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Reprinted from Living in a Chemical World Volume 534 of the Annals of the New York Academy of Sciences June 30, 1988 Are There Thresholds for Carcinogenesis?a The Thorny Problem of Low-Level Exposure ARTHUR C. UPTON Institute of Environmental Medicine New York University Medical Center New York, New York 10016 INTRODUCTIOht Few issues in health policy are more contentious than the choice of the appro- priate dose-incidence model for use in estimating the risks of cancer associated with low-level exposure to a carcinogen. The notion that there may be no thresh- old fDr carcinogenic effects-namely, that some degree of risk may be associated with the lowest dose of carcinogen-seems to contradict everyday experience that teaches us that essentially no other type of insult produces a lasting injury unless it exceeds some threshold of severity. In the past, toxicological risk assessments have traditionally been based on the concept of a no-effect level. The applicability of this concept to mutagenic effects, however, came to be questioned by the middle of the century.' Since then, the applicability of the concept to carcinogenic effects-which likewise may conceiv- ably be mediated through effects on individual cells, rather than groups of cells- also has been challenged. i[n principle, of course, it is not possible to prove or disprove the existence of an absolute threshold for carcinogenesis. Hence the argument for or against the threshold hypothesis must be based on theoretical as well as empirical evidence.2 Some of the salient lines of evidence are summarized briefly in the following. BIOLOGY OF CARCINOGENESIS Unicellular, Monoclonal Origin of Cancer The monoclonal origin of cancer is suggested by enzymological studies of human tumor cells, in which X-linked glucose-6-phosphate dehydrogenase has been used as a marker.3 Similar evidence has come from studies of chemically induced tumors of chimeric mice, in which glucose phosphate isomerase has been used as a marker.4,5 Cytogenetic analysis of tumor cells has also suggested their monoclonal nature.6 The evidence that cancer usually originates from a single precursor cell im- plies, as does the heritable nature of the malignant phenotype, that appropriate damage to one cell alone may suffice to increase the probability of the disease in a a F'reparation of this report was supported in part by Grants ES 00260 and CA 13343 from the U.SI. Public Health Service and Grant SIG-9 from the American Cancer Society. 863

864 ANNALS NEW YORK ACADEMY OF SCIENCES suitably susceptible individual. The data also indicate, however, that more than one alteration is necessary to convert a normal cell into a cancer cell, as discussed below. Multicausal, Multistage Nature of Carcinogenesis Clinical, pathological, and experimental data imply that cancer evolves through at least three successive stages: initiation, promotion, and progression.7 Initiation Initiation, which starts the process, does not itself suffice to cause neoplasia but predisposes the affected cell and its progeny to subsequent steps in carcino- genesis. The initiated cell may not be recognizable as such, however, or may never form a tumor, unless it is further altered by subsequent tumor-promoting stimulation. The mechanism of tumor initiation remains to be established; but sonne type of mutational process is suggested by the evidence that 1) initiation is relatively prompt and irreversible; 2) most ultimate carcinogens are mutagens; 3) the frequency of cell transformation induced by a given carcinogen usually is highest if exposure to the agent occurs just before or during the DNA synthetic phase of the cell cycle8; 4) DNA to which a chemical carcinogen is bound can serve as a template for DNA replication9; 5) after exposure to a carcinogen, DNA synthesis and subsequent cell division "fix" the potential for neoplastic change'o; 6) in a given biological system, the carcinogenic potency of an initiating chemical is generally correlated with the extent to which it binds covalently to DNA, and witlr, the nature of the resulting reaction products; and 7) susceptibility to cancer is increased in persons who are deficient in DNA repair.l I The frequency of neoplas- tic transformation is far higher, however, than that of single gene mutation in cells exposed to genotoxic carcinogens12•13; hence, the data implicate multiple onco- genic sites, damage of the genome at sites unlikely to be repaired (for example, tandem repeats), or genetic damage other than point mutations. i4 The specific genes that may be involved are only beginning to be defined but appear to include antioncogenes as well as oncogenes.'i It is noteworthy, furthermore, that activa- tion and expression of more than one oncogene appears to be necessary for cell transformation in vitro.1s Tumor Promotion Tunlor promotion is the process that results in the additional change, or changes, necessary to cause the neoplastic transformation of an initiated cell. In contrast to initiation, which can result from a single exposure to an appropriate tumor-initiating agent, tumor promotion requires repeated and sustained stimuli. Although tumor promotion has been demonstrated in a number of tissues, its mechanisms have thus far been studied systematically only in a few model sys- tems. In one of these, the mouse skin model, nanomolar concentrations of the tumor•promoting phorbol ester 12-0-tetradecanoylphorbol- I 3-acetate induce stim- ulation of 1) macromolecular synthesis; 2) hyperplasia; 3) polyamine synthesis; 4) prosta;eandin synthesis; 5) protease production; 6) alterations of cell membrane enzymes and glycoproteins; 7) induction of sister-chromatid exchanges; 8) altered

UP'I'ON: THRESHOLDS FOR CARCINOGENESIS? 865 differentiation; and 9) modified responses to other growth-controlling factors.16 Such effects have also been demonstrated, in vivo and in vitro, in cells of various other species, including man. As yet, however, it is not known whether any of these responses is critical for tumor promotion. Furthermore, although all tumor- p:"omoting agents induce cellular proliferation in their respective target tissues, each appears to be relatively tissue-specific. The capacity of the promoters to induce pleiotropic effects at nanomolar concentrations and their discrete struc- ture-activity relationships implicate a hormone-like mode of action.1-19 Agents that possess initiating activity as well as promoting activity can cause neoplasia by themselves if given in sufficient doses. The effects of such "com- plete" carcinogens can be enhanced, however, by various other agents that are not active by themselves but can potentiate the effects of carcinogens if given simultaneously with them.20 Such "co-carcinogens," which include certain phe- ncls, aliphatic hydrocarbons, and aromatic hydrocarbons, are prevalent in the environment and appear to act by altering the uptake, distribution, andlor metabo- lism of carcinogens, or by enhancing the susceptibility of the target cells or host.20 Tumor promoters have thus traditionally been considered to act predominantly through epigenetic mechanisms.'$ Recently, however, the production of indirect da~,nage to DNA, resulting in mutations and chromosome aberrations, has been implicated in a growing number of instances21-23 ; for example, target organ-spe- cific DNA adducts have been identified in association with the carcinogenic ef- fects of diethylstilbesterol on the hamster kidney.24 Tumor Progression Tumor progression is a process through which successive alterations in neo- plastic cells give rise to increasingly autonomous clonal derivatives.u The precise nature of such alterations remains to be determined, but mutations and chromo- some aberrations have been tentatively implicated.7 Tumor progression may be acc,elerated by repeated exposure of neoplastic cells to carcinogenic stimuli or by sele ction pressures that favor the outgrowth of increasingly autonomous subpopu- lations of cells. EPIDEMIOLOGIC DATA ON DOSE-INCIDENCE RELATIONSHIPS IN HUMANS In contrast to the hundreds of chemicals that have been observed to possess oncogenic activity in laboratory animals, less than three dozen are known to induce cancer in man.26 In few cases, moreover, are the relevant epidemiological data adequate to characterize the relationship between cancer incidence and the dose of a given carcinogen, except in a semiquantitative way. Analysis of the dose-incidence relationship is less difficult with ionizing radia- tion than with carcinogenic chemicals because dosimetry with radiation is not complicated in the same way by pharmacokinetic variables. Furthermore, inci- dence data for irradiated populations are available over a wide range of radiation doses,z'.zII whereas comparable dose-incidence data for chemicals are generally lacking. In no case, however, do the data suffice to define the dose-incidence relationship in the low dose domain or to exclude the possibility of a threshold. Hence assessment of the carcinogenic risk associated with low-level exposure to

866 ANNALS NEW YORK ACADEMY OF SCIENCES any carcinogenic agent must depend on extrapolation from observations at higher dose levels, based on assumptions about the relevant dose-incidence relation- ships and mechanisms of carcinogenesis. The extrapolation models generally used for estimating the carcinogenic risks of low-level irradiation are of the nonthreshold type; that is, the linear non- threshold model or the "linear-quadratic" nonthreshold model is usually used.28•=9 The strongest epidemiological evidence in support of these models con- sists of (1) the large excess of acute leukemia and other juvenile malignancies that is associated with a dose as low as 1-5 rad in utero3o.31; (2) the excess of thyroid 16701 ATOMIC BOMB SURVIVORS g 200 1950•19)4 400 MASSACHUSETTS ¢ a FLUOROSCOPY W Z 150 U Q 300 e U N U f- N 6 Q ¢ 1IM1 ~ 200 m W 0 m LL W Z SA ~ v 100 Z 1 3 1 4 5 O k 5 0 1 I 1 U Y 2 - 0 Rsd U Rd1 2 100 200 300 400 500 600 100 200 300 400 5oG 600 BREAST DOSE BREAST DOSE BREAST DOSE kUMIER OF FLUOROSCOIIC EXAMINAT10H3 FIGURE 1. Incidence of cancer of the female breast as a function of dose in A-bomb survivors, in women treated with X-rays for acute postpartum mastitis, and in women subjected to multiple fluoroscopic examinations of the chest during treatment of pulmonary tubercuaosis with artificial pneumothorax. (Reproduced from Boice er a0' with permission from the Radiological Society of North America.) tumors that occurs following epilating irradiation of the scalp for tinea capitis in childhood, which is associated with an average dose to the thyroid gland of only 6-8 rad;3z•33 (3) the excess of breast cancers (FIG. 1) in (a) women exposed to A- bomb radiation, (b) women given therapeutic irradiation for postpartum mastitis, (c) women who received multiple fluoroscopic examinations of the chest during the treatment of pulmonary tuberculosis with artificial pneumothorax, and (d) women exposed occupationally to external gamma radiation in the painting of

UlP7CONc THRESHOLDS FOR CARCINOGENESIS? 867 soo ANFCUAL 400 INCIDENCE stuod7tdirsd f9r at. PER Io00o0 00 MEN 200 100 0 10 lo ]0 40 S® DOSE RATE (citarsceas o+akad p.r dt.y) FIGURE 2. Incidence of lung cancer in regular smokers of cigarettes in relation to the number of cigarettes smoked per day. (Reproduced from Doll35 with permission.) luminous clock and instrument dials, which is similar in all four groups, irrespec- tive of the marked differences among the groups in the duration of exposurezs,34; and (4) the excess of leukemia in A-bomb survivors, which is evident at doses below 0.25 Gy.28,29 The data from each of these studies, although not adequate to precisely define the shape of the dose-incidence curve in the low dose domain, are compatible with linear nonthreshold functions for each of the neoplasms in question. SV/ VVith respect to the carcinogenic effects of chemicals, as opposed to ionizing J~- radiation, quantitative dose-incidence information for human populations is far more, limited. Nevertheless, considerable information is available for a few chemi- cals, one of them being cigarette smoke, which contains thousands of compounds, including initiating agents as well as promoting agents. In cigarette smokers, the incidence of lung cancer (FtG. 2) increases as a function of the average number of cigarettes smoked per day raised to a power of 1.8.36 Similarly, in chemists who were employed as distillers of 2-napthylamine, the cumulative incidence of cancer of the urinary bladder increases steeply with the duration of occupational exposure, approaching 100% in those who were exposed for 5 ydars or longer (FIG. 3). 100 0 FIGURE 3. Cumulative incidence of tumors of the uri- nary bladder, at 30 years after start of exposure in 78 distillers of 2-naphthylamine and benzidine, in relation to duration of occupational exposure. (Reproduced from Saffiotti3' [based on data from Williams38] with permission from the International Agency for Research on Cancer.) 1\ 00 `I 2 3 4 5~ Durotion of Ezposure (yrs)

3b$ ANNALS NEW YORK ACADEMY OF SCIENCES In asbestos workers, likewise, the incidence of lung cancer and of inesothe- lioma appears to increase linearly with the intensity and duration of exposure.39 It is noteworthy, furthermore, that in cigarette smokers who have also been exposed occupationally to asbestos, the carcinogenic effects of cigarette smoke and asbes- tos appear to interact multiplicatively rather than additively (TABLE 1). Also noteworthy is the fact that the excess of lung cancer in ex-smokers decreases rapidly after cessation of smoking,41 suggesting that cigarette smoke affects primarily on late stages of carcinogenesis, acting as a promoting agent. This situation contrasts sharply with that in irradiated28 or asbestos-exposed42 populations, in whom the risk of lung cancer persists long after exposure. EXPERIMENTAL DOSE-EFFECT DATA Carcinogenesis in LaBoratory Animals The neoplasms induced experimentally in animals of different species vary widely in dose-incidence relationships. Although neoplasms of virtually every TABLE 1. Age-Standardized Lung Cancer Death Rates for Cigarette Smoking, Occupational Exposure to Asbestos Dust, or Both Group Exposure to Asbestos? History of Cigarette Smoking? Death Rate Mortality Difference Mortality Ratio Control No No 11.3 0.0 1.00 Asbestos workers Yes No 58.4 +47.1 5.17 Control No Yes 122.6 +111.3 10.85 Asbestos workers Yes Yes 601.6 +590.3 53.24 NOTE: Age-standardized lung cancer death rates are rates per 100,000 man-years stan- d«rdized for age on the distribution of the man-years of all the asbestos workers. Number of lung cancer deaths based on death certificate information. (Adapted from Selikoff./0) type have been induced in one experiment or another, all types of neoplasms are not elicited in animals of any one species or strain. In fact, certain types of neoplasms actually decrease in frequency with increasing dose of whole-body in'adiation (FiG. 4). Among chemically induced neoplasms, the observed variations are attribut- able in part to pharmacokinetic differences affecting the dosage of carcinogen to diff-.rent cells and subcellular targets. Such an explanation cannot account, how- ever, for the observed variations in dose-incidence relations among radiation- induced neoplasms, which remain largely unexplained. Because of the multi- causal, multistage nature of carcinogenesis, and the fact that the mechanism of carcinogenic effects is not the same in all instances, some diversity of dose- incidence relationships is to be expected. Obviously, the observed dose-incidence curves cannot all be represented by the same mathematical function. Nevertheless, the following generalizations emerge from the data: (1) a carcinogenic-induced elevation in the age-specific incidence of a particular neoplasm may or may not result in an increase in the final cumulative incidence of tumors, depending on the survival of the population at risk; (2) chemicals differ greatly in carcinogenic effectiveness, with the result that

UP;CON: THRESIiOLDS FOR CARCINOGENESIS? 869 1000 2000 3000 4000 5000 DOSE (rods) FIGURE 4. Dose-incidence curves for different neoplasms in animals exposed to external radiation: (A) myeloid leukemia in X-irradiated mice (Upton et al.43); (B) mammary gland tumors at 12 months in gamma-irradiated rats (Shellabarger et al.44); (C) thymic lymphoma in X-irradiated mice (Kaplan and Brown45); (D) kidney tumors in X-irradiated rats (Malda- gue46); i(E) skin tumors in alpha-irradiated rats (percentage incidence x 10) (Burns et al.°7); (F) sk.in tumors in electron-irradiated rats (percentage incidence x 10) (Burns et al.'~; and (G) reticulum cell sarcoma in X-irradiated mice (Metalli et al.48). (Modified from reference 49. Reproduced from Upton'-' with permission from the Elsevier/North-Holland Publishing Company.) the daily dose required to double the risk of neoplasia varies among different chemic;~ls by more than six orders of magnitude (FIG. 5); (3) with ionizing radia- tion, the dose-incidence curve for high linear energy transfer radiation generally rises more steeply with dose and is less dependent on the dose rate than is the curve for low linear energy transfer radiation54; (4) for many types of neoplasms, the incidence passes through a maximum at some intermediate dose and de- creases with further increase in the dose (FIG. 4); (5) the median time of tumor ~ ~ a TCDD FIGURE 5. Range of carcinogenic potency in male rats. (Reproduced from Gold et al.50 with permission from the National Institute of Environmental Health Sciences. ) ~AttinomycinD ~ 4llotosin Bt - Bis-(chloromethyl) ether l0onq r -- I Stenqmotocystin ~0BCP ~-- Diethyistitbestrol Procorbozine. HCJ EDB 2-AAF Auromine-0 Anil ine. HCI -rDDT .2 ,4,6-Trlchl orophenol , Metronidozole C-~FDdC RedNo.t ~ o c 109 FD 8 C Green No.I

87® ANNALS NEW YORK ACADEMY OF SCIENCES appearance t under conditions of daily exposure tends to vary inversely with the daily dose d according to the function dt" = constant (1) where n is greater than one55; (6) because radiation or a given chemical can often influence carcinogenesis through more than one mode of action, at least at high dose levels, the dose-incidence curve may reflect a combination of initiating effects, promoting effects, and anticarcinogenic effects, depending on the particu- lar agent, dose, and exposure conditions; (7) the combined effects of different agents may be additive, synergistic, or antagonistic, depending on the agents in question and the conditions of exposure56; (8) at low-to-moderate dose levels, the effects of a complete carcinogen can generally be accentuated by appropriate tumor-promoting stimuli, which unmask initiating effects of the carcinogen that would otherwise remain unexpressed; and (9) under conditions in which initiating Dose-Response for Initioting Acirvity of Benzo(a)pyrene 1.0 C ff' 0 0001{ , d .. i -i 0.01 0.1 10 t0 t00 BaP (µ9) FIGURE 6. The yield of skin papillomas per mouse versus dose per application, after single and multiple doses of benzo- [alpyrene. After treatment, 5 µg of 12-O-tetradecanoylphorbol- 13-acetate was typically applied three times per week. (Repro- duced from Bums and Alberts' with permission from Mary Ann Liebert, Inc.) effects are promoted to full expression, they generally increase as a linear non- threshold function of the dose of the initiating agent (FIG. 6). A number of experiments have been carried out with laboratory animals to characterize the dose-incidence curve in the low dose domain. In the largest of the experiments to date, performed with BALB/c female mice exposed to 2- acetylaminofluorine in the diet, the incidence of hepatomas increased as a linear nonthreshold function of the daily dose, whereas the dose-incidence curve for t.umors of the urinary bladder approached a quasithreshold and resembled a hockey stick in shape (FIG. 7). Comparably large experiments have not been carried out with ionizing radiation, but the combined results of a number of s4zable studies in mice, rats, and dogs imply that for most types of tumors (malig- nant as well as benign) the carcinogenic effectiveness per unit dose of X-rays and gamma rays is generally reduced at low doses and low dose rates, whereas that of high linear energy transfer radiations remains constant or may even be enhanced at low doses and low dose rates (FIG. 8), arguing against the likelihood of a threshold in such instances.z'.57,5s

UPTON: THRESHOLDS FOR CARCINOGENESIS? 871 00 r 75 FIGURE 7. Cumulative incidence of neoplasms of the liver and urinary bladder in female BALB/c mice exposed to 2- acexylaminofluorine at various concentrations in the diet for up to 33 months. (Reproduced from Littlefield et al.s2) Cell Transformation in Vitro i ~ 8iobee P 25 i P7 0 0 50 100 150 Dose (ppm) The neoplastic transformation of cells in vitro is not a perfect model of carci- nogenesis in vivo, but the two systems have enough features in common at the ceiflular level so that cell transformation can be exploited to identify carcinogenic agents and explore their mechanisms of action. Although few detailed dose- response curves for cell transformation have been published, the effects of ben- zo[a]pyrene and ionizing radiation have been studied systematically. With ben- zo[a]pyrene, the logarithm of the frequency of morphological transformation in Syrian hamster embryo cells increases linearly with the logarithm of the dose59,60; the slope of the dose-effect curve suggests a one-hit model for this response except at the highest doses, where the deviation from linearity is attributable to cytot:oxicity.61 A one-hit model also holds for transformation by the combined effects of X-rays and benzo[ajpyrene.61 For Syrian hamster embryo cells transformed by X-rays, the logarithm of the transformation frequency per surviving Syrian hamster embryo cell appears to increase curvilinearly with the logarithm of the dose from I rad to 150 rad, but a linear response with a slope of one cannot be excluded. It is noteworthy, further- more, that an increase in the frequency of cell transformation is detectable at a dose ~of only I rad.62 Dose-response curves for X-ray-induced transformation of C3F[ 10T1/2 cells show an exponential increase in transformation frequency (foci per su)rviving cell),63,64 with a doubling dose that is higher (about 100 rad) than the doubling dose in hamster cells (about 10 rad). The effects of fractionating or protracting the dose of radiation vary with the experimental conditions in question. With a total dose of less than 100 rad, frac- tionation has been observed to enhance its transforming effectiveness,65--67 whereas the opposite effect has been observed with higher doses (300-800 rad) 63,sa.s6.s1 The transforming effectiveness of gamma radiation has generally FIGURE 8. Dose-response curves depicting the incidence of tumors in laboratory animals in relation to the dose and dose rate of high and low linear energy transfer radiation. (Reproduced from Thomson et al.s' [also in Updon et al.54}.) DosE-->

872 ANNALS NEW YORK ACADEMY OF SCIENCES been observed to diminish with protraction whereas that of high linear energy transfer, radiation has been enhanced.69 Few comparable split-dose experiments have been performed with chemicals. Two doses of N-acetoxy-2-fluorenylacetamine administered 2-24 hr apart, how- ever, were observed to yield a higher frequency of transformation with Syrian hamster embryo cells than the same total dose administered at once. In contrast, methyl-N'-nitro-N-nitrosoquanidine, mitomycin C, and ultraviolet light were less effective if delivered in split doses than in a single dose. The effectiveness of methyl methanesulfonate was unaffected by dose fractionation.70 The morphological transformation of C3H 10T1/2 cells in vitro, like that of cells in vivo, is not a one-step process. The first step appears to be rapid event," occurring with one-hit kinetics in a high percentage of carcinogen-exposed cells.''--74 The second step appears to be either a further qualitative change, occur- ring at a low frequency during the growth or confluence of the cells,'}-76 or an amplification of the transformed phenotype, possibly by release of the cells from inhibitory effects of neighboring nontransformed cells.72,n,78 Clearly, further data will be needed to elucidate the mechanism of in vitro transformation and its relevance to carcinogenesis in vivo. Mutations and Chromosome Aberrations In view of the putative roles of mutations and chromosome aberrations as mechanisms of carcinogenesis, the dose-response relationships for these changes must be considered in assessing the risks associated with low-level exposure to carcinogens. The changes in DNA that are induced by ionizing radiation and genotoxic chemicals, which include single-strand and double-strand breaks, base altera- tions, cross-linkage, and other modifications, can result from traversal of the cell nucleus by a single ionizing particle79 or from interaction of the DNA with a single electrophilic molecule.80,81 Although a dose of low linear energy transfer radiation that is lethal to 50% of dividing cells (that is, 2.5 Sv) causes hundreds of DNA strands breaks per cell, much of the damage is reparable, depending on the effec- tiven.ess of the cell's repair processes.79 Such homeostatic repair processes are thought to enable the average cell to repair thousands of lesions in its DNA that occur "spontaneously" each day through the effects of natural background radia- tion, free radicals, and other degradative processes.82,83 In spite of repair, how- ever, the persistence of residual damage or the occurrence of lesions resulting from misrepair can give rise to mutations or chromosome aberrations or both, the frequency of which will depend on the amount and severity of DNA damage. The frequency of mutations at the guanine (hypoxanthine) phosphoribosyl transferase locus in human lymphocytes increases as a linear, nonthreshold func- tion of the X-ray dose over the range from 50 to 220 mSv, amounting to about six mutations per 106 cells per Gy, whether the dose is delivered in several fraction- ated exposures or in a single brief exposure.84 In X-irradiated mouse spermatogonia in vivo, the frequency of specific locus mutations increases as a linear-quadratic function of the dose, amounting to approximately six mutations per 106 cells per locus per Sv at low-to-intermediate doses and dose rates; with fast neutrons, the frequency of mutations increases more steeply, as a linear nonthreshold function of the dose, and independent of the dose rate.56