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Cancer Investigation, 7(3), 267-276 (1989) CONTROVERSIES IN BASIC SCIENCE The Question of Thresholds for Radiation and Chemical Carcinogenesis Arthur C. Upton Institute of Environmental Medicine New York University Medical Center New York, New York INTRODUCTION Selection of the dose-incidence model that is appro- priate for predicting the risks of cancer from low-level exposure to a given carcinogen is among the most con- tentious issues in public health. Although the existence of a threshold in the dose-effect relationship is well documented for many, if not most, types of toxicological effects, the exi stence of a threshold for the mutagenic effects of ionizing radiation (1-3) and of certain chemicals (4,5) has been questioned since the middle of the century. More recently, the existence of a threshold for car- cinogenic effecm also has been seriously questioned, since carcinogenesis may, likewise, be envisionei to result from effects on indivichlat cells rather than groups of cells (6-8). Because in principle it is not possible to prove or disprove the existence of a threshold for carcinogenesis, the argument for or against the threshold hypothesis must be based on theoretical as well as empirical evidence (7,8). Some of the cogent data and concepts are surveyed in the following. Copyright © 1989 by Marcel Dekker, Inc. 267 BIOLOGY OF CARCINOGENESIS Monoclonal, Multicausal, Multistage Nature of Cancer The evidence that cancer usually originates from a single transformed cell (9-11) implies that appropriate damage to one cell alone may suffice to increase the prob- ability of neoplasia in a suitably susceptible individual. A single alteration, however, apparently does not suffice to convert a normal cell into a cancer cell. On the con- trary, cancer typically appears to evolve through a suc- cession of stages; for example, initiation, promotion, and progression (12,13). The mechanism of initiation remains to be established, but some type of mutational change is implicated by evidence that: (i) the initiating event is relatively prompt and irreversible (14,15); (ii) most ultimate carcinogens are mutagens (16); (iii) the frequency of cell transforma- tion that is induced by a given carcinogen is usually max- imal if exposure to the agent occurs just before or during 2025545949

L 268 DNA synthesis (17); (iv) the carcinogenic potency of an initiating chemical is generally correlated with the extent to which it binds covalently to DNA and with the nature of the resulting DNA adducts; and (v) DNA to which a chemical carcincgen is bound can serve as a template for DNA replication (18) which, along with subsequent cell division, is necessary to "fix" the potential for neoplastic change (19); (vi) susceptibility to cancer is increased in persons who are deficient in their capacity to repair DNA damage (20). Whatever the nature of the mutational change may be, it results in a frequency of initiation that is orders of magnitude higher than the rate of mutations at any given genf: locus (21,22), implying that multiple oncogenic sites, damage to the genome at sites unlikely to be repaired (e.;;., tandem repeats), or genetic damage other than point mutations are likely to be involved (14). The specific genes that are affected may be presumed to include antioncogenes as well as oncogenes (Table 1). Initiation can thus be envisioned to result either from the homozygous inactivation or deletion of an antioncogene, or from the aberrant activation of an otherwise normal proto-oncogene, through aneuploidy, chromosomal re- arrangement, or point mutation. For neoplastic transfor- mation, as opposed to initiation, the activation of a single oncogene alone appears to be insufficient (13). Although initiation can result from only one exposure to an appropriate: initiating agent, tumor promotion typically requires repeated and sustained exposures to an appropriate promoting agent, although low doses of the agent may suffice. In two-stage mouse skin carcinogene- sis, for example, nautomolar concentrations of 12-O-tetra- decanoyl phorbol-13-acetate (TPA) are sufficient to Table 1 Comparative Properties of Oncogenes and Antioncogenes Oncogenes Gene active Specific translocations Translocations not hereditary Dominant Tissue specificity may be broad Antioncogenes Gene inactive Deletions or invisible mutations Mutations hereditary and nonhereditary Recessive Considerable tissue specificity Especially leukemias ,and lymphomas Source: From Ref. 20. Solid tumors (e.g., Wilm's, retinoblastoma) Upton promote the effects of radiation or chemical initiators, causing concomitant stimulation of: (i) macromolecular synthesis; (ii) hyperplasia; (iii) polyamine synthesis; (iv) prostaglandin synthesis; (v) protease production; (vi) alterations of certain cell membrane enzymes and glyco- proteins; (vii) induction of sister-chromatid exchanges; (viii) altered differentiation; and (ix) modified responses to various growth-controlling factors (23). Whether any one of these changes is critical for tumor promotion, however, is not clear. Traditionally, TPA and other tumor-promoting agents have been considered to act predominantly through epigenetic mechanisms (24,25), but recent observations indicate that some of these agents can damage DNA indirectly (26-29) implying that such genotoxic effects also may be involved in promotion. Tumor progression, the process through which suc- cessive generations of neoplastic cells give rise to increas- ingly autonomous clonal derivatives (30), has been at- tributed at least in part to mutations and chromosome aber- rations (15). The process can be accelerated, however, by selection pressures that favor the outgrowth of pro- liferative subpopulations, including repeated exposure to growth-stimulating agents and carcinogens (15,30). I EMPIRICAL DOSE-INCIDENCE RELATIONSHIPS FOR CARCINOGENSIS Although hundreds of chemicals have been found to be oncogenic in laboratory animals, less than three dozen have been observed to be capable of inducing cancer in humans (31). With few exceptions, moreover, the relevant data are not sufficient to characterize the dose-incidence relationship except in a semiquantitative way (8). With ionizing radiation, for which the dosimetry is less complicated by pharmacokinetic variables than is the dosimetry for most chemicals, dose-incidence data are available over a relatively wide range of radiation doses (32,33). At best, however, the data do not suffice to define the dose-incidence relationship in the low-dose domain. Assessment of the carcinogenic risks associated with low- level irradiation must thus depend on extrapolation from observations at higher levels of exposure, based on assumptions about the relevant dose-incidence relation- ships and mechanisms of carcinogenesis. The extrapolation models that are used for estimating the carcinogenic risks of low-level irradiation generally assume a linear nonthreshold relationship between risk and dose in the low-dose domain, although the data do not exclude a threshold (8,33,34). Among the lines of epidemiological evidence that are consistent with a

Thresholds for Radiation and Chemical Carcinogenesis nonthrehsold relationship are: (i) a 25-50% excess of leukemia in c;hildren exposed to diagnostic x-rays in utero, in whom the radiation dose is estimated to have averaged less than 50 mGy (35,36); (ii) an excess of thyroid tumors in persons who received therapeutic irradiation of the scalp in childhood for tinea capitis, in whom the dose to the thyroid gland is estimated to have averaged no more than 60-80 mGy ;37,38); (iii) a dose-dependent excess of breast cancer, of essentially the same magnitude for a given dose, in: (a) women exposed to A bomb radiation, (b) women given therapeutic irradiation of the breast for postpartum mastitis, (c) women who received multiple fluoroscopic examinations of the chest during the treat- ment of pulmonary tuberculosis with artificial pneumo- thorax, and (d) women exposed to external gamma radia- tion in the painting of luminous clock and instrument dials (33,39); and (iv) a dose-dependent excess of leukemia in A bomb survivors, which is evident at doses below 300 mGy (33,34). [n each of the above populations, the dose- incidence data in low-to-intermediate dose range are com- patible with a linear nonthreshold relationship for the neoplasms in question. Comparable data, moreover, are available for certain radiation-induced neoplasms in laboratory animals (8,32,40,41). As concerns the car- cinogenic effects of chemicals, quantitative dose-inci- dence data for humans are extremely limited, with few exceptions. A iloteworthy exception is cigarette smoke, the major cause of lung cancer. In cigarette smokers, the incidence of lung cancer increases as a function of the number of cigarettes smoked per day raised approximately to a power of 1.8 (42). Furthermore, the absence of any clear indication of a threshold in the dose-incidence curve soo ANNUAL 400 ENCIDENCE sta,auai:.d fer ,se t'ER 100000 300 MEN 200 too t9 20 30 40 DoSE RArE tcila,atta Wnok.dp.reayl Figure 1. Annual in:idence of lung cancer in regular cigarette smokers, in relation to the number of cigarettes smoked per day. (From Ref. 61.) 269 100f- ffW C tii = z W > ~ p40 ~ 20 T T 1 T 1 2 3 4 5 LENGTH OF EXPOSURE (yrs) Figure 2. Cumulative incidence of cancer of the urinary bladder in 78 distillers of /3-naphthylamine and benzidine. (From ReF. 8, based on data from Ref. 62.) (Fig. 1) is consistent with epidemiological data implying that the risk of lung cancer can be increased even in nonsmokers by passive exposure to cigarette smoke over prolonged periods (43). Other populations for which the dose-incidence data are compatible with a nonthreshold type of response in- clude groups of chemists who were employed as distillers of 2-naphthylamine. In one such group, the cumulative incidence of cancer of the urinary bladder was observed to increase with the duration of occupational exposure, approaching 100% in workers who were exposed for five years or longer (Fig. 2). In asbestos workers, likewise, the rates of lung cancer and mesothelioma appear to increase linearly with the in- tensity and duration of exposure (44). Furthermore, in asbestos workers who smoke cigarettes, the combined car- cinogenic effects of asbestos and cigarette smoke appear to be multiplicative rather than merely additive (Table 2), implying that the two agents exert their effects through complementary rather than similar mechanisms. With respect to the mechanism of cigarette smoke- induced carcinogenesis, it is noteworthy that the excess of lung cancer in ex-smokers stops rising relatively promptly after cessation of smoking (45), suggesting that cigarette smoke affects primarily late stages of car- cinogenesis. The carcinogenic effects of cigarettes thus stand in contrast to those of radiation (33) and asbestos (46), which continue to become manifest for decades after exposure.

270 Upton Table 2 Age-Standardized Lung Cancer Death Rates as Affected by Cigarette Smok-ing, Occupational Erposure to Asbestos Dust, or Botha Exposure to asbestos History of cigarette smoking Death rate Mortality difference Mortality ratio No No 11.3 0.0 1.00 Yes No 58.4 +47.1 5.17 No Yes 122.6 + 111.3 10.85 Yes Yes 601.6 +590.3 53.24 aAge-standardizz.d lung cancer death rates are rates per 100.000 man-years standardized for age on the distribution of the man-years of all the asbestos workers. Number of lung cancer deaths based on death certificate information. Source: From Ref. 59. Because of the multicausal, multistage nature of carcin- ogenesis and the fact that the mechanism of carcinogenesis is not the same for all cancers and all agents, some diver- sity of dose-incidence relationships is to be expected. The neoplasms that a;re induced by a given chemical in dif- ferent tissues or in animals of different species also may vary in dose-incidence relationships because of pharmaco- genetic and pharmacokinetic differences affecting the dosage of carcinogen to different target cells (47). The observed age- md tissue-dependent variations in dose- incidence relationhips among radiation-induced neoplasms are largely unexplained as yet (41), but differences in cell proliferation kinetics and homeostatic ability (including capacity to repair DNA damage) may constitute poten- tial sources of such variation (20). To explore the dose-incidence curve for carcinogenesis at low doses, a number of large-scale experiments have been carried out vtith laboratory animals. In the largest of these to date, the incidence of hepatomas in mice was observed to increase with the concentration of 2-AAF in the diet even at the lowest dose level tested (Fig. 3), whereas the dose•-incidence curve for tumors of the urinary bladder was quasithresholded (Fig. 3). This con- trast in dose-incidence curves may have resulted from dif- ferences between thie liver and the bladder in the metabol- ism of 2-AAF among other explanations. Because a given carcinogen may influence the probabil- ity of neoplasia through more than one type of effect, at least at high dose levels, its dose-incidence curve can reflect differing combinations of initiating effects, pro- moting effects, and anticarcinogenic effects, depending on the dose and other circumstances. The combined effects of multiple agents may, likewise, be additive, synergistic, or antagonistic, depending on the agents in question and the conditions of exposure. At low to moderate dose levels, the effects of a complete carcinogen can general- ly be accentuated by appropriate tumor-promoting stimuli, which unmask initiating effects that would otherwise re- main unexpressed (Fig. 4). It is noteworthy, moreover, that under conditions in which initiating effects are pro- moted to full expression they often increase as a linear nonthreshold function of the dose of the initiating agent (Fig. 4). Furthermore, whereas the carcinogenic effec- tiveness per unit dose of x-rays and gamma rays tends to 100r- 75 ~ Liver I / 1 ~ / °.~ ~50 .E Bladder 25 - / / / 0 i ®0 50 100 150 Concentration of 2-AAF in the Diet (ppm) , Figure 3. Cumulative incidence of tumors of the Gver and of the urinary bladder in female BALB/c mice exposed to 2-acetylaminofluorene (2-AAF) at various concentrations in the diet for up to 33 months. (From Ref. 63.). ,

Thresholds for Radiation and Chemical Carcinogenesis %'isx(1iie1) o 7Y.(1/91) " g63 Y.(1t/167) (0$4) . 50 100 150 200 250 300 3S0 400 450 Radiation dose (r) Figure 4a. Cumulative incidence (in percent) of leukemia in C57BL mice in relation to the dose of whole-body x-radiation administered in a single exposure (--o-o-), with or without subsequent injections of urethane (-x-x-). (Reproduced from Ref. 64.) Figure 4b. Cumultui,ve incidence of carcinomas of the skin in mice exposed once weekly to benzo(a)pyrene (BaP), with or without subse- quent exposure to 12-C'-tetradecanoyl phorbot-13-acetate (TPA) twice weekly. Doses refer to the amount of B(a)P applied to the skin each week. (Reproduced from Ref. 65.) decrease with decreasing dose and dose rate, that of high- LET radiation tends to remain constant or even increase (Fig. 5) (32,40,4I)~. 271 400 A/n, 24 FR. (A) / 60 FR. (a) Figure 5. Life shortening (all causes) in male B6CF, mice in relation to the total dose of single, fractionated (FR), or continuous whole-body neutron- or gamma-irradiation. (Reproduced from Ref. 66.) Cell Transformation In Vitro The neoplastic transformation of cells in vitro, although not strictly analogous to carcinogenesis in vivo, provides a model system that can be helpful in identifying carcino- genic agents and exploring their mechanisms of action. Few detailed dose-response curves for cell transforma- tion have been published as yet, but the morphological transformaton of Syrian hamster embryo cells by ben- zo(a)pyrene (BAP) (48,49) is consistent with one-hit kinetics except at cytotoxic dose levels (50). A one-hit model also holds for the transformation of such cells by the combined effects of x-rays and BAP (50). With x-rays alone, the frequency of transformation per surviving cell is increased by a dose as low as 10 mGy, above which it appears to increase curvilinearly with the dose up to 1.5 Gy; however, a linear increase over the same dose range cannot be excluded (51). Although the rate of transformation per unit dose typically decreases on pro- traction or fractionation of exposure to gamma rays, it may increase on protraction or fractionation of exposure to fast neutrons (Fig. 6). In C3H101/2 cells irradiated in vitro-as well as in thyroid and mammary "clonogens" irradiated in vivo (52)-"initiation" appears to occur with a frequency as high as 0.01-0.1 per cell per Gy (53) and to increase as a linear nonthreshold function of the dose (Fig. 7). The subsequent, final transforming event in such cells is far rarer, however, occurring at a rate of only 10-6 to 10-7 per cell generation (53,54).

272 15 38cGy/mm 0O86cOf/m,n JANUS NEUTRONS SLOPE (:Gy-'1 59fix10'1 53.0x10-1 IOTV2 CELLS COR COEF 0.990 0 998 SLOPE RATp(-/el 1 8.89 0 086cGy/min ' 38CGy /min LL 0 20 40 60 DOSE,cGy 100 Figure 6. Frequency of neoplastic transformation in C3H IOTI/2 cells exposed to fission-spectrum neutrons. Dashed lines indicate linear regres- sions fitted to the initial portions of the dose-effect curves. (Reproduced from Ref. 67.) Interpolation and Extrapolation Models Although the relation between the incidence of neoplasia and the dose of carcinogen is known to vary with the type of neoplasm, the carcinogen, and other variables, the dose-incidence relationships at low doses is not known precisely for any neoplasm or carcinogen. The risks of low-level exposure to a cancer-causing agent can thus be assessed only through interpolation or extrapolation from effects observed at higher levels of exposure. For many of the neoplasrns induced by ionizing radiation, the dose- incidence relaticn generally conforms to the patterns il- lustrated in Figure 8, which are consistent with those to be expected if the probability of carcinogenesis could be increased in a suitably susceptible individual by an appro- priate mutation or chromosomal aberration in a single somatic cell. Under this assumption, the dose-incidence curve for high-L',ET radiation would be expected to con- form, in general, to the expression: I = C + aD (1) where I is the incidence at dose D, C is the incidence in nonirradiated can:rols, and the coefficient a is a constant; similarly, for low-LET radiation, the dose-incidence curve would conform, in general, to the expression: I = (C + aD + bD2)e-(p°+q°2) (2) where the symobls are comparable to those above, ex- cept for a different value of the coefficient a and the ad- dition of the coefficients b, p, and q (55). Upton 70 t1 G 30 C a) ~ ti 20 C O ~ a E 10 ~ ~ ~ H 0 •- Dose (Rods) Figure 7. Dose-response relationship for the induction of neoplastic transformation in mouse 10T1/2 cells by x-rays alone (o), or by x-rays followed by phorbol ester, starting 48 h after irradiation and continued for the full 6-week expression period (•). No increase in transforma- tion frequency was detected following exposure to phorbol ester alone. (Reproduced from Ref. 68.) While many of the observed dose-incidence curves con- form to the latter pattern, the curve for radiation-induced breast cancer appears more nearly linear, as noted above. To allow for uncertainty about the shape of the dose- incidence curve at low doses and thus to obtain a range of reasonable risk estimates, alternative models (Figs. 9 and 10) have been used in assessing the risks of low-level exposure to carcinogens. Most such models treat carcino- genesis as a multicausal, multistage process. Depending on the particular model that is used for interpolation or extrapolation, however, the estimated risk at low doses can vary by order of magnitude (e.g., Table 3). The linear (one-hit) model for interpolating between the lowest dose

Thresholds for Radiation and Chemical Carcinogenesis P(d) 30-5 '+ 1Figure 8. Estimated risk of liver cancer, p(d), in relation to the dose of aflatoxin, d, as de:ermined with different dose-incidence models; i.e., OH, one-hit model; MS, multistage model; W, Weibull modelt MH multihit model; and MB, Mantel-Bryan (log-probit model). (From Ref. 56.) Table 3 Estimated Risk cf Cancer of the Human Urinary Bladder from Dail,v Ingestion of 0.12 g of Saccharin Method of transspecies scaling and of high- to low-dose extrapolation Lifetime cases per million exposed Rat dose adjusted to human dose by surface area rule Single-hit model 1,200 Multistage model (with quadratic term) 5 Multihit model 0.001 Mantel-Bryan probit model 450 Rat dose adjusted to human dose by mg/kg/day equivalence Single-hit modet 210 Multihit model 0.001 Mantel-Bryan prot» t model 21 Rat dose adjusted to human dose by mg/kg/lifetime equivalence Single-hit model 5,200 Multihit model 0.001 Mantel-Bryan probit model . 4,200 Source: From Ref. 60. 273 g:ner.)forrn all killing atterwrttes F (D) F(D) -(ap + at D+aZD2IeKP(-dt D-QZD2) Dose, D F(D)=aa+nZD2 WMrauc Dou,D F(D)-aa+etD tineu Oose, 0 F{D)=ca+crD.c~D~ hner auadytic Figure 9. Dose-response curves for four different mathematical models relating cancer incidence to radiation dose which were evaluated by the National Academy of Sciences Advisory Committee on the Biological Effects of Ionizing Radiation. (From Ref. 33.) - high dose rate - - - iow dose rate DOSE -~- Figure )[0. Diagrammatic representation of char.tcterisdc dose-response curves, relating the incidence of tumors in laboratory animals to the dose and dose rate of high-LET (-) radiation and low-LET (---) radiation. (Reproduced from Ref. 69.) at which a significantly increased incidence has been observed and the baseline (zero dose) incidence is general- 1y thought to overestimate the risk at low doses (8,56), and thus to provide an "upper limit" estimate of risk, with the lower limit of the range extending to zero.

274 Although the mechanisms of action of carcinogens of different types are still to be defined precisely, the ex- isting data suggest that a linear nonthreshold interpola- tion model may be appropriate only for an initiating agent or a complete carcinogen, and that a model yielding a smaller estimate of the risk at low doses is more likely to be appropriate for a promoting agent. Similarly, for a chemical that is activated through nonlinear metabolic processes (57) or that acts through toxic effects elicited only at relatively high doses (e.g., immunosuppression) (58), a thresholad or quasithreshold dose-incidence model is likely to be more appropriate. In view, howt:ver, of the existence within the human population of individuals who vary widely in their suscep- tibility to cancer, as well as those who are at different stages of carcingenesis as a result of the action of other cancer-causing agents or risk factors, it is assumed that a carcinogen may pose some degree of risk to the popula- tion at any dose, by exerting carcinogenic effects that are additive with those which account for the "spontaneous" baseline incidence of cancer (Fig. 11). Hence, unless an agent can be shown to act through effects that are not addi- tive with those which account for the "spontaneous" baseline incidence of cancer, a nonthreshold model is generally recommended for assessing the carcinogenic risks of the agenr for public health purposes. ProbabNRy of Cancer Figtnre 11. Diagram illustrating the expected increment in risk of cancer resulting from a lowdose of a hypothetical carcinogen. Because cellular effects similar to those of the carcinogen may be produced in its absence by "background" mechanisms, the effects resulting from low doses of that carcinogen may be additive with those resulting from other "back- ground" risk factors, thu;; causing an increase in the risk that is propor- tional to the dose. (From Ref. 70.) Upton ACKNOWLEDGMENT Preparation of this report was supported in part by Grants ES 00260 and CA 13343 from the U.S. Public Health Service and Grant S[G-9 from the American Cancer Society. The author is grateful to Mrs. Lynda Witte for assistance in the prepar- tion of this report. Address reprint requests to Arthur C. Upton, Institute of Environmen- tal Medicine, New York University Medical Center. 550 First Avenue. New York, New York 10016. REFERENCES 1. Muller HJ: The manner of production of mutations by radiation. In Radiation Biology Vol. 1. High Energy Radiation. Edited by A Hollaender. McGraw-Hill, New York, 1954, pp 475-626. 2. United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). Report of the Scientiftc Committee on the Effects of Atomic Radiatiott. United Nations, New York, 1958. 3. United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). Genetic and Somatic Effect of Ionizing Radiation. Report to the General Assembly, with Annexes. United Nations, New York, 1986. 4. Auerbach C: Chemical mutagenesis. Biol Rev 24:355-391, 1949. 5. Ehling UH, Averbeck D, Cerutti PA, et al: Review of the evidence for the presence or absence of thresholds in the induction of genetic effects of genotoxic chemicals. International Commission for Pro- tection Against Environmental Mutagens and Carcinogens. ICPEMC Publication No. 10. Mutat Res 123:281-341, 1983. 6. Lewis EB: Leukemia and ionizing radiation. Science 125:965-975. 1957. 7. Scherer E, Emmelot P: Multihit kinetics of tumor cell formation and risk assessment of low doses of carcinogen. In Carcinogens: Identification and Mechanisms ofActron. Edited by AC Griffin, CR Shaw. Raven Press, New York, 1979, pp 337-364. 8. Zeise L. Wilson R, Crouch EAC: The dose response relationships for carcinogens: a review. Env Health Perspect 1988 73:259-306, 1987. 9. Fialkow PJ: Clonal origin of human tumors. Biochim Biophys Acta 458:283-321, 1979. 10. Ponder BAJ: Genetics and cancer. Biochim Biophys Acta 605: 368-410, 1980. 11. Sandberg AA: A chromosomal hypothesis ofoncogenesis. Cancer Genet Cytogenet 8:277-285, 1983. 12. Farber E, Sarma DSR: Biology of disease. Hepatocarcinogenesis: A dynamic cellular perspective. Lab Invest 56:4-22, 1987. 13. Nicholson GL: Tumor cell instability, diversification, and progres- sion to the metastatic phenotype: from oncogene to oncofetal ex- pression. Cancer Res 47:1473-1487, 1987. 14. Barrett JC, Crawford BD, Ts'o POP: The role of somatic muta- tion in a multistage model of carcinogenesis. In Mammalian Cell Transformation by Chemical Carcinogens. Edited by N Mishra, VC Dunkel, M Mehlman, Senate Press, Princeton Junction, NJ. 1980, p 467. 15. Farber E: Cellular biochemistry of the stepwise development of cancer with chemicals: G.H.A. Clowes Memorial Lecture. Cancer Res 44:5463-5474, 1984. 16. Barrett JC, Elmore E: Comparison of carcinogenesis and muta- genesis of mammalian cells in culture. In Handbook ofErperimental

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