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{ UPTON: THRESHOLDS FOR CARCINOGENESIS? 873 The frequency of chromosomal aberrations in human lymphocytes irradiated iin vitro increases as a linear-quadratic nonthreshold function of the dose, approx- icnating 0.1 aberration per cell per Sv in the low-to-intermediate dose region.85 The dose required to double the frequency of aberrations in such cells can thus be calculated to approximate 0.05 Sv.86 With high linear energy transfer radiation, the frequency of aberrations increases more steeply, as a linear nonthreshold function of the dose, and irrespective of the dose rate.ss,as Dose-response relationships for chemically induced mutations and chromo- somal aberrations are less well defined than those for ionizing radiation, in part because of the greater diversity of types and mechanisms of chemically induced DNA damage. Chemical mutagenesis and clastogenesis involve complex pro- cesses, including pharmacokinetic variables (uptake, transport, distribution, and excretion), metabolic activation and detoxication, and various reactions leading to the production of DNA lesions and their subsequent repair-misrepair. Each of these steps may conceivably involve first-order kinetics at low doses and hence be linear, so that in principle the overall process may be linear and not approach a threshold. Even if mutagenesis at low dose levels involved only linear processes, the slope of the resulting dose-response relationship could be orders of magnitude shallower than the slope at high dose levels, so that the dose-response curve could appear to reach a threshold or a quasithreshold.87 In fact, nonlinear mecha- nisms are likely to operate in at least some of the transport, metabolism, elimina- tion, and repair processes that are involved in mutagenesis,8' and it is noteworthy that a single step involving a threshold in such a sequence could give the overall process a threshold. Hence, in view of the complexity of the many processes involved in chemical mutagenesis, it is not astonishing that the dose-response curves for mammalian cells exposed in vitro have been observed to include re- sponses that appear to involve linear nonthresholds as well as quasithresholds.88 Whether any of the responses truly involves a threshold, however, cannot be det:e rmined from existing data. Other factors complicating assessment of the practical implications of dose- response data for chemical mutagenesis are the fact that chemicals vary more than a millionfold in mutagenic potency and the fact that the magnitude of the variation among chemicals also differs depending on the types of cells and indices of mutagenicity in question.88,89 FACTORS MODIFYING THE DOSE RESPONSE A variety of factors are known to affect dose-incidence relationships in car- cinogenesis.4D These include, among others, variables influencing the susceptibil- ity of exposed individuals (for example, genetic background,I ' age at exposure,2S immunological reactivity,91 differences in DNA repair capacity,8S and differences in drug metabolism9z,93). The capacity to metabolize a chemical can vary among humans by more than 100-fold11 and among species by more than 1000-fold.93 In any one person, moreover, the balance between toxification and detoxification may be highly dose-dependent.95 As a result, the effective dose of a substance to its biological target may differ substantially among persons at a given ambient exposure level. Also of potential importance in modifying the dose-response relationship for a given carcinogen are the effects of other physical or chemical agents. The interac-

874 ANNALS NEW YORK ACADEMY OF SCIENCES tive effects of these agents may be additive, synergistic, or inhibitory, depending on the agents in question and the conditions of exposure.56,116 In a number of instances, appropriate stimulation by a tumor-promoting agent has been observed to convert a curvilinear dose-incidence response involving a threshold into a linear response not involving a threshold (FIG. 9). HEALTH POLICY IMPLICATIONS The problem of risk assessment for purposes of public health policy is compli- cated by the fact that cancer arises through successive stages, each of which may be affected differently and in as yet unpredictable ways by a given agent. No single process is known to be applicable to all carcinogens, all types of cancer, and all persons at risk. Most multistage models assume, however, that 1) a normal cell must undergo two or more stochastic and essentially irreversible changes to become transformed into a cancer cell; 2) one or more of the changes may be inherited via the fertilized egg (zygote); and 3) it is the clonal proliferation of a 5µq TPA 2x/week \0 P : i-NoTPA 10 20 30 40 50 so 70 BoP (µg/week) FIGURE 9. The incidence of carcinomas of the skin after 350 days of treatment in mice exposed to a weekly dose of benzo[alpyrene on Mon- day, with or without 5.0 Ecg of 12-O-tetradeca- noylphorbol-13-acetate on Wednesday and Friday. Doses refer to the amount of ben- zo[alpyrene given per week. The treatments were started at 56 days of age. (Reproduced from Burns and Albert" with permission from Mary Ann Liebert, Inc.) single cell in which all the necessary changes have occurred that ultimately gives rise to a cancer.97 According to such a model, any agent that directly or indirectly increases the probability of any one of the changes may be a carcinogen because such an agent would increase the likelihood that a cell will ultimately acquire all of the changes necessary for transformation. The model also implies that the changes necessary for malignant transformation must occur in the proper se- quence, because some carcinogenic stimuli act only on early stages while others act on later stages, and that carcinogens that affect different stages in the process can be multiplicative rather than merely additive in their combined effects. In the absence of definitive human data, risk assessment must depend on other types of evidence (for example, on the results of bioassays in laboratory animals or on short-term tests for carcinogenicity). Under such circumstances, risk as- sessment is complicated by questions about 1) the reliability of the test system for predicting risks to humans (quantitatively as well as qualitatively); 2) the repro- ducibility of the test results; 3) the influence of species differences in pharmaco- kinetics, metabolism, hoemostasis, repair rates, life span, organ sensitivity, and baseline cancer rates; 4) the influence of differences in dose, dose rate, and routes

UPTON: THRESHOLDS FOR CARCINOGENESIS? 875 of exposure; 5) the significance of benign, as opposed to malignant, tumors; 6) the precise nature of the dose-incidence relationship; and 7) the significance of nega- tive results. On r.he basis of present knowledge, the carcinogenicity of an agent for human tissue cannot be predicted accurately by extrapolation from animal data. A chemi- cal that causes tumors in a particular organ of one species may cause tumors in another organ, or no tumors at all, in other species; for example, bioassay results in the mouse have been predictive for the rat in only about 80% of cases, and vice versa.18-99 The problem is complicated further by the fact that the human popula- tion is exposed to myriads of agents interacting in various ways, whereas animals in the standard bioassay are ordinarily exposed to only one agent at a time. ' The dose-incidence models used by national and international experts for estimating the carcinogenic risks of low-level ionizing radiation are generally of the nomthreshold type.27-29•10° The models also allow, however, for the fact that the magnitude of risk per unit dose appears to vary with the form of cancer, sex, age at irradiation, type of radiation (linear energy transfer), dose, and dose rate. In view ~of these differences, each type of neoplasm is generally considered indi- vidually, with efforts to integrate insofar as possible all relevant epidemiological and experimental data. Although the relation between incidence and radiation dose is known to vary from one type of neoplasm to another, the observed effects of dose rate and linear energy transfer on the dose-incidence relation generally conform to the patterns illustrated in FIGURE 8, which are consistent with those expected if one were to assume that carcinogenesis could be initiated in a suitably susceptible individual by a mutation or chromosomal aberration in a single somatic cell. According to this interpretation, the dose-incidence curve for high linear energy transfer radia- tion woul.d be expected to conform, in general, to the expression I = C + aDE-pD (2) where I is the incidence at dose D, C is the incidence in nonirradiated controls, and a and p are constant coefficients; for low linear energy transfer radiation, the dose-incidence curve would conform, in general, to the expression , I=(C+aD+bD2)e-(pD+qD2) (3) where the symbols are comparable to those above, except for different values of the coefficients a and p and an additional coefficient q.tol Although many of the observed dose-incidence curves conform to the pat- terns described above, the curve for breast cancer appears more nearly linear, and the curve for osteosarcomas induced by radium-226 appears more nearly qua- dratic.28 'Because of the complex, multicausal, multistage nature of carcinogene- sis, no one simple model is likely to characterize the dose-incidence relation over a wide range of doses and exposure conditions. At intermediate-to-high doses, a complete carcinogen can be expected to exert promoting effects as well as initiat- ing effects on tumor formation through alterations in cell population kinetics and other changes. At still higher doses, the response can be expected to saturate because of cytotoxicity. In vie w of uncertainty about the shape of the dose-incidence curve at low doses and low dose rates, various hypothetical models have been used in an effort to arrive at a range of estimates for assessing the risks of low-level radiation (FIG. 10) and chemicals (FIG. 11).

876 ANNALS NEW YORK ACADEMY OF SCIENCES I goneral form a all killing ,~, anenutte: F(D) ~ c F(D) - (ap +c~ D+a2D2l&xp(-Qr D -AZD2) - Doa.. D Do+., D Dou, D a FIGURE 10. Dose-response curves for four different mathematical models relating cancer incidence to radiation dose. (Reproduced from Reference 28 with permission from the National Academy Press.) Criteria to aid in the evaluation of epidemiological and experimental data on the carcinogenicity of chemicals have been formulated by the International Agency for Research on Cancer,26 the Interagency Regulatory Liaison Group,102 and the Office of Science and Technology Policy.103 These criteria include defini- tions for weighing the adequacy of the data (for example, definitions of "sufficient evidence" and. "limited evidence" 26). In situations where there is sufficient evi- dence for the carcinogenicity of a chemical in'laboratory animals but not in humans, the compound is assumed to present a carcinogenic risk to humans, although the magnitude of the risk cannot be estimated with precision.26 Although Ibioassay and short-term "screening" tests may give information on the mode of action of a chemical, such tests are considered to provide no more than support- i.ng evidence of carcinogenicity and not to provide sufficient evidence by them- selves. Estimation of carcinogenic risks on the basis of animal data, however good the an imal data may be, is fraught with uncertainty. Although a chemical with carcin- ogenic potency in one species (such as aflatoxin Bi) is likely to be carcinogenic in another, the procedure for extrapolating across species involves assumptions about species differences in metabolism and appropriate scaling factors for dose and time. Various attempts have,been made to determine correct scaling factors based on pharmacokinetic data,95 but the question remains unresolved.

UPTON: THRESHOLDS FOR CARCINOGENESIS? In addition, a dose-response model must be used for interpolating between the lowest dose at which a significantly increased incidence has been observed and the base.line (zero dose) incidence. For this purpose, a linear, nonthreshold (one- hit) dose-incidence model is generally recommended, although such a model cannot be verified experimentally.104 This type of model gives higher estimates, however, than other models (FIG. 10 and TABLE 2). Hence it is usually thought likely to overestimate the risk at low doses and is thus often considered to esti- mate the "upper limit" of risk. Evidence concerning the modes of action of different classes of carcinogens (initiatoi,s, promoters, co-carcinogens, and complete carcinogens) suggests that a linear nonthreshold model may be appropriate only for initiating agents and com- plete carcinogens, whereas models yielding smaller estimates of risks at low doses might represent more accurately the dose-incidence relationships for other classes of carcinogens. For some types of carcinogens, thresholds might even be envisioned to exist because of relevant pharmacokinetic factors. For example, some chemicals that must be activated metabolically to become carcinogenic may be handled through nonlinear metabolic processes, with the result that thresholds for their carcinogenic effects may exist.87 In addition, some agents may act through i.oxic or systemic effects that are produced only at high doses (for exam- ple, those causing carcinogenic effects on the mucosa of the urinary bladder in association with cystitis and urinary tract calculi,106 or those acting through im- munosuppressive effects.91 If it can be shown, however, that a chemical acts through mechanisms that are shared by agents that contribute to the baseline incidence of "spontaneously P(d) g0'5 -4 10-8 -~ ( -F - 1 I 10~2 102 d FIGURE iCl. Estimated risk of liver cancer, P(d), in relation to dose of aflatoxin, d, as determined with different dose-incidence models. The models for the different curves are as follows: OH, one-hit model; MS, multistage model; W, Weibull model; MH, multihit model; MB, Mantel-Bryan (log-probit model). (Reproduced from Krewski and Van Ryzin10' with permission from the Elsevier/North-Holland Publishing Company.)

878 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE 2. Estimated Human Risks from Ingestion of 0.12 G/Day of Saccharin Method of High- to Low-Dose Extrapolation Lifetime Cases per Million Exposed Cases per 50 Million per Year Rat dose adjusted to human dose by surface area rule Single-hit model 1,200 840 Multistage model (with quadratic term) 5 3.5 Multihit model 0.001 0.0007 Mantel-Bryan probit model 450 315 Rat dose adjusted to human dose by mg/kg/day equivalence Single-hit mod'eI 210 147 Multihit model 0.001 0.0007 Mantel-Bryan probit model 21 14.7 Rat dose adjusted to human dose by mg/kgl lifetime equivalence Single-hit model 5,200 3,640 Multihit model 0.001 0.0007 Mantel-Bryan probit model 4,200 2,940 NOTE: Adapted from Reference 105. occurring" cancer, then exposure to only a small dose of the chemical can be expected to increase the incidence by some finite amount.z•107-109 For this reason, the use of a nonthreshold model is generally recommended in risk assessment when the mode of action of the carcinogen in question is not known. CONCLUSIONS The possibility that there may be no threshold for the induction of some forms of cancer by ionizing radiation or certain chemicals, at least in appropriately susceptible individuals, is suggested by (1) evidence that most cancers arise from a single transformed cell; (2) the heritable nature of the transformed phenotype; (3) the association between neoplastic transformation and specific mutations or chromosomal aberrations; (4) the correlation between carcinogenicity and geno- toxicity; (5) the nature of the observed dose-response relationships for mutations, chromosomal aberrations, and cell transformation in vitro; and (6) the nature of the dose-incidence relationships for certain neoplastic lesions in vivo. At the same time, however, carcinogenesis appears to be a multistage process involving the stepwise evolution of increasingly autonomous cells in which the outcome is influenced by such variables as age, genetic constitution, physiological state, metabolism, and homeostatic interactions within and among tumor-forming cells and normal cells. Other variables that complicate analysis of dose-incidence relationships are (1) poorly defined interactions among cancer-causing agents, which may be additive, multiplicative, or antagonistic in their combined effects; (2) the fact that the human environment contains myriads of agents, many of which are known to modulate the effects of others; (3) the existence of nonlinear kinetics in the metabolism of certain chemical carcinogens; and (4) evidence that some agents act primarily through mechanisms that presumably operate only at high dose levels.

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