risk decreased with increasing age
at exposure. Irradiation at 64 weeks yielded fewer carcinomas
than in controls, with an excess normalized risk of 0.6 forboth 1 and 2 Gy. The excess normalized risk was 1080 inoestrogen-treated controls compared with untreated animals.
The findings indicated that administration of oestrogen
increased the radiation sensitivity of the mammary gland inyoung animals considerably. Administration of oestrogeninfluenced the shape of the dose-response curve for radiationinduced mammary cancer in young rats. In untreated animals
there was a linear dose-effect relationship, whereas inoestrogen-treated ones the relationship could onlybe describedby a quadratic function. In older rats, radiation dose-effectrelationships in oestrogen-treated and non-treated animals
were best fitted by linear relationships. The reduced risk ofradiation exposure at mid-life was observed in oestrogentreated and control rats.
161. The influence of androgens in the development ofradiation-induced thyroid tumours in male Long-Evansrats was investigated by Hofmann et al. [H12]. Wheneight-week-old male rats were treated with radiation(1.5 MBq Na131I), thyroid follicular adenomas andcarcinomas were observed at 24 months of age with a highincidence, 94%. Castration of males prior to irradiation
significantly reduced this tumour incidence to 60%. Whentestosterone was replaced in castrated, irradiated male rats,
differentially increased incidences of thyroid tumours
occurred, depending on the time interval for hormone
replacement. Immediate (age 26 months) or early (age612 months) testosterone replacement at approximate
physiological levels led to thyroid follicular tumour
incidences of 100% and 82%, respectively, whereasintermediate (1218 months) or late (1824 months)
testosterone treatment led to only 70% and 73% incidences,respectively. Continuous testosterone replacement (224months) in castrated, irradiated male rats raised the thyroid
tumour incidence to 100%. Only the two 100% values are
significantly different from the value of 60% in castrated
irradiated animals not receiving testosterone replacement.
Since elevated TSH is a reported requisite for thedevelopment of radiation-associated thyroid tumours, theeffects of testosterone on serum thyroid-stimulating
hormone levels were examined. Mean serum thyroidstimulating hormone values in all irradiated animal groups
were significantly elevated and well above those in agematched, non-irradiated animals at 6, 12, 18, and 24
months. Serum thyroid-stimulating hormone levels werehigher in continuous testosterone-replaced irradiatedcastrates than in intact, irradiated males but lower in
irradiated castrates without testosterone treatment. Interval
testosterone replacement in castrated male rats was generally
associated with increased serum thyroid-stimulating
hormone levels during the treatment interval and withlowered thyroid-stimulating hormone levels after
discontinuation of testosterone treatment, particularly in
irradiated rats. However, when irradiated, castrated males
received late (age 1824 months) testosterone replacement,
there was no elevation of thyroid-stimulating hormone at

254

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

the end of the treatment interval. Thus an indirect effect of
testosterone via early stimulation of thyroid-stimulating
hormone may be at least partly responsible for the highincidence of radiation-induced thyroid tumours in malerats.
162. Watanabe et al. [W28] examined the influence of sex
hormones on the induction of intestinal metaplasia by x rays
in five-week-old Crj:CD(SD) rats of both sexes. At the age offour weeks, the animals were gonadectomized and given
testosterone or DES in the form of subcutaneous implants
containing 0.252.5 mg hormone. One week later, they were
irradiated with x rays to give two doses of 10 Gy to the gastricregion at a three-day interval, for a total of 20 Gy. Six months
after radiation exposure, the incidence of intestinal metaplasia
with alkaline phosphatase (ALP) positive foci in males was
significantly higher than in females, in orchidectomized
males, or orchidectomized plus DES-treated rats. The
incidence of intestinal metaplasia with ALP-positive foci innormal females appeared lower than in ovariectomized
females and was increased by treatment with testosterone ordecreased by DES. Numbers of foci of intestinal metaplasia
with Paneth cells and total numbers appeared to increase inmales treated with DES. These results suggest a promotingrole for testosterone in the development of radiation-induced
ALP positive lesions and alsoindicate considerable differencesamong intestinal metaplasia subtypes in their response tohormone stimulation.
163. Rat prostate tumours after androgen ablation by
castration showed an increase, from 0.4% to 1.0%, of theapoptotic index as determined by the TUNEL assay. The
apoptotic index did not vary significantly over time after
castration. Irradiation of intact rats to 7 Gy resulted in an
apoptotic response of 2.3%. When castration was initiatedthree days prior to irradiation, peak levels of 10.1% for the
apoptotic response were recorded. Androgen restoration
with testosterone implants restored the intact animal
response [J10].
164. In conclusion, it can be said that many hormones are
powerful regulators of cell proliferation and programmed
cell death in specific tissues and organs. The resulting
influence on radiation risk per unit dose is well proven (seealso Table A.1). An important part of differences in risks
linked to gender or age may be traced to hormones acting
as endogenous growth factors.
2. Viruses, bacteria, and genetic sequences
165. Viruses, bacteria, and microbial genetic sequences
have been shown to play an important role in thepathogenesis of animal tumours. Human malignancies
such as Burkitt lymphoma and T-cell leukaemia are causedby the Epstein-Barr virus [L12] and the retrovirus HTLV-1[D4], respectively, and a variety of carcinomas, including
cervical, skin, anal, and others, by papilloma viruses[H13]. Hepatitis type B and C virus, the bacterium
Heliobacter pylori, some parasites such as Opistorchis
viverrini and Schistosoma haematobium are proven or

putative causes of hepatoma, gastric cancer, cholangiocarcinoma and urinary bladder cancer, respectively [M64].
One mechanism of interaction might be the inhibition of
DNA repair by viral proteins. The HBV protein HBx was
shown to interact with cellular DNA repair capacity in a
p53-independent manner after ultraviolet C irradiation[G37]. Interaction of cancer viruses with radiation may
also occur by mutation or translocation of dormant viralsequences. In a multi-stage process, virally infectedorganisms may also be much more susceptible to radiationinduced cancer if a virus is causing or facilitating one of
the genetic transformations leading to the outbreak ofmalignancy. Astier-Gin et al. [A25] investigated the roleof retroviruses in murine radioleukaemogenesis in C57B1
mice. The protocol associated the injection of a non-pathogenic retrovirus (T1223/B virus) and a dose from x rays (2× 1.75 Gy), which alone was non-leukaemogenic in thissystem. Thymic lymphomas induced by the combined
effect of virus and irradiation or irradiation alone wereanalysed for MuLV proviral organization and RNA
expression with the Southern or Northern blotting
techniques, respectively. The active involvement of theretrovirus was shown by the detection of a recombinant
provirus in the chromosomal DNA of every tumour
induced by the combined treatment with virus andradiation. No specific site in the genome was found forprovirus integration and no relationship was observed
between viral RNA expression and tumour induction.
Trisomy 15 was observed in all metaphases irrespective of
the protocol of tumour induction. The G-banding techniquerevealed an extra band in several thymic lymphomas
induced by irradiation and T1223/B virus injection. This
complex pattern of viral behaviour may pose great
obstacles for diagnosis and for the elucidation of risk fromcombined exposures.
3. Miscellaneous factors
166. Many other sometimes poorly defined biologicalmaterials have also been shown to influence the responseof organisms to ionizing radiation. For example, themodulating effect of microbial substances on survival after
acute radiation doses in mammals (mice, rats, dogs, sheep,and monkeys) was studied by Andrushenko et al. [A12].
The highest protection was found for some vaccines containing inactivated bacteria and given before the radiation
exposure. Polysaccharides, lipopolysaccharides, and proteinlipopolysaccharide complexes were also able to increase
the radioresistance. The mechanisms involved in themodulation of the status and the number of stem cells of
the immune system remains to be elucidated. Such effects
might also be of importance at low exposure levels, e.g. formalignancies of the haematopoietic system.
167. To test the hypothesis that low-dose radiation, suchas is used for diagnosis, may act as a co-carcinogen in
inflammatory bowel disease, Weinerman et al. [W2]induced inflammation with DMH in a mouse system tostudypotential sensitization towards the radiation exposure(see Section B.1 for genotoxic action of DMH). Four

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

groups of BALB/c mice (a control, DMH, DMH plus lowdose radiation, and low-dose radiation) were studied. No
protective or carcinogenic effects of the radiation in combination with DMH were found compared with DMH
alone. This type of negative experimental finding isdirectly important for radiation protection of the patient, inthat individuals with inflammatory bowel disease undergo
many diagnostic x-ray examinations throughout life.
168. A strong effect was, however, found from theinteraction of ionizing radiation with surgical procedureson the stomach. Griem et al. [G15] followed patients withpeptic ulcer who had received radiotherapy to control
excessive gastric acid secretions, a method used between
1937 and 1965 (mean dose to the stomach = 14.8 Gy). The
mortality study involved 3,609 patients; 1,831 were treatedwith radiation and 1,778 were treated by other means.
Compared with the general population, patients treatedwith or without radiation were at significantly increased
risk of dying of cancer and non-malignant diseases of thedigestive system. Radiotherapy was linked to significantly
elevated relative risk for all cancers combined (RR = 1.53;
95% CI: 1.31.8). Radiotherapy and surgery together
increased the rate of stomach cancer (RR = 10) above thesum of individual effects. There is no specific informationon co-carcinogenic mechanisms in the post-surgicalreaction of stomach tissue or on tumour location.
169. The influence of pre-immunization with a rectal
extract on radiation-induced carcinoma of the rectum wasstudied by Terada et al. [T37] in 47-week-old A/HeJ
mice. The animals received 40 Gy (20 Gy per week fromx rays) in the pelvic region with or without two priorinjections of rectal extract from adult animals of the samestrain emulsified with complete Freud’s adjuvant. Aftereight months, rectal adenocarcinomas were observed insignificantly higher numbers in pre-immunized micecompared with non-immunized animals (62% vs. 18%).
The results indicate that local immunological reactions
sensitize to the carcinogenic action of ionizing radiation.
170. Finally, the effect of psychosocial factors such as fear,
anguish, and chronic stress on the health status ofindividuals and populations, both in psychosomatic
expressions and in the subjective perception of radiationexposed persons, is clearly an important problem during
and after accidents and cases of environmental
contamination, such as seen in areas affected by the
Chernobyl accident [I13]. However, a review of these
aspects is beyond the scope of this Annex and involves
professional disciplines outside the realm of UNSCEAR.
Despite the attention given by the media to the potential
deleterious effects of ionizing radiation in combination
with conventional industrial pollutants in such instances,
little scientific information is available on specificexposure situations. Some potentiallyimportant modifying
factors are discussed in connection with dietary factors in
Section B.6.

255

D. COMBINED MODALITIES IN
RADIATION THERAPY
171. A large number of chemotherapeutic drugs are used inclinical cancer therapy in combination with radiation. The
main ones in use or about to be used are described in thisSection, with emphasis on the mechanisms of interaction
between the drugs and radiation to reveal possible
mechanisms of interaction between chemical agents andradiation under environmental and normal occupationalsettings. The main findings relating to modes of action andcombined effects are summarized in Table 6. However, it
should be clearly noted here that the final goal of tumourtherapy-related studies is tumour control and therefore cell
death (apopotosis, necrosis) or cell inactivation (loss of
proliferative capacity, differentiation, senescence). These
effects are mostly deterministic and often mechanisticallydifferent from the stochastic radiation effects that are ofconcern in radiation protection. Therefore, highly sigmoidal
dose-effect relationships and considerable threshold doses arefound for the contribution of many of these agents to theinteraction with radiation. Several groups of agents are also
covered in the preceding sections, e.g. alkylating agents under
the heading “genotoxic chemicals”.
1. Alkylating agents, nitrosoureas, andplatinum coordination complexes
172. Alkylating agents were among the first compounds
found to be useful in cancer chemotherapy, and because of
their variety and relative tumouricidal selectivity, theyremain important components of many modern
chemotherapeutic regimens. Although the alkylating
agents are a diverse series of chemical compounds, they all
have the common property of displaying a positively
charged, electrophilic alkyl group capable of attacking
negativelycharged, electron-rich nucleophilic sites on most
biologic molecules, thereby adding alkyl groups to oxygen,nitrogen, phosphorus, or sulphur atoms. Theirchemotherapeutic usefulness derives from their ability toform a variety of DNA adducts that sufficiently alter DNA
structure or function, or both, so as to have a cytotoxic
effect [L37]. Many of the pharmacologically useful agents
undergo a complex activation process.
173. The most common site of DNA alkylation is the N-7
position of guanine. Alterations at this position are relatively
silent in their effect on DNA function, because these adducts
do not interfere with the base-pairing scheme. In contrast,
adducts at the N-3 position of cytosine, the O-6 position of
guanine, and the O-4 position of thymidine interfere with the
Watson-Crick base-pairing scheme and are therefore likely tointerfere with fidelity of replication and transcription, leading
to mutagenicity and cytotoxicity. In addition to directinterference with replication and transcription, the formationof DNA adducts leads to a variety of structural lesions,
including ring openings, base deletions, and strand scissions[B41, F20, H20]. Many of the DNA adducts and lesions arefurther acted on by repair enzymes that can restore theintegrity of the DNA, or if the repair process is only partially

256

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

completed, they can cause additional DNA damage, such as
the creation of apurinic or apyrimidinic sites or DNA strand
breaks. Bifunctional alkylating agents with the capacity togenerate two electrophilic groups and to form two adducts arecapable of forming DNA-interstrand and DNA-protein crosslinks that interfere directly with DNA replication, repair, andtranscription [L38].
174. Alkylating agents are cell-cycle-dependent but notcell-cycle-specific. They exert their cytotoxic effects oncells throughout the cell cycle but have quantitatively
greater activity against rapidly proliferating cells, possibly
because these cells have less time to repair damage before
entering the vulnerable S phase of the cell cycle [T19].
Cells in which cross-links occur accumulate and die in theG2 phase of the cell cycle. Persistent DNA strand breaks
may result in lethal chromosomal damage in the mitotic
phase of the cell cycle.
(a) Nitrogen mustards (mechlorethamine,
melphalan, chlorambucil, cyclophosphamide)
175. Nitrogen mustard, originally studied for its potential
as a vesicant in chemical warfare, is a highly reactive
analogue of sulphur mustard and was the first alkylating
agent introduced into clinical therapy [G22]. Exposure tothese alkylating agents results in the formation of simple
DNA adducts, DNA-interstrand cross-links, and DNAprotein cross-links [E12].
176. Experimental investigations of interactions between
derivatives of nitrogen mustard and radiation in vitro showedthat these interactions are additive, independent of sequenceof treatment with the two agents, and not markedlyinfluenced
by the interval between treatments [D14, H21]. An isobolic
analysis confirms the additivity, although when radiation
precedes the mustard by 4 hours, the effect is on the borderlineof supra-additivity, indicating that the two agents may share
a common mechanism of cell killing [D14]. Neither radiosensitization nor interference with sublethal damage repair has
been implicated in these interactions. Hetzel et al. [H21],
examining the effects of combined treatment on V79 cellspheroids, put forth the interesting proposal that theenhancement seen with the nitrogen mustard derivative
chlorambucil in combination with irradiation may be related
to its ability to alter the internal oxygen profile in spheroids,
resulting in partial reoxygenation.
177. The main use of nitrogen mustards was in thetreatment of lymphomas, breast and ovarian cancer, andcancers of the central nervous system. A prospective
randomized study examined whether MOPP (nitrogenmustard, vincristine, procarbacine prednisone) therapy
alone is superior to combined modality treatment ofextended field radiation and MOPP in patients with
Hodgkin’s disease [O13]. No significant differences werenoted between the combined modality therapy and therapy
with MOPP alone. However, overall toxicity was different.
Viral and fungal infections occurred more frequently in the
combined modality. In an overview by Cuzick et al. [C44]of post-operative radiation therapy of breast cancer, nodifference was seen in mortality over the first 10 years
between patients treated with radiation therapy. After 10
years, however, there was a lower survival associated withradiation therapy. In recent years, chemotherapy has beenfavoured for breast cancer treatment. However, the use of
post-operative radiation therapy needs to be reconsideredin patients who receive adjuvant chemotherapy and inwhom drug resistance develops, leading to failure ofchemotherapy. By decreasing the local tumour burden,
adjuvant radiation therapy may decrease the probability ofdrug resistance and increase the probability of cure in thosepatients [H42].
(b) Nitrosoureas
178. The chloroethylnitrosoureas, including 1,3-bis(2chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3cyclohexyl-1-nitrosourea (CCNU), and methyl-CCNU (MeCCNU), are highly lipophilic and chemically reactive
compounds that are clinically active against a variety oftumours (reviewed in [L39]). Chemical decomposition ofthese agents in aqueous solution yields two reactive intermediates, a chloroethyldiazohydroxide and an isocyanategroup [C30, M40]. The latter react with amine groups ina carbamoylation reaction. The isocyanates are believed todeplete glutathione, inhibit DNA repair, and altermaturation of RNA. The chloroethyldiazohydroxide undergoes further decomposition to yield reactive chloroethylcarbonium ions that form a variety of adducts with all four
DNA bases and the phosphate groups of DNA. Of major
importance in the antitumour effects of nitrosoureas is theformation of DNA interstrand cross-links, as demonstrated
by the close correlation between cross-link formation andcytotoxicity [E13, K30, L40]. Alkylation seems to be themore important feature of direct nitrosourea action.
179. Additive or greater-than-additive responses have beenrecorded in in vitro and animal studies, with the greatest
enhancement associated with the presence of the drug insome experiments before irradiation and in others after
irradiation. Deen and Williams [D15] provided an isobolic
analysis of the effects of combined BCNU-radiation treatment of 9L rat brain tumour that suggested some concentration dependence of these interactions. At two levels of
BCNU (1 and 7.5 mg ml1) all data points fell within the
additivity envelope, indicating similar mechanisms ofaction for the drug and radiation, but at other levels (3 and5 mg ml1) supra-additivity was noted, suggesting thatalternative mechanisms might be operating. CCNU
resulted in less interaction than did BCNU [D16], a finding
confirmed by the study of Kann et al. [K31] on L1210
cells. In experiments comparing the radiosensitizing effects
of four nitrosoureas, the compound without alkylating
activity, 1,3-bicyclohexyl-1-nitrosourea (BCyNU), was themost effective sensitizer [K31]. BCyNU was reported to
selectively inhibit glutathione reductase activity [M41].
Kann et al. [K31] concluded that because the agent without
alkylating activity was the most potent radiation synergist,
alkylation was not involved in the enhancing effect, which

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

may relate instead to differential repair-inhibiting activity.
However, even if the enhancement of the cytotoxic effectsof ionizing radiation could be ascribed to repair inhibition,
details of the mechanisms of cell killing are still not clear,because inhibition of DNA repair by the nitrosoureas was
not complete and permanent [K32]. Rather, their effect was
to slow the rate of strand rejoining, prolonging the period
when numerous unjoined breaks are present, and lethality
was considered a consequence of this prolongation.
180. Controlled clinical trials have demonstrated the efficacy
of nitrosoureas combined with irradiation as adjuvant therapy
for glioblastoma and anaplastic astrocytoma (reviewed in[L39]).
(c) Platinum coordination complexes
181. Cisplatin and its analogues are an important group ofagents now in use for cancer therapy [R15]. Cisplatin (cisdiaminodichloroplatinum (II)) can bind to all DNA bases,
but in intact DNA, there appears to be preferential binding
to the N7 positions of guanine and adenine [B42, M42,
P12]. Cisplatin binds to RNA more extensively than toDNA, and to DNA more than to protein [P13]. In thereaction of cisplatin with DNA or other macromolecules,
the two chloride ligands can react with two different sites
to produce cross-links [E14, E15, F21, F22]. Studies of theeffects of platinum DNA binding on the three-dimensional
structure of the DNA double helix revealed that theplatinum lesions cause bending of the DNA double helix,
suggesting that the stereochemistry of the platinum
molecule is maintained and that DNA is modified in its
three-dimensional conformation [R14]. The cytotoxicity ofcisplatin against cells in culture has been found to berelated directly to total platinum binding to DNA and tointerstrand and intrastrand cross-links. Intrastrand
guanine-guanine cross-linkage inhibits DNA replication[G23, P14]. Diaminocyclobutane-dicarboxylatoplatinum(II), carboplatin, and other cisplatin analogues appear tohave subcellular mechanisms of action similar to cisplatin.
They form lesions with DNA that are recognized by
antibodies reacting with cisplatin-DNA lesions [P15].
182. More than two decades ago, Zak and Drobnik [Z6]reported an apparent interaction between cisplatin andionizing radiation after whole-body irradiation of mice.
Since then, cisplatin has been reported to enhance thecytotoxicity of radiation in a number of studies in both cell
culture and tumour-bearing animals (reviewed in [B26,
B43, C31, D17, D18, D19, H22, H23]). Isobolic analysis
provides some evidence that this interaction can be supraadditive [D20]. A pronounced inhibition of repair of bothradiation-induced potentially lethal damage and sublethal
damage by platinum drugs has been demonstrated inseveral cell lines [B44, C32, D18, D21, O3, Y8]. The
survival curves of cells exposed to platinum compounds
have either a reduction or no shoulder, and this effect isinterpreted as evidence for the inhibition of sublethal
damage repair by platinum because of the role of sublethal
repair in the formation of the shoulder of the radiation

257

survival curve. The enhanced killing of irradiated cells byplatinum compounds may be due to an enhanced
production of DNA double-strand breaks. Repair of DNAplatinum adducts results in a gap that, in association withradiation-induced DNA single-strand breaks (rejoining of
which is retarded by platinum compounds), produces new
DNA double-strand breaks [Y9, Y10].
183. DNA-protein cross-links and the binding of highmobility-group proteins to DNA-platinum lesions seem toplay a role in the radiosensitizing mechanism of cisplatin
at moderate doses in hypoxic cells [K33, S50, S51, W16].
However, comparable in vivo experiments with RIF-1
tumours in mice failed to show the preferential radiosensitization of hypoxic cells at low radiation doses bycisplatin [S52]. Herman et al. [H24] provided evidence that
intracellular pH is an important variable in the action of
cisplatin as a radiosensitizer of hypoxic cells using murine
fibrosarcoma cells in vitro. Radiosensitization of cancer
cells in vitro and as spheroids was observed when platinum
drugs were delivered before and during irradiation. In
addition, enhanced cell killing was demonstrated whenthese drugs were added immediately after irradiation(reviewed in [B26, H22, H23, S53]).
184. A number of animal in vivo studies have reported
sequence-dependent positive interactions between the twomodalities. Increased lifespan was reported when cisplatin
was administered before whole abdominal irradiation ofKrebs II ascitic carcinoma-bearing mice compared withcisplatin after irradiation [J8]. Supra-additivity wasreported when cisplatin was given before x rays in SCCVII[T20, Y11] and RIF-1 [L41] or simultaneously in SCCVII
and RIF-1 carcinoma-bearing mice [K34].
185. The platinum coordination complexes are the mostimportant group of agents now in use for cancer treatment.
They are curative in combination therapy for testicularcancer and ovarian cancer and play a central role in thetreatment of lung [A29, K52, S54, S84, T21], head andneck [A27, B45, C33, C34, H25, O4, S55], brain [S56,
S57], and bladder cancers [C35].
2. Antimetabolites(a) Antifolates
186. Despite the clinical importance of antifolates incancer therapy, there are only a limited number of reportsof experimental data relating to interactions ofmethotrexate and radiation in vitro. Early studies of Berry[B47, B48] suggested that methotrexate might be useful asa radiosensitizer, with the greatest enhancement occurringwith a cytotoxic drug concentration or in hypoxic cells.
Enhancement was influenced by the proliferation status of
the cells, and although stationary-phase cells showed anenhanced response to radiation, this was accompanied bya decreased response to methotrexate, which cancelled any
gain [B49]. The synergistic effects between methotrexate
and radiation can be explained by impaired DNA repair

258

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

owing to depleted intracellular pyrimidine and purine pools[A19]. Methotrexate cytotoxicity may also result fromdrug-induced single- and double-strand breakage of DNA[B46]. These breaks appear come from the methotrexateinduced depletion of intracellular nucleotide pools, with
impairment of the ability to repair DNA damage. Synergistic
effects were observed only when drug and radiation were
given at the same time. Radioprotective effects of methotrexate were observed when it was administered hours before
radiation treatment (see paragraph 119).
187. Effects of intracerebral injections of methotrexate,
whole-brain radiation, or a combination of both were analysed
on intracerebrally implanted RT-9 gliosarcoma in male CDFisher rats. Methotrexate alone and radiation alone eachprolonged survival moderately. Combined methotrexate andradiation caused a significant prolongation of survival in all
animals [W17].
188. In acute lymphoblastic leukaemia, current treatment
is divided into four phases: remission induction bychemotherapy; central nervous system preventive therapy
by radiation or combined modality treatment (radiation
plus methotrexate); consolidation; and maintenance withchemotherapy. However major adverse effects of central
nervous system preventive therapy have been documented,
including CT-detected brain abnormalities, impaired
intellectual and psychomotor function, and neuroendocrine
dysfunction. These adverse effects have been attributed
mainly to radiation therapy[R16, S58]. Several approaches
have been tested to decrease adverse effects, including
reduction of cranial irradiation from 24 to 18 Gy in regimens
using cranial radiation plus intrathecal chemotherapy withmethotrexate or the use of triple intrathecal chemotherapy
with methotrexate alone or with methotrexate, cytarabine, andhydrocortisone (reviewed in [P28]).
(b) Pyrimidine analogues and precursors
189. Deoxyuridine analogues that increase radiosensitivity
include 5-bromodeoxyuridine (BrUdR), 5-fluoro-2'deoxyuridine (FUdR), and 5-iododeoxyuridine (IUdR). In
these compounds, a halogen atom replaces the hydrogen at
the 5 position on the pyrimidine ring of deoxyuridine.
Because the van der Waals radii of bromine (1.95 Å) andiodine (2.15 Å) resemble closely the methyl group of
deoxythymidine (2.00 Å), BrUdR and IUdR are moreaccurately referred to as thymidine analogues. FUdR is
considered a uridine analogue because the van der Waals
radius of the fluorine atom (1.35 Å) most closely resembles
hydrogen (1.20 Å) [S59]. The biological effects of FUdR
are significantly different from those of BrUdR/IUdR andwill be discussed separately.
190. 5-Fluorouracil (5-FU) and FUdR are thefluoropyrimidines of greatest clinical interest. The fluoropyrimidines require intracellular activation to exert theircytotoxic effects. They are converted by multiple
alternative biochemical pathways to one of several active
cytotoxic forms. Incorporation of 5-FU into DNA inhibits

DNA replication and alters DNA stability by producing
DNA single-strand breaks and DNA fragmentation [C36].
Fluoropyrimidines may also induce DNA strand breaks
without being directly incorporated into DNA, possibly
through the inhibition of DNA repair as a result of dTTP
(deoxy-thymidine-triphosphate) depletion [Y12].
191. The synthesis and antitumour activity of 5-FU was
initially described by Heidelberger et al. in 1957 [H26].
Complete tumour regression was observed in mice bearing
sarcoma tumours after 5-FU and radiation, not observed
after each treatment alone [H27]. In mice with a transplanted leukaemic cell line, 5-FU and radiation interacted
synergistically when the drug was given before and afterradiation, with the effects being most noticeable in thelatter situation [V8]. Squamous-cell carcinoma responses
in mice from combined exposures were dependent on total
drug doses; however, the response was independent of theschedule of drug administration and consistent only withan additive effect [W18].
192. In vitro studies of Nakajima et al. [N15] with mouse
L cells showed an enhanced effect of combined treatmentof 5-FU and radiation on cell survival. The results suggestthat maximum enhancement occurred when drug-treatedcells were irradiated in the S phase and also confirmed theimportance of post-irradiation drug treatment. Enhancement was dependent on drug concentration, increasing
with increased dosage, and on treatment duration. The
prolonged temporal requirement and the cytotoxic dose of
5-FU for the induction of sensitization following x-ray
exposure implicates incorporation of 5-FU into RNA as animportant mechanism involved in the combined effect.
193. A series of in vitro combined treatments using
ionizing radiation and 5-FU on the human adenocarcinoma
cell lines HeLa and HT-29 were performed by Byfield et al.
[B50]. Based on these experiments they concluded that (a)sensitization occurred only with post-irradiation drug
treatment, with prior exposure to 5-FU being strictly
additive; (b) enhanced cell killing could not be explainedby drug-induced additional acute damage or inhibition ofsublethal damage repair; (c) the effect is maximized if cells
are exposed to 5-FU for prolonged periods following
irradiation; and (d) the concentration of 5-FU required forthese effects is associated with dose-limiting toxicity inclinical studies.
194. Attempts to define more clearly the mechanism ofinteraction of 5-FU have used the derivative FUdR, which
may limit the complex effects of 5-FU. Radiosensitization
by FUdR in human colon cancer cells (HT-29) was
critically dependent on the timing of exposure, being mostmarked when irradiation occurred 812 hours after
exposure to a clinicallyachievable drug concentration, withno effect resulting when the cells were irradiated first[B51]. FUdR impaired sublethal damage repair in a dosedependent manner but had no effect on the induction of
double-strand breaks [H28]. Sensitization correlated withthymidylate synthase inhibition [B51] and depletion of

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

dTTP pools [H28] and was blocked by co-incubation withthymidine [B51]. These findings strongly suggest that
FUdR acts by inhibiting thymidylate synthase.
195. In more recent studies by Miller and Kinsella [M43],
a 2-hour exposure to low doses of FUdR resulted inextended thymidylate synthase inhibition after the drug
was removed (up to 30 hours after treatment). Although
the enzyme was nearly completely inhibited (>90%), an
increase in radiosensitivity of cells was not evident until 16hours after removal of the drug. Therefore, no direct
correlation between thymidylate synthase inhibition andradiosensitization was observed. Parallel analysis of cellcycle kinetics showed that cells accumulated during theearly S phase after drug exposure and the rise and fall ofradiosensitivity of the entire cell population over time
followed the change of proportion of cells in early S phase[M44], a relatively radiosensitive phase of the cell cycle[T22]. These data suggest that radiosensitization by FUdR
is in part caused by alterations in cell kinetics and aredistribution of cells through the cell cycle.
196. Concomitant radiotherapy with 5-FU has been
evaluated in patients with cancers of the oesophagus,
rectum, anus, bladder, and advanced laryngeal tumours(reviewed in [K35, M45, O5]). A recent consensus
conference at the National Institute of Health reviewed thedata from clinical trials and has recommended combined
post-operative 5-FU and radiotherapy as the most effective
management of patients with stage II or III surgicallyresected rectal cancer [N16]. In general, 5-FU andradiation in combined modality treatment is superior toradiation alone in the treatment of intestinal tumours.
197. Differential sensitization of tumours with bromated oriodinated pyrimidines, including BrUdR and IUdR, has been
observed. These analogues influence only proliferating cells
and may therefore preferentially sensitize rapidly growing
tumours surrounded by more slowly proliferating normal
tissue. BrUdR and IUdR are readily incorporated into the
DNA of mammalian cells. The incorporation follows thethymidine salvage pathway. The extent of thymidine
replacement in DNA, however, is not simply a function ofcompetition within the salvage pathway, because the preferredpathway for thymidine incorporation is through the de novo
synthesis of pyrimidine nucleotides.
198. Steric hindrance resulting from analogue incorporation
into DNA appears minimal. In contrast, the physicochemical
properties of altered DNA are influenced by thymidine
replacement. Incorporation of BrUdR increases the forces thatbind the strands of DNA together [P16]. This may alter DNA
transcription and replication. The affinity of chromosomal
proteins for BrUdR- and IUdR-substituted DNA is increased.
This increased affinity has been associated with therepression or induction of cellular proteins, receptors, andgrowth factors (reviewed in [M45]). BrUdR and IUdR
cause a dose-dependent delay of cells in the S and G2
phases of the cell cycle, as demonstrated in human ileal
and spleen cells in vitro [P17].

259

199. The physicochemical properties of IUdR- andBrUdR-containing DNA have been implicated in its
increased sensitivity to radiation. The large, highlyelectronegative halogen atoms greatlyincrease the cross-sectional
area available for trapping radiation-produced electrons. In
addition, migration of absorbed energy to a halogenatedbase has been demonstrated [F23, L43]. Highly reactive
uracilyl radicals may result from these reactions.
200. Erikson and Szybalski [E16, E17] reported radiosensitization of human cells exposed to BrUdR and IUdR.
These studies revealed that the incorporation of halogenated pyrimidine radiosensitizes the cell through a direct
effect on DNA [S59]. It was demonstrated that BrUdR
resulted in greater thymidine replacement than did IUdR.
However, IUdR was a more effective sensitizer to x rays,even at lower levels of incorporation [E18, M46, M47].
The distribution between the two DNA strands was not a
critical factor in radiosensitization. Sensitization was also
shown to be independent of the presence of oxygen [H29].
201. Recent analysis of radiosensitization by IUdR and
BrUdR in two exponentially growing human colon cancer
cell lines (HCT116 and HT29) using the linear-quadraticmodel revealed that an increase in the initial slope of thecell survival curve is the predominant mode of radiosensitization [M46, M47]. This suggests that the radiosensitizing effect may be the result of an increase in theamount of initial DNA damage. However, other recent invitro studies with plateau-phase cells (CHO cells) suggestthat IUdR and BrUdR are, in fact, potentially lethal
damage repair inhibitors [F27, W19]. These different
proposed mechanisms of radiosensitization of BrUdR and
IUdR in exponentially growing and plateau-phase cells are
not inconsistent and may reflect a bimodal mechanism.
202. Significant systemic toxicity was noted in animals[B52, G24] and humans, suggesting minimal tumour
selectivity for these analogues. For clinical investigations
in humans, therefore, tumours were selected that weresurrounded by practically non-proliferating normal tissue(brain, bone, and muscle), thereby limiting the incorporation of BrUdR and IUdR into normal cells within theirradiated volume (reviewed in [M44]).
203. Gemcitabine (2',2'-difluorodeoxycytidine) is a new
antimetabolite. It is a pyrimidine analogue and appears toprevent the addition of other nucleotides by DNA
polymerase (masked chain termination) and to impair
DNA repair. Gemcitabine has been shown to be a potent
radiosensitizer in a variety of tumour cell lines, including
HT-29 colorectal carcinoma, pancreatic cancer, breast,
non-small-cell lung and head and neck cancer cell lines. It
was most effective when administered prior to radiation.
For most cell lines, sensitization was evident at noncytotoxic concentrations of gemcitabine. For most celllines, the primary radiosensitizing effect seems to be
associated with depletion of endogeneous nucleotide pools[L54, S83, S85]. Radiosensitization by gemcitabine wasobserved in mice bearing tumours in vivo [M70]. In

260

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

clinical trials gemcitabine seems to be a powerful radiation
enhancer in the treatment of non-small-cell lung cancer[H51, V13].
(c) Hydroxyurea
204. Hydroxyurea, a relatively simple compound, is a
representative of a group of compounds that have as theirprimary site of action the enzyme ribonucleoside
diphosphate reductase. This enzyme, which catalyses thereactive conversion of ribonucleotides to deoxyribonucleotides, is a crucial and rate-limiting step in thebiosynthesis of DNA. The mechanism of cytotoxicity fromhydroxyurea is related to direct inhibition of DNA
synthesis and repair. Hydroxyurea causes cells to arrest at
the G1/S phase transition [S61]. It selectively kills cells
synthesizing DNA at concentrations that have no effect on
cells in other stages of the cell cycle [S62].
205. Additive or greater-than-additive responses havegenerally been reported for combined treatment withhydroxyurea and ionizing radiation in vitro. Phillips andTolmach [P7], using synchronized HeLa cells, reported
that enhancement occurred only when the drug was present
post-irradiation. They demonstrated that hydroxyurea
inhibits potentially lethal damage repair. In synchronized
V79 cells, hydroxyurea treatment was necessarybefore andafter irradiation to be effective as a radiosensitizer [S61].
Sensitizing by hydroxyurea resulted from its inhibitory
action at the G1/S-phase transition or its lethal action
during the S phase. Kimler and Leeper [K36] showed thatthe enhancement of radiation-induced lethality observed
when hydroxyurea was present after irradiation was
specific for G1 and S phase cells, but that the drug did notinterfere with recovery from radiation-induced divisiondelay in the G2 phase. Non-cytotoxic doses of hydroxyurea
significantly increased the early S-phase population in ahuman bladder cancer cell line (647V) [K37]. Exposure tothese non-toxic concentrations of hydroxyurea before
irradiation resulted in radiosensitization. In the humancervix carcinoma cell line Caski, the radiosensitizing effectof hydroxyurea was mainly due to a significantly longer G2
block, indicating effects on DNA repair [K2].
206. Hydroxyurea has been shown to inhibit the repair ofradiation-induced single-strand breaks in HeLa cells. The
time course for repair of radiation-induced, single-strand
DNA breaks is partially inhibited by exposure tohydroxyurea before and after irradiation [F24]. The effectsof hydroxyurea on DNA repair after UV irradiation havealso been studied, and depletion of the triphosphate pools(except dTTP) appears to be responsible for the observed
alterations in DNA repair and enhanced cytotoxicity[C38].
207. Piver et al. [P18] showed a significant reduction in
the radiation dose needed to control mammary tumours inmice when hydroxyurea was given with fractionatedradiation exposure. However, a similar study on implanted
squamous cells of cervix carcinoma in nude mice showed
no radiosensitizing effect [X3].

208. Clinical trials of hydroxyurea radiosensitization have
involved patients with head and neck malignancies [R17,S63] and primary brain tumours [L27]. The most convincing trials, suggesting radiosensitization and improved local
control with hydroxyurea, involved patients with uterine
cervical carcinoma [P19, P20, S64]. Although these studies
suggested improved results, none of the trials considered
cell cycle times of the tumour and normal tissue orhydroxyurea concentrations in the relevant tissues [S65].
(d) Other antimetabolites
209. Other antimetabolites of clinical relevance are arabinose
nucleosides, including cytarabine (cytosine arabinoside,
ara-C); ara-C, a cytidine analogue, has important clinicalactivity against acute myelocytic leukaemia. It is an inhibitorof DNA synthesis and kills cells selectively during the S phaseof the cell cycle. Synergism between cytarabine and anumber of antitumour agents, including alkylating agents,
platinum coordination complexes, and topoisomerase II
inhibitors, has been observed in vitro and in animalmodels. ara-C enhances the activity of these compounds by
inhibiting the repair of strand breaks associated with theseagents.
210. Another class of antimetabolites is the purine
analogues, including 6-mercaptopurine and 6-thioguanine,
which act as guanine analogues, and adenosine analogues,
including arabinofuranosyladenine (9-ß-arabinofuranosyladenine, ara-A). All these compounds have antileukaemic
activity and are used in combination chemotherapy. Their
activity is directed against DNA replication and repair.
211. The potential of the thymidylate synthase inhibitor
tomudex to interact with ionizing radiation was assessed byTeicher et al. [T38]. Tomudex (1 µM) decreased theshoulder of the radiation survival curve in both oxygenatedand hypoxic HT-29 cells (human colon carcinoma) andSCC-25 cells (squamous-cell carcinoma of the head andneck), respectively. The effect was more significant inoxygenated cells. In tumour-bearing animals, tomudex incombination with radiation showed an additive to supraadditive effect on tumour control. The interaction effectwas dependent on the fractionation schedule of drug andradiation. In each assay, the results obtained with tomudex
were equal to or exceeded the results of comparable
experiments with 5-fluorouracil.
3. Antitumour antibiotics
212. Anthracyclines such as doxorubicin, daunomycin,
epirubicin, and idarubicin cause a range of biochemical
effects in tumour cells. The antitumour activityand toxicity
are the result of free-radical formation and/or triggering of
topoisomerase-II-dependent DNA fragmentation. The
enzyme is prevented from finishing its cycle with the religation of the broken strands. In addition, the alteration of
the DNA helical structure that occurs on DNA intercalation by anthracyclines may trigger enhanced topoisomerase II activity. The net result is that addition of

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

anthracyclines to tumour cells dramatically increases
protein-associated breaks. There is strong evidence that the
topoisomerase II mechanism is the means by which
doxorubicin and other anthracyclines kill leukaemia andlymphoma cells.
213. As a second mechanism, anthracyclines are able toform oxygen radicals. Evidence suggests a role for anthracycline-induced radical formation by virtue of its killing ofovary, breast, and colon tumour cells. Much of thisevidence depends on the key roles that gluthathione andgluthathione peroxidase playin detoxifying hydrogen peroxide and organic peroxides. Doxorubicin is an inhibitor of
mitochondrial and cell respiration and reduces oxygen
consumption by cells in the outer layers of the tumour.
This may lead to improved oxygenation and radiosensitivity of hypoxic areas of the tumour [D23]. On the other
hand, aclarubicin, an anthracycline differing in its sugar
moiety from doxorubicin, was shown to exert its enhancement effect on x-ray-induced cell killing in HeLa cells only
when given after radiation exposure (5 µg ml1 for onehour) [M63]. The authors hypothesized that this potentiation, which is visible through 10 cell divisions, is due tothe interaction between radiation and drug damage, amechanism probably relevant only for very high acuteexposures.
214. Bleomycin is another important antitumour antibiotic. Its action has been associated primarily with its
ability to produce single- and double-strand breaks in
DNA. The sequence of events leading to DNA breakage
begins with the metabolic activation of bleomycin. The
activated agent binds to DNA as the result of intercalation.
Highly toxic oxygen intermediates, such as the superoxideor hydroxyl radicals, are then formed that attack DNA.
There is indirect evidence that the same processes required
to repair ionizing radiation damage also are used inbleomycin repair [C39]. The lesions caused by bleomycin
include chromosome breaks and deletions, very similar to
the action of ionizing radiation. Bleomycin is therefore
called a radiomimetic drug. There is, however, some basesequence specificity for the site of DNA cleavage. Bleomycin binds preferentially to the DNA strand opposing thesequences GpT and GpC to attack and cleave the strand at
the 3' side of G [P21, S66]. A primary point of attack in
non-mitotic cells is considered to be the link regions ofDNA between nucleosomes [K38].
215. The effects of the interaction between bleomycin andionizing radiation on cell survival have been reported torange from additive to greater than additive [B26, H30,
T28, T29]. These effects are schedule-dependent, withmaximum interaction occurring when there is only a shorttime interval between administration of the two agents orwhen they are administered simultaneously, possibly
reducing the extent of repair of any induced damage orsimilar lesions by these two agents. Although both ionizing
radiation and bleomycin induce G2 arrest, their damage isindependent and purely additive [K39]. Thus, in contrast
to the sometimes greater-than-additive effects observed for261

cell lethality, bleomycin and radiation do not interact in the
induction of cell-cycle blocks.
216. Bleomycin and radiation have been combined
frequently in the treatment of head and neck cancer. There
are several randomized clinical trials (reviewed in [S67]),
some of which showed a benefit in response rate and/or
survival; others, however, including the largest trials, did
not reveal any benefit from the use of bleomycin andradiotherapy.
217. Mitomycin C is a bioreductive alkylating agent thatis inactive in its original form but is activated to analkylating species by reduction of the quinone and subsequent loss of the methoxy group. Recent studies indicatethat bifunctional alkylation by mitomycin C occurs
preferentially in a reducing environment [T23]. In an
aerobic environment, the reduction of mitomycin C
initiates a chain of electron transfers that leads ultimately
to the formation of toxic hydroxyl and superoxide radicals[B54].
218. Mitomycin C significantly reduced the radiationresistant subpopulation of KHT carcinomas growing intramuscularly in C3H/HeJ mice when administered 24 hours
before radiation. Isobolic analysis indicated that this treatment combination led to supra-additive cell killing in thetumour [S68]. Combined treatment with mitomycin C andradiation of C3H mouse mammary carcinoma in vivo
showed that the drug significantly enhanced the radiationinduced growth delay when administered before radiation[G25]. Isobolic analysis revealed that pre-irradiation
treatment with mitomycin C resulted in a supra-additive
response, whereas post-irradiation treatment resulted inonly an additive response. The enhancement appeared to berelated to both a direct radiosensitization and a pronounced
cytotoxic effect of the drug against radioresistant hypoxic
cells.
219. A randomized trial using mitomycin C with radiotherapy for head and neck cancer showed a disease-freesurvival benefit [W21]. However, a high incidence ofpulmonary complications was reported. Mitomycin C is
included in many multimodality therapy regimens for
gastrointestinal tumours in combination with 5-FU andradiation. For cancers of the anal region, chemotherapy
with 5-FU and mitomycin C plus irradiation have been
widely accepted as the conventional treatment for mostpatients, and surgery may not be required in many cases(reviewed by [S14]).
220. Actinomycin D (dactinomycin) binds to DNA byintercalation. The intercalation depends on a specificinteraction between the polypeptide chains of the antibiotic
and deoxyguanosine and blocks the ability of DNA to act
as a template for RNA and DNA synthesis [R17]. The
predominant effect is selective inhibition of DNAdependent RNA synthesis. In addition to these effects,
actinomycin D causes single-strand breaks in a manner
similar to doxorubicin.

262

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

221. Experimental investigations of interactions between
actinomycin D and radiation were reviewed by Hill andBellamy [B26, H30]. The overall conclusion of this review
was that irrespective of the sequence employed, the two
agents are at least additive and that a shorter rather than alonger interval between the two agents is the most beneficial. Because actinomycin D generally leads to adecreased Dq and a decrease in split-dose recovery, it
inhibits sublethal damage repair but does not effectpotentially lethal damage repair in exponentially growing
cells. This increased radiation damage expression, resulting from residual non-repaired single- and double-strand
breaks in DNA, has been proposed as a possible
mechanism for interaction between actinomycin D andionizing radiation.
222. Actinomycin D is effective in the treatment of Wilms’
tumour, Ewing’s sarcoma, embryonal rhabdomyosarcoma,
and gestational choriocarcinoma. It enhances radiation
effects in clinical therapy when both are given simultaneously. When given after radiation therapy, actinomycin
D, like doxorubicin, can recall the irradiation volumes by
erythema of the skin or by producing pulmonary reactions[D32]. It is not known whether this is due to interaction
between the damage done by radiation and that by the drug
or whether it represents only additivity of the effects. The
recall effect can be observed even after a period of several
months between radiation and drug treatments.
4. Microtubule poisons223. Many antineoplastic agents currently in use arebiosynthetic products and were initially isolated fromplants [D24]. In this Section, the mode of action alone andin combination with radiation of the vinca alkaloids,
epipodophyllotoxins, and taxanes is reviewed.
224. Vinca alkaloids are present naturally in minutequantities in the common periwinkle plant, Catharanthus
roseus. Vincristine (VCR), vinblastine (VBL), desacetyl
vinblastine (vindesine), and vinorelbine are in clinical use.
Vinca alkaloids exert their cytotoxic effects by binding toa specific site on tubulin and preventing polymerization of
tubulin dimers, disrupting the formation of microtubules[M55]. The binding occurs at sites that are distinct frombinding sites of other antimicrotubule agents, such ascolchicine, podophyllotoxin, and paclitaxel [B55].
225. The effect of single doses of VCR on mice spermatogonia was investigated by Hansen and Sorensen [H31], andthe influence of these drugs on the radiation response ofmurine spermatogonial stem cells was examined. VCR
significantly reduced the survival in the differentiatedspermatogonia and to a lesser extent in the stem cells. VCR
radiosensitized spermatogonial stem cells, with the effectbeing most prominent when it was administered after
irradiation. Grau et al. [G26] evaluated the interaction
between VCR and x rays in a murine C3H mammary
carcinoma and its surrounding skin. VCR caused atemporary blockage of cells in the mitotic phase. The

tumour control studies, however, showed a lack ofcorrelation between the VCR-induced accumulation of cells
in the G2/M cell-cycle phase and enhancement of tumour
radiation response. Nevertheless, pre-irradiation VCR
caused radiosensitization in both tumours and skin,whereas post-irradiation VCR mostly resulted in responses
equal to radiation only.
226. The effect of combining VBL and ionizing radiation on
tumour response was investigated in CDF1 mice bearing theMO4 mouse fibrosarcoma [V9]. Different treatment schedules
for the combination of VBL and radiation all resulted inadditive tumour responses. The maximum percentage oftumour cells that could be accumulated in mitosis by a single
intravenous bolus of VBL was around 13%. The results showthat this will probably be insufficient for significant radiation
enhancement.
227. Paclitaxel (commercial name Taxol), another microtubule poison, was first isolated from the Pacific yew, Taxus
brevifolia. Paclitaxel promotes microtubule assembly in vitro
and stabilizes microtubules in mouse fibroblast cells exposedto the drug [S69, S70]. It binds preferentially to microtubules
rather than to tubulin dimers [P22]. Although the binding site
for paclitaxel on microtubules is distinct from the binding site
for exchangeable guanosine triphosphate (GTP) and forcolchicine, podophyllotoxin, and VBL, the specific bindingsite for paclitaxel on microtubules has not been identified.
Unlike other antimicrotubule agents such as colchicine andthe vinca alkaloids, which induce microtubule disassembly,
paclitaxel shifts theequilibrium towards microtubule assemblyand stabilizes microtubules. Distinct morphological effects
suggest that paclitaxel adversely affects critical microtubule
functions during interphase and mitosis. Paclitaxel belongs to
the group oftaxanes, microtubuli stabilizing agents containing
a taxane ring. Microtubuli stabilizing agents without taxane
ring are called taxoids.
228. Choy et al. [C40] evaluated the possible radiosensitizing effects of paclitaxel on the human leukaemic cellline (HL-60). When HL-60 cells were treated with paclitaxel,
up to 70% of them were blocked in the G2/M phase. Isobolic
analysis of the data revealed that the combined effects ofionizing radiation and paclitaxel fell within the range between
additivity and synergism. Reasoning that paclitaxel could
function as a cell-cycle-selective radiosensitizer, Tishler et al.
[T24, T25] examined the consequences of combined
drug/radiation exposures on the radioresistant human grade 3
astrocytoma cell line, G18, under oxic conditions. Survival
curve analysis showed a dramatic interaction between
paclitaxel and ionizing radiation, with the degree of enhanced
cell killing dependent on paclitaxel concentration and on thefraction of cells in the G2 or M phases of the cell cycle.
229. Three human ovarian cancer cell lines were used to
examine the radiosensitizing effects of paclitaxel: BG-1,
SKOV-3, and OVCAR-3 [S71]. Paclitaxel was found tohave a significant radiosensitizing effect on all cell lines.
Proliferating cells were more sensitive to paclitaxel,radiation, and the combination than confluent cells.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

Treatment of proliferating cells with paclitaxel 48 hours
prior to irradiation had a greater radiosensitizing effectthan treatment 24 hours prior to irradiation.
230. Liebmann et al. [L44] examined the radiosensitizing
effects of paclitaxel in four cell lines: MCF-7, A549,
OVG-1, and V79. All cell lines developed a G2/M block
after paclitaxel exposure. Paclitaxel acted as a radiosensitizer in human breast cancer cells (MCF-7), in humanovary adenocarcinoma cells (OVG-1), and in Chinese
hamster lung fibroblast cells (V79). However, paclitaxel
was unable to enhance the radiation sensitivity of humanlung adenocarcinoma cells (A549). Paclitaxel increased thelinear component of the radiation survival curves in all celllines. The quadratic component was unaffected by paclitaxel
in the rodent cell line. The cells that were sensitized toradiation by paclitaxel had a relatively small baseline linear
component, while A549 cells had a large linear component.
Asynchronous and synchronous cells from carcinomas of thehuman uterine cervix were irradiated alone and after
paclitaxel treatment [G27, T39]. Irradiating paclitaxel-treatedcells resulted in a strictly additive response, like the response
in lung adenocarcinoma cells and in contrast to the earlier
supra-additive results with astrocytoma cells, breast cancer
cells, and ovarian cancer cells. Paclitaxel affected the cervicalcarcinoma cells at stages of the cell cycle other than G2/M.
This may explain the failure to observe paclitaxel radiosensitization with these cells, and it may indicate thatpaclitaxel has a multiplicity of actions, with differences in
effectiveness likely between cells of different origins. Similarcell-line-specific results on the cell-cycle specificity of the
combined paclitaxel radiation effects were reported for othertumour cell lines. In non-synchronized and synchronizedhuman fibroblasts, however, the combined effect was additive
to even subadditive [G36]. Subadditive effects on cell survival
between radiation and paclitaxel were reported for the humanlaryngeal squamous-cell carcinoma cell line SCC-20 [I15].
231. Besides having cell-cycle effects, paclitaxel is able toinduce apoptosis by a p53-independent mechanism. On amolecular level, paclitaxel effects primarily involve phosphorylation of the product of the bcl-2 gene downstream of
p53 [M68].
232. Hei et al. [H32, H33] assessed the potential oncogenic
effects of paclitaxel either alone or in combination withgamma irradiation in C3H10T½ cells. In contrast to humancells in vitro, the mitotic block induced by paclitaxel in 10T½
cells was only partial. While paclitaxel was ineffective intransformant induction, it enhanced the oncogenic transforming potential of gamma rays in a supra-additive manner.
233. In vivo experiments with animal tumours showed thatenhanced tumour radiosensitivity after paclitaxel treatment
was attributable to two distinct mechanisms. Paclitaxel wasable to enhance the radioresponse of apoptosis-sensitive and-resistant tumours but not the normal tissue radioresponse,
thus providing true therapeutic gain. Tumour reoxygenation
and antiangiogenic properties occurring as a result of
paclitaxel-induced apoptosis in apoptosis-sensitive tumours

263

and mitotic arrest after paclitaxel treatment in apoptosisresistant tumours are twodistinct radiosensitizing mechanismsof paclitaxel [M67]. In mice bearing spontaneous mammary
carcinoma, paclitaxel and radiation interacted in a supraadditive manner in controlling tumour growth. However, no
supra-additive response has been observed in normal tissue,
indicating a favourable therapeutic gain [C48].
234. Antitumour activity of paclitaxel has been observed inadvanced ovarian cancer and metastatic breast cancer. The
initial activity reported in refractory ovarian cancer has now
been confirmed in three subsequent studies with response rates
ranging from 21% to 40% (reviewed in [Y13]). Significant
activity (56%62%) has also been observed in metastatic
breast cancer [H34]. Docetaxel, a paclitaxel derivative, hasbeen shown to be 100-fold more potent than paclitaxel in
bcl-2 phosphorylation and apoptotic cell death [H50]. The
radiosensitizing activity of docetaxel has been reported inclinical trials with head and neck cancer [S79] and nonsmall-cell lung cancer [G38, O20].
5. Topoisomerase poisons235. Epipodophyllotoxins from extracts of the mandrake
plant (Podophyllum pelatum) have been used for medical
purposes for centuries as cathartics or as treatment forparasites or venereal warts. Podophyllotoxin, an antimitotic
agent that binds to a site on tubulin distinct from thatoccupied bythe vinca alkaloids or paclitaxel, was identified
as the main constituent possessing cytostatic activity. These
early tubulin-binding podophyllotoxins possessed a prohibitively high degree of clinical toxicity. For example, aconsiderable risk of pneumonitis was observed following
irinotecan and radiotherapy for lung cancer [Y15]. However, two glycosidic derivatives of podophyllotoxin,
etoposide (VP-16) and teniposide (VM-26), have verysignificant clinical activity against a wide variety ofneoplasms. Their main target is DNA topoisomerase II.
236. Epipodophyllotoxins were found to arrest cells in thelate S or early G2 phase of the cell cycle rather than theG2/M border that would have been expected of an
antimicrotubule agent [K40]. It was noted that these agents
had no effect on microtubule assembly at concentrations
that were highly cytotoxic [L45]. It was subsequently foundthat these drugs produced DNA strand breaks in intact
cells but that these effects were not seen when theepipodophyllotoxins were incubated in vitro with purified
DNA, suggesting that direct chemical cleavage in DNA
was not occurring [W20]. The epipodophyllotoxins exert
their cytotoxic effects by interfering with the scissionreunion reaction of the enzyme DNA topoisomerase II[Y10]. The enzyme binds to DNA covalently and forms
single-strand, protein-associated breaks. On a molar basis,
teniposide is approximately 10 times more effective thanetoposide at inducing DNA strand breaks [L46]. In
addition, the epipodophyllotoxins inhibit the catalytic or“strand-passing” activity of topoisomerase II that permits
the enzyme to catenate DNA circles and disentangle
topologically constrained DNA.

264

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

237. Isobolic analysis of the combined modality treatmentof etoposide and radiation on asynchronous growing V79
fibroblasts showed that considerable potentiation occurs
upon concomitant radiation/drug exposure [G28]. Synergistic cell killing was observed as radiation was applied
before or concomitantly with etoposide. Rapidly repairable
radiation-induced DNA damage was fixed into lethal
lesions by etoposide, giving rise to supra-additive interaction under concomitant radiation/drug exposure. The
shoulder of the radiation survival curve was eliminated. A
second interaction mechanism was that cells arrested in theG2 phase of the cell cycle byirradiation were hypersensitive
to the cytotoxic effects of the drug. Recently, Goswami etal. [G29] reported that the synthesis of topoisomerase II issuppressed as cells accumulate in G2 following irradiation.
Ng et al. [N17] investigated the ability of etoposide topotentiate the x-irradiation response and to inhibit the
repair of potentially lethal damage and sublethal damage
in confluent cultures of a radioresistant (Sk-Mel-3) and aradiosensitive (HT-144) human melanoma cell line. In
both cell lines, etoposide inhibited sublethal damage repair;
however, in contrast to camptothecin, a topoisomerase I
inhibitor, it also inhibited potentially lethal damage repair
in HT-144 cells but not in the radioresistant cell line
Sk-Mel-3.
238. Non-cytotoxic concentrations of etoposide (1.7 mM)
caused little or no effect in V79 cells when combined withradiation [S72]. Even at highly toxic doses of etoposide,human bladder carcinoma cells were not radiosensitized by
the drug [M48]. Etoposide and teniposide have demonstrated highly significant clinical activity against a wide
varietyof neoplasms, including non-Hodgkin's lymphomas,
germ-cell malignancies, leukaemias, and small-cell lungcarcinoma [B56, O6, W22].
239. Camptothecin, a heterocyclic alkaloid, and its
analogues are inhibitors of topoisomerase I and possess
antitumour activity. Camptothecin was first isolated fromthe stem wood of Camptotheca acuminata, a tree native tonorthern China. Characterization of the molecular structure ofcamptothecin critical for antitumour activity has led to thedevelopment of the camptothecin analogue topotecan andothers with greater solubilityand improved therapeutic indicesin preclinical models.
240. DNA topoisomerase I is the unique target for
camptothecin [S73]. Topoisomerase I transiently breaks asingle strand of DNA, thereby reducing torsional strain andunwinding DNA ahead of the replication fork. Human
DNA topoisomerase I binds to its nucleic acid substratenon-covalently. The bound enzyme then creates a transient
break in one strand and concomitantly binds covalently to
the 3'-phosphoryl end of the broken DNA strand. Topoisomerase I then allows passage of the unbroken DNA
strand through the break site and religates the cleaved
DNA. Camptothecin blocks the topoisomerase I in the form
that is covalently bound to DNA [C41]. Camptothecininduced DNA strand breaks have been detected frequently
at replication forks close to growth points. The cytotoxicity

of camptothecin, a highly S-phase-specific agent, may be
explained by the collision of drug-stabilized topoisomerase
I-DNA complexes with moving replication forks, leading
to replication arrest and conversion of topoisomerase-Ibound transient DNA strand breaks into persistent breaks[H35]. A direct stereospecific interaction between camptothecin and DNA topoisomerase is essential for the radiosensitizing effect of the inhibitor [C47].
241. Exposure to camptothecin under conditions of lowdose-rate irradiation (1 Gy h1) induced the accumulationof cells in the S phase in V79 and HeLa cells. Isobolic
analysis of survival data consistently showed supraadditivity of cell killing in both cell lines upon concomitant
exposure to camptothecin and low-dose-rate irradiation.
Cytokinetic cooperation appears to be the main determinant of cell survival in treatments associating camptothecin and radiation in growing cells. Non-cytotoxic
concentrations of camptothecin produced a reproducible
effect at x-ray doses of up to 2 Gy; however, like cells
treated with etoposide at non-toxic concentrations, theradiation survival curves for drug-treated and untreated
V79 cells were comparable at higher radiation doses [S72].
X-irradiation of camptothecin-treated SV40 transformed
normal (MRC5CVI) and ataxia-telangiectasia (AT5BIVA)
fibroblast cells resulted in additive prolongation of S-phasedelay in MRC5CVI cultures and additive effects for cellkilling in both cell lines [F11]. Hypersensitivity of
AT5BIVA to camptothecin was not attributable to elevatedlevels of complex trapping.
242. HT-29 human colon adenocarcinoma cells growing
in spheroids were more resistant to both SN-38, a metabolite of a derivative of camptothecin (irinotecan: CPT-11),
and radiation than HT-29 monolayers. SN-38 at a subtoxic
concentration (2.5 µg ml1) increased the lethal effects ofradiation on spheroids in a supra-additive manner but only
acted additively on monolayers. The mechanism of radiosensitization of SN-38 is due to the inhibition of potentiallylethal damage repair in spheroids [O18]. In both small-celllung cancer and small-cell/large-cell lung carcinoma xenografts, combination treatment with SN-38 and radiation
resulted in a significant tumour regression compared withthe use of SN-38 or radiation alone [T40].
243. Gamma-rayirradiation of AS-30D rat hepatoma cells
followed by a 2-hour exposure to camptothecin in vitro wasfound to act additively at low radiation doses and synergistically at higher radiation doses, as shown by isobolic
analysis [R18]. Treatment of established ascites tumours inrats with either camptothecin or 131I-labelled monoclonal
antibody RH1, which specifically localizes in hepatoma
ascites, prolonged rat survival but was ineffective at curing
animals of tumours. In contrast, combined therapy consisting of camptothecin followed by the injection of 131Ilabelled monoclonal antibody RH1 cured 86% of animals.
These results suggest that topoisomerase I inhibitors may
be useful for increasing the efficacy of radioimmunoconjugates for the treatment of cancer.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

244. Subtoxic concentrations of topotecan potentiatedradiation-induced killing of exponentiallygrowing Chinese
hamster ovary or P388 murine leukaemia cultured cells[M49]. Survival curve shoulders were reduced; the slopesof the exponential portions of the curves were slightly
decreased. Potentiation of radiation-induced cell killing by
topotecan was absolutely dependent on the presence of the
topoisomerase I inhibitor during the first few minutes after
irradiation. A dose-dependent reduction in cell survival
was obtained with a 4-hour exposure of topotecan
following irradiation of human carcinoma cells in culture
and murine fibrosarcoma in mice [K22]. No enhancementof cell killing was seen when cells were treated with thedrug before irradiation. In vivo tumour studies showed asignificant radiosensitizing effect of topotecan that wasdependent on both drug dose and time sequence (before
irradiation). There was no enhanced skin reaction
following the combined treatments [K22].
6. Bioreductive drugs
245. The oxygenation status of clonogenic cells in solid
tumours is believed to be one of the main factors adversely
affecting tumour response in radiotherapy. In totally
hypoxic cells, the radiation dose must be raised by a factor
as great as 3 to achieve the effects obtained in fully oxic
cells. The presence of 2%3% of such resistant cells may
double the total radiation dose required for eradication of
all tumour cells [G30, T26]. It appears that solid tumours
can contain two distinct classes of hypoxic cells: chronically and transiently hypoxic cells [C42, T26]. However, inclinical radiotherapy, treatment is usually sufficiently
protracted to allow a significant re-oxygenation.
246. Results of clinical studies on the use of hyperbaric
oxygen in combination with radiotherapy to increase
oxygenation of hypoxic tumour cells have been conflicting.
Nine randomized trials have been reported, of which only
three gave statistically significant positive results for theuse of hyperbaric oxygen, particularly in tumours of thehead and neck region and advanced carcinoma of thecervix [D25, D26, F13, W23]. A second approach towards
increased delivery of oxygen to tumours involved the useof erythrocyte transfusions. Retrospective studies of cancer
patients with anaemia showed some indications for anegative correlation between anaemia and the outcome ofradiotherapy [B57, D27, D28]. Use of the perfluorochemical oxygen-carrying emulsion Fluosol-DA and 100%
oxygen as an adjunct to radiotherapy is a third approach toincreased oxygen delivery. Clinical trials in the treatmentof head and neck cancer showed a benefit of this combinedmodality treatment [L42, R19].
247. Clinical trials with hypoxic cell radiosensitizers rely
on a different approach [R2]. Drugs that replace oxygen inchemical reactions that lead to radiation-induced DNA
damage are used as adjuncts to radiotherapy. These drugs
sensitize hypoxic tumour cells to radiation but do notsensitize normal tissue, which is already maximallysensitized by oxygen. Hypoxia-directed drugs would have265

limited use as single agents, because they would not
destroy the normally oxygenated tumour cells; however,they could be extremely valuable in combination withradiotherapy or drugs that selectively kill aerobic cells.
Optimal use of hypoxia-directed drugs would therefore
require the development of regimens in which concomitanttherapies with agents attacking each cell population werecombined effectively to eradicate all the different cell
populations within the tumour. Drugs that are selectively
toxic to hypoxic cells should be relatively non-toxic tohealthy normal tissue, which is generally well perfused andwell oxygenated.
248. Bioreductive drugs are activated by metabolic
reduction in tumour cells to form highly effective cytotoxins. Tumour selectivity exploits the presence of hypoxia
in tumours, since oxygen can reverse the activating step,
thereby greatly reducing drug activity in most normal
tissues. Selectivity can also depend on the level ofexpression in tumour cells of the particular reductase forwhich the drug can act as a substrate. These include DTdiaphorase, various P450 isozymes, cytochrome P450
reductase, xanthine oxidase, and doubtless other enzymes
as well.
(a) Quinone alkylating agents249. Quinone alkylating agents, as well as various nitro
compounds and the benzotriazine di-N-oxides, have theability to undergo metabolic reduction in such a way as toselectively kill hypoxic cells. When quinones are reduced
under normal aerobic conditions, the cell is placed inoxidative stress due to a process known as redox cycling[P23, T27]. Although oxidative stress due to cycling is
considered important in the toxicity of quinones and otherredox labile agents to normal oxic cells, this pathway is infact less damaging than the highly toxic metabolites that
predominate in hypoxic cells. This is partly because of theprotective enzymes that detoxify superoxide, that is,superoxide dismutase and catalase. Another pathway that
protects oxic cells from the toxic action of quinones is
direct reduction by DT-diaphorase. Unlike other reductases, DT-diaphorase catalyses a concerted two-electron
reduction step, which is therefore not reversible by oxygen.
Radiosensitizing effects of EO9, an analogue of mitomycin C, and porfiromycin, another quinone alkylating
agent, were reported in experimental animal tumour
models [A26, R20].
(b) Nitroimidazoles
250. Nitroimidazoles are reduced intracellularly, but in theabsence of adequate supplies of oxygen they undergo
further reduction to more reactive products [E9]. The
formation of these products is initiated by an enzymemediated single-electron reduction of the nitro group to afree radical that is an anion at neutral pH. The reduction
pathway can proceed in successive steps past the nitroradical anion (one electron addition), the nitroso (twoelectrons), and the hydroxylamine (four electrons) to266

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

terminate at the relatively inactive amine derivative (sixelectrons). In aerobic conditions the predominant reaction
is redox cycling through radical anion analogous toquinone bioreduction, and oxidative stress may result fromthis pathway. The precise molecular nature of the covalent
reaction products that predominate under hypoxia have notbeen identified, but these products almost certainly derivefrom the nitroso- or hydroxylamine reduction level or theirring cleavage products such as glyoxal [W24].
251. The first compound tested in clinical trials was the
5-nitroimidazole, metronidazole [D29, U16, U17]. It was
selected because of its known activity both in vitro [C37,
F25] and in experimental murine tumours [B58, R21,
S74]. Misonidazole was the first in a series of 2-nitroimidazole compounds to be used in the clinic. Because the2-nitroimidazole compounds are more electron-affinitive
than metronidazole, they are more efficient as hypoxic cellsensitizers. Misonidazole was shown to be more efficient
as a radiosensitizer in experimental tumour systems thanmetronidazole [F26]. Clinical experience with misonidazole as a radiosensitizer showed some benefit of the drugin some head and neck cancer and pharyngeal cancer
studies [D12, D22, F14, O7]. However, clinical use islimited because it induces cumulative peripheral neuropathy.
252. Neurotoxicity is linked to the lipophilic properties of
the compound [B59, B60, B61]. The less lipophilic
misonidazole analogue SR 2508 (etanidazole), withradiosensitizing activity comparable to that of misonidazole, was subsequently used. Adding etanidazole toconventional radiotherapy was beneficial for patients who
had squamous-cell carcinoma of the head and neck without
regional lymph node metastasis [L34]. Nimorazole, aweakly basic 5-nitroimidazole with an electron affinity
lower than that of the 2-nitroimidazoles, was evaluated ina randomized trial in patients with squamous-cellcarcinoma of the larynx and pharynx [O8]. Results
demonstrated a statistically significant improvement inlocoregional control. Nimorazole was much less toxic thanetanidazole, and the toxicity was reversible.
253. RSU 1069 is the leading compound of dual-function
hypoxic cell radiosensitizers. It is a 2-nitroimidazole
containing a monofunctional, alkylating aziridine ring.
RSU 1069 has radiosensitizing properties and can be up to
100 times more toxic to hypoxic cells than to aerobic cells[S75]. The increased differential toxicity compared withthat of other simple nitroimidazoles is due to the alkylating
function in the molecule [W25]. Following bioreduction,
therefore, the drug is converted into a bifunctional agent
that can cause both DNA strand breaks and cross-links [J5,O9, S76, W26]. In mice injected with RSU 1069, aerobic
cells exhibited large numbers of DNA single-strand breaks,
while toxic DNA interstrand cross-links were produced
only in hypoxic cells. Cells from bone marrow and spleen
showed extensive numbers of DNA single-strand breaks
but minimal cross-linking compared with tumours [O10].
However, clinical testing revealed severe gastrointestinal

toxicity at doses below those needed for therapeutic benefit[H17].
254. A series of pro-drugs (e.g. RB 6145) have beendeveloped that release RSU 1069 spontaneously underphysiological conditions [J6]. In vitro and in vivo animal
data showed that the hypoxic cell specificity and cytotoxic
activity are retained but that at the same time the acutetoxicity is reduced in animal models [A23, C4, C11, C44,
S77, S78]. The efficacy of the combined treatment of
SCCVII transplantable tumours is significantlyhigher thanthat of treatment with radiation alone.

(c) Benzotriazine di-N-oxides
255. Brown and collaborators [M50, Z7, Z8] introduced
the benzotriazine di-N-oxide tirapazamine (SR 4233) andanalogues into the field of bioreductive drugs. Like thenitro compounds and quinones, the benzotriazine di-Noxides are reduced to one-electron reduced free radicals[B62, K10, L30, Z9]. Tirapazamine is highly efficient inkilling hypoxic cells in vitro and in vivo [B53, K9, Z7, Z8].
Unlike the toxicity of other bioreductive drugs studied, thetoxicity of SR 4233 does not level off at normal oxygen
concentrations but continues to decrease as the oxygen
concentration increases.
256. The drug appears to induce DNA strand breaks bymeans of an oxidative damage to pyrimidines [E8].
Analysis of DNA and chromosomal breaks after hypoxic
exposure to SR 4233 suggests that DNA double-strand
breaks are the primary lesion causing cell death [B28].
More DNA single-strand breaks and a greater
heterogeneity in DNA damage were observed in tumour
cells than in spleen and marrow cells of mice exposed totirapazamine, consistent with the presence of hypoxic cells
and the greater bioreductive capacity of tumours [O10].
257. SR 4233 is also extremely active when used in
combination with fractionated radiation schedules [B62,
E8, Z9]. This enhancement is seen when SR 4233 is given
before and after irradiation [Z10]. In two animal tumour
models (KHT and SCCVII), SR 4233 with radiation
produced a significantly greater enhancement than did
nicotinamide with carbogen, a combination that has beenshown to improve tumour oxygenation. In RIF-1 tumour,which has the lowest hypoxic fraction of the three, theresponse was comparable for the two modalities [D8]. SR
4233 was able to enhance the tumour growth delay
produced by radioimmunotherapy in severe combined
immunodeficient phenotype mice with human cutaneous
T-cell lymphoma xenografts [W27]. In a study by Lartigau
and Guichard [L29], the pO2 dependence of the survival ofthree human cell lines (HRT 18, Na11+, and MEWO)
exposed to tirapazamine (SR 4233) alone or combined withionizing radiation, was studied in vitro. There was amarked increase in cell killing when tirapazamine wascombined with radiation, compared with either
tirapazamine or radiation given alone.

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267

Glossary
Absolute risk

Excess risk attributed to an agent and usually expressed as the numeric difference
between exposed and unexposed populations (e.g. five cancer deaths over a lifetime
per 100 people, each irradiated with 1 Sv).

Additivity

Effect of a combined exposure equaling the sum of the effects from single-agent
exposures.

Absorbed dose

Chemicals The amount of an applied dose of chemical absorbed into the body or
into organs and tissues of interest.
Radiation The average energy imparted to matter in an element of volume byionizing radiation divided by the mass of that element of volume. The SI unit for
absorbed dose is joule per kilogramme (J kg1) and its special name is gray (Gy).

Alkylating agentscompounds that transfer an alkyl group to DNA.

Antagonism

General A combined effect of two or more interacting agents that is smaller than
the addition of the single-agent effects with known dose-effect relationships.
Chemical antagonism or inactivation Chemical reaction between two compounds
to produce a less toxic product. Example: toxic metal and chelator.
Dispositional antagonism Alteration of absorption, biotransformation, distribution, or
excretion of one agent in such a way that the time-concentration product in the target
organ is diminished. Example: prevention of absorption with charcoal.
Functional antagonism Two agents balance each other by producing opposite effects
on the same function. Example: drug with vasodepressing side effect and vasopressor.
Receptor antagonism Competitive binding to the same receptor producing a smaller
effect. Examples: oxygen in carbon monoxide poisoning, ethanol in methanol
poisoning.

Antioxidants

substances preventing oxidation.

Biochemical effect monitoring

Monitoring of biochemical and molecular effects, i.e. changes in sequence, structure,
and/or function of biologically relevant molecules caused by an exposure to an agent
or a mixture of agents. Biochemical effect monitoring determines tissue dose or
biologically effective dose. Examples are direct measurement of DNA adducts andstrand breaks. Biochemical effect monitoring takes into account individual differencessuch as genetic background and deficiencies in DNA repair. A disadvantage is thedifficulty of directly monitoring changes in target cell populations. Most analyses are
therefore done on surrogate tissue such as blood cells.

Biological indicator

Measurable biological effect that is clearly, specifically, and quantifiably related
to an exposure.

Biological effective dose

Biological effect in cells or tissues at risk with direct relevance to the initiation or
progression of a disease; see also Biochemical effect monitoring.

Biological monitoring

Continuous or repeated monitoring of potentially toxic agents or their metabolites
in cells, tissues, body fluids, or excretions (internal dose). Biological monitoringtakes into account individual differences in absorption or bioaccumulation of
agents in question. It has the advantage of being comparatively easy to monitor.

Biomonitoring

monitoring the environment or a population with biological markers.

CarcinogenAn agent, chemical, physical, or biological, that can act on living tissue in such a
way as to cause a malignant neoplasm.
Solitary or complete The agent does not need additional action of further
exogenous cancer risk factors to cause a neoplasm.
Indirect or precarcinogen The agent has to be transformed to its active molecularform (ultimate carcinogen) in the metabolism.

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ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

Co-factor

A substance or agent that acts with another substance to bring about certain effects; e.g.
coenzyme, a low-molecular entity needed for enzymatic activity of the apoenzyme.

Combined effect

The joint effects of two or more agents on the level of molecules, cells, organs, andorganisms in the production of a biological effect.

Concentration additivity

Combined effect is predicted by addition of concentrations of different agents on
a normalized concentration-effect graph; valid in the case of isoaddition. In thiscase, a combined effect can arise even when all single-agent concentrations are
below their threshold for the endpoint under study (see also effect additivity).

Confounder

A variable that can cause or prevent the outcome of interest, is not an intermediatevariable, and is not associated with the factor under investigation. Such a variablemust be controlled in order to obtain an undistorted estimate of the effect of thestudy factor(s) on risk.

Confounding

A situation in which the effects of two processes are not separated. The distortionof the apparent effect of an exposure on risk brought about by the association withother factors that can influence the outcome. Distortion of a measure of the effectbecause of the association of exposure with other factor(s) that influence theoutcome under study (WHO).

Dependent action

Action of two and more agents, in which the effect of a second agent depends onthe effect of a first agent. Dependent action leads to combined effects different fromheteroadditivity.

Deterministic effect

Effect on sufficient proportion of cells to disrupt tissue or organ function. The
probability of causing observable damage will be zero at small doses but will
increase steeply to unity above a threshold. Above the threshold, the severity ofdamage will also increase with dose. Examples include cataracts, skin erythema,and stem-cell depression in bone marrow or the small intestine.

Dose

Radiation See Absorbed dose radiation.
Chemicals The amount of a chemical administered to an organism. See also
Absorbed dose chemicals.

Dose-response relationshipThe relationship between the magnitude of exposure to a chemical, biological, or
physical agent (dose) and the magnitude or frequency and/or severity of associatedadverse effects (response).

Effect additivity

Combined effect is predicted by adding the effects of different agents; valid in thecase of heteroaddition. In this case, the combined effect is zero as long as all
single-agent concentrations are below their threshold.

Environmental monitoring

Quantitative determination of a potentially detrimental agent in the environment(external dose).

Epigen, epigenetic

Changes in an organism brought about by alterations in the expression of genetic
information without any change in the genome itself; the genotype is unaffectedby such a change but the phenotype is altered.

Exposure

Concentration, amount, or intensity of a particular physical or chemical orenvironmental agent that reaches the target population, organism, organ, tissue, or cell,usually expressed in numerical terms of substance concentration, duration, andfrequency (for chemical agents or microorganisms) or intensity (for physical agents
such as radiation). In the radiation field, exposure may also denote the electrical chargeof ions caused by x or gamma rays per unit mass of air; however the term is used in its
more general sense as described here.

External dose

Radiation Dose from an external radiation source; obtained from being within aradiation field.
Chemicals Concentration of an agent in an exposure medium, i.e. air or water; seealso Environmental monitoring.

Genotoxicity

Ability to cause damage to genetic material. Such damage may be mutagenic
and/or carcinogenic.

ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

269

HazardSet of inherent properties of a substance, mixture of substances, or a process
involving substances that, under production, usage, or disposal conditions, makeit capable of having adverse effects on organisms or the environment, dependingon the degree of exposure; in other words, a source of danger.

Heteroadditivity

Additive effect from two independently acting agents with different modes ofaction and therefore different dose-effect relationships. See also Effect additivity.

Initiator

In the multi-stage model of carcinogenesis, initiators are defined by their ability to
induce persistent changes (probably due to genotoxic effects) in the cell (initiation).
If there is subsequent promotion, these changes may result in tumour formation.

Independent action

Action of two and more agents in which the effect of one agent is independent of
the effect of the other agent. Independent action leads to combined effects definedas heteroadditive.

Interaction

Combined, mutual effects between agents on a molecular and/or cellular levelwithin a short time.

Internal dose

Radiation Dose from radioactive material deposited in the body.
Chemicals (a) Amount of a chemical recently absorbed; measured, e.g. as metal
concentration in blood; (b) amount of chemical stored in one or several bodycompartments or in the whole body (body burden); used mainly for cumulative
toxicants; (c) in the case of ideal biological monitoring, amount of active chemical
species bound to the critical sites of action (target dose; e.g. carbon monoxidebinding to haemoglobin).

Isoadditivity

Additive effect from two similarly acting agents or from two increments of thesame agent on an upward bent dose-effect relationship. See also Concentration
additivity. On a descriptive level without detailed information about dose-effectrelationships, isoadditivityis sometimes indistinguishable from supra-additivity orsynergism.

Mitogens

substances with a mitogenic effect on cells.

Multiplicative response

Effect of two agents for which the single-agent response coefficients or relative
risks have to be multiplied to describe the combined response.

MutagenA substance that can induce heritable changes (mutations) of the genotype in a cell
as a consequence of alteration or loss of genes or chromosomes (or parts thereof).

Mutation

A hereditary change in genetic material. A mutation can be a change in a single
base (point mutation) or a single gene or it can involve larger chromosomal
rearrangements such as deletions and translocations.

Non-genotoxic effect

Effect of an agent at the cellular, organ, or organism level without direct effects on
the genome such as DNA damage.

Non-stochastic effectsee Deterministic effect.

No Observed Adverse Effect Level(NOAEL)The greatest concentration or amount of a substance, found by experiment orobservation, that causes no detectable adverse alteration of morphology, functional
capacity, growth, development, or lifespan of the target organism under definedconditions of exposure. Alterations of morphology, functional capacity, growth,development, or lifespan of the target can be detected at this level but may be
judged not to be adverse.

No Observed Effect Level(NOEL)The greatest concentration or amount of a substance, found by experiment orobservation, that causes no detectable adverse alteration of morphology, functional
capacity, growth, development, or lifespan of the target organisms distinguishable
from those observed in normal (control) organisms of the same species and strain
under the same defined conditions of exposure.

Potentiation

Synergism.

Precursor

Substance from which another, usually more biologically active, substance isformed.

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ANNEX H: COMBINED EFFECTS OF RADIATION AND OTHER AGENTS

Progression

Increase in autonomous growth and malignancy; used in particular to describe thetransition from benign to malignant tumours and the progression of malignancy.
There are probablynumerous stages of progression during neoplastic development.
The process of progression features in the general model of carcinogenesis as wellas in the multi-stage model.

Promoter

Risk factors of cancer that are capable of triggering preferential multiplication ofa cell changed by initiation. Often, following initiation, a long-term action on thetarget tissue is necessary. Promoters often cause enzyme induction, hyperplasia,
and/or tissue damage. The essential primary effects are considered to be reversible.
As a rule, promoters do not bind covalently to cell components and do not exert an
immediate genotoxic action.

Relative risk (RR)

Ratio between the cancer cases in the exposed population to the number of casesin the unexposed population. A relative risk of 1.5 indicates a 50% increase incancer due to the agent under consideration. Excess relative risk (ERR) is RR  1.

Sensitizer

An agent or substance that is capable of causing a state of abnormal responsivenessin an individual. In most cases, initial exposure results in a normal response, but
repeated exposures lead to progressively strong and abnormal responses.

Stochastic effect

Effect of an agent on a cell of a random or statistical nature in which the cell is
modified rather than killed. If this cell is able to transmit the modification to later
cell generations, any resulting effect, of which there may be many different kinds
and severity, are expressed in the progeny of the exposed cell. The probability ofsuch a transmittable effect resulting from an exposure to a genotoxic agent
increases with increments of dose, at least for doses well below the threshold for
deterministic effects. The severity of the damage is not affected by the dose. Whenthe modified cell is a germ cell, the stochastic effect is called a hereditary effect.

Subadditivity

Less than additive; effect of a combined exposure being less than the sum of effects
from single-agent exposures.

Supra-additivity

More than additive; effect of a combined exposure exceeding the sum of the effects
from single-agent exposures.

Synergism

A combined effect of two or more interacting agents that is greater than the
addition of the single-agent effects with known dose-effect relationships.

Target (biological)

Any organism, organ, tissue, or cell that is subject to the action of a pollutant orother chemical, physical, or biological agent.

Threshold dose

The minimum dose that will produce a biological effect. Dose below which noeffects occur (“true”, mechanistically derived threshold) or are measurable(apparent threshold). For a given agent there can be multiple threshold doses, inessence one for each definable effect.

Tissue dose

Local dose in an organ or a functional or structural entity of an organ. See also
Absorbed dose and Internal dose-chemicals.

Topoisomerase

ubiquitous enzymes that alter DNA configuration or topology.

Toxicity

Capacity of an agent to cause injury to a living organism. Toxicity can only be
defined in quantitative terms with reference to the quantity of substanceadministered or absorbed, the way in which this quantity is administered (e.g.
inhalation, ingestion, or injection) and distributed in time (e.g. single or repeateddoses), the type and severity of injury, and the time needed to produce the injury.

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271

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P26

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