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~ . - " ~9:~5 XE489 177 ~l HUTAT RES-FUND HOL 11 97 [C]ELSEVZER SOIENCE BV POIR NE Fundamental and Molecular Mechanisms of Mutagenesls ! ELSEVIER Mutation Research 376 (1997) 177-184 Aromatic amine DNA adduct formation in chronically-exposed mice: considerations, for human comparison , Miriam C. Poirier a, , Frederick A. Beland b National Cancer Institute, Bldg. 37 Rm. 3B25, MSC-4255, 37 Convent Drive, NIH, Bethesda, MD 20892-4255, USA b National Center for Toxicological Research, Jefferson, AR 72079, USA Abstract Lifetime chronic exposure of mice to the aromatic amines 4-aminobiphenyl (ABP) and 2-acetylaminofluorene (AAF) produces liver and urinary bladder tumors. In parallel experiments, DNA adduct levels in target tissues reach a steady-state (a balance between adduct formation and removal) after about four weeks of either AAF or ABP ingestion. For these and other carcinogens, steady-state DNA adduct levels most frequently increase linearly with dose, but the formation of tumors also depends upon a variety of factors, including the proliferative capacity of the target tissue, the sex of the animal, genotoxic properties of the specific adducts formed, and other unknown events. Chronic dosing experiments in animal models are of interest for human risk assessment because human exposure is typically intermittent, involving repeated However, it is to be that in genetically-diverse human population, where the lifetime > 70 expected exposures. a averages years, the relationship between tumorigenesis and DNA adduct formation will be relatively more complex than that observed in mice. From our studies of chronic ABP exposure in male mice, we have obtained the daily dose of ABP and the steady-state level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) adduct associated with a 50% mouse bladder tumor incidence. Our attempt at a human extrapolation for adducts and urinary bladder cancer in smoking males (20-40 cigarettes/day) is based on the ABP dose per cigarette, values for the dG-C8-ABP adduct in bladder biopsies of lifetime heavy smokers at age ~ 70, and the smoking-related bladder tumor incidence (absolute lifetime risk) for Caucasian males in the United States aged 65-84 years. The extrapolation has produced two major predictions, one related to adduct formation and the other related to tumorigenesis. First, the observed level of smoking-related dG-C8-ABP in DNA of human bladder epithelium, expressed as a function of daily ABP intake, is about 3500-times higher than similar data for mice, which suggests that humans may perform the biotransformation of ABP more efficiently than mice. Second, at a similar bladder tumor incidence, mouse bladder contained adduct concentrations that were much higher than those observed in human bladder; for example, at a 2.6% tumor incidence, mouse bladder contained an average of 55.5 fmol dG-CS-ABP/Ixg DNA (1850 adducts/10s nucleotides), while bladders from Caucasian male smokers contained an average of 0.036 fmol dG-C8-ABP/I~g DNA (1.2 adducts/10s nucleotides). This suggests that factors other than ABP-DNA adducts, such as adducts of other carcinogens, the influence of promoters, and synergistic effects of all of these factors contribute substantially to smoking-related bladder cancer in humans. Ke~.ords: 4-Aminobiphenyl; Cancer; Lifetime exposure; Bladder; Mouse 1 i " Correspqnding author. Tel.: + 1 (301) 402-1835; Fax: + 1 (301) 496-8709; e-mail: poirierm@dc37a.nci.nih.gov 0027-5107/97/$17.00Copyright © 1997 Elsevier Science B.V. All dghtsrese~ed. PH S0027-510~(97)00041-9 THIS ARTIOLE 15 FOR INDIVIDUAL USE ONLY AND HAY HOT BE FURTHER REPRODUOED OR STORED ELECTRONICALLY NZTHOUT NRITTEN PERHISSZON FROH THE COPYRIGHT HOLDER. UNAUTHORIZED REPRODUCTION HAY RESULT IN FIHANCIAL AND OTHER PEHALTZE$.

178 M. C. Poirier, F.A. Beland / Mutation Research 376 (1997) 177-184 1. Introduction If DNA adduct levels in humans are to be applied for the prediction of human cancer risk, it may be useful first to explore the relationship between adduct levels and tumorigenesis in animal models before making human comparisons. This study is an attempt to bring together current knowledge concerning the dose of a chemical carcinogen, tumor incidence, and DNA adduct formation in the same target tissue. The chemical carcinogen in question is 4-aminobiphenyl (ABP) and the comparison will be made for urinary bladder cancers in mice given ABP chronically in the drinking water (Poirier et al., 1995) and humans inhaling ABP in cigarette smoke (Patrianakos and Hoffman, 1979; Poirier and Beland, 1992). The anal- ysis is based on certain assumptions, with particular caveats that will be discussed in detail, since the conclusions are only as sound as the underlying data. However, more than providing a definitive statement on mouse-human extrapolation, hopefully this exer- cise will constitute a framework for further thought and discussion. 4-Aminobiphenyl is a potent urinary bladder car- cinogen for both humans (Clayson, 1981) and mice (Schieferstein et al., 1985). Widespread chronic hu- man exposure occurs through cigarette smoking (Patrianakos and Hoffman, 1979), and studies in mice have demonstrated tumorigenesis resulting from continuous lifetime administration in the drinking water (Schieferstein et al., 1985). In this report, data for tumorigenesis and DNA adduct formation in mice chronically-exposed to 2-acetylaminofluorene (AAF) and ABP will be reviewed. In addition, smok- ing-related ABP doses, human bladder DNA adduct levels, and incidence of smoking-related bladder tu- morigenesis in 65-84-year-old Caucasian males in the United States have been obtained from the litera- ture. Finally, the three parameters (chronic dose, DNA adduct level, and tumor incidence) have been compared and extrapolated to elucidate species- specific mechanisms of ABP genotoxicity. Admit- tedly, the extrapolation presented must contain omis- sions; nonetheless, it constitutes an attempt in an iterative process designed to evaluate interspecies comparisons of DNA adduct determinations and. their use in human cancer risk estimation. This prototype analysis has novel conclusions and demonstrates an approach that may eventually be informative for other classes of chemical carcinogens, such as the aflatoxins, heterocyclic amines, and polycyclic aro- matic hydrocarbons. 2. Chronic dosing of aromatic amine carcinogens in mice 2.1. Kinetics of DNA adduct formation and retnoval during chronic carcinogen administration In studies of chemical carcinogens in which a single concentration of compound is given chroni- cally, the DNA adduct levels increase rapidly at first, but a plateau is obtained when the processes govern- ing DNA adduct formation and those responsible for adduct removal reach equilibrium (Poirier and Be- land, 1992; Poirier and Beland, 1994). The magni- tude of the plateau reflects the concentration of chronically-administered carcinogen. For example, in livers of mice given AAF for 56 days at a concentra- tion of 30 mg AAF/kg diet, the adduct plateau was at 32 fmol adduct/l~g DNA, while at 150 mg AAF/kg diet, the plateau was at 105 fmol/~zg DNA (Poirier and Beland, I994). At doses between 30 and 150 mg/kg, the adduct levels at equilibrium are predicted to vary between 32 and 105 fmol/p.g DNA and be proportional to dose. A dose-response profile for multiple doses can be generated by plot- ting DNA adduct levels at a single time point as a function of the carcinogen' concentration for each dose administered (Poirier and Beland, 1992). Six DNA adduct dose-response profiles have been obtained with aromatic amines; these include livers and bladders of male and female mice given ABP chronically in the drinking water (Poirier et al., 1995) and livers and bladders of female mice given AAF in the diet (Poirier et al., 1991). For both compounds DNA adducts were determined in livers and bladders after 28 days of exposure to several doses. In the livers and bladders of female mice exposed to AAF, adduct levels increased linearly with dose through the entire dose range. A similar trend was observed in livers of male mice given ABP. In the bladders of male mice and the livers of female mice given ABP, there was also a linear increase in adduct formation at the four lowest ABP | i | ! ! I I li II

i i i i i i i ! M. C. Poirier, F.A. Beland / Mutation Research 376 (1997) 177-184 179 doses. In the bladders of female mice, the dG-C8- ABP adduct levels reached a plateau at low doses. Thus, linearity for DNA adduct formation, particu- larly in the lower dose range, was observed in five of the six different dose-response profiles (Poirier and Beland, 1994). 2.2. Mouse tumorigenicity studies The DNA adduct studies described above were modeled on tumorigenicity experiments in which mice were given either AAF in the diet (Staffa and Mehlman, 1979) or ABP in the drinking water (Schieferstein et al., 1985) chronically at several doses for a lifetime. Tumors were observed in livers and bladders. Profiles for tumorigenicity as a func- tion of increasing carcinogen concentration were lin- ear only in livers of female mice exposed to AAF. Low tumor incidences (< 15%) were observed in bladders of female mice given ABP and in livers of male mice given ABP. In bladders of male mice exposed to ABP and female mice exposed to AAF, tumorigenesis was negligible at the lowest doses but increased rapidly at the highest doses; in the case of AAF, this has been attributed to the induction of cell Table 1 Correlation between dose, dG-C8-ABP (mean + SEM) and tumor incidence in bladders of male mice given ABP in the drinking water ABP in ABP dose a Bladder Bladder drinking water dG-CS-ABP tumors (ppm) (wg/kg b.wt./day) (fmol/Ixg DNA) (%) 28 5600 37.7 + 27.9 0% 6300 b 55.5 2.6% 55 11000' 114.64-8.6 17.6% 1 I0 22000 138.8 4- 7.7 48.4% 22400 140 50% a Assuming a 25 g male mouse consuming 5 ml of water per day; b.wt, body weight. b Values in bold were obtained directly from curves of dose vs. adducts and dose vs. tumor incidence (not shown)~ CA value of 2.56% was used for the smoking-related human bladder tumor incidence, but it was not possible to read so accurately from the mouse curves; therefore the incidence used for the mice was 2.6%. 2.3. DNA adducts and tumorigenesis in bladders of male mice given ABP The mouse-human comparison to be generated in this paper will focus on tumorigenesis and DNA adduct formation in bladders of male mice given proliferation at higher doses of carcinogen (Cohen ABP in the drinking water. Bladder tumors were and Ellwein, 1990). Taken together, the data suggest observed by Schieferstein et al. (1985) in male that DNA adducts may constitute a necessary pre-re- BALB/c mice given ABP at six dose levels in the quisite for tumorigenesis, but that other contributing drinking water for 24 months. DNA adducts were I 1 I 1 1 1 1 factors include cell proliferation, sex of the animal, tissue specificity, and additional unknown events. =~ 80 ' "~ 60 20 50 100 150 200 fmol dG-CS-ABP//ag DNA Fig. 1. Relationship between the levels of dG-C8-ABP in bladder DNA of male BALB/c mice administered ABP for 28 days in the drinking water (Poirier 6t al., 1995) and the reported tumor incidence in male BALB/c mice administered the same doses of ABP for 14 to 24 months (Schieferstein et al., 1985). examined in bladders of similarly-treated mice of the same strain given ABP for 28 days. Fig. 1 shows the relationship between bladder tumor incidence and dG-C8-ABP adduct formation at doses of 0, 7, 14, 28, 55, Ii0 and 220 ppm (Schieferstein et al., 1985; Poirier et al., 1'995). Adduct and dose values at tumor incidences of 2.6% and 50% were obtained from the curves of dose vs. tumors and dose vs. adducts, as published previously (Poirier and Beland, 1994; Poirier et al., 1995; Schieferstein et al., 1985); the values are shown in Table 1. 3. Chronic dosing of ABP in humans through smoking 3.]. Overall strategy The strategy employed for the human analysis was to ascertain the smoking-related lifetime abso- i

180 M. C. Poirier, F.A. Beland / Mutation Research 376 (1997) 177-184 lute bladder cancer risk (bladder tumor incidence) for Caucasian males in the United States, aged 65-84 years, who smoked I-2 packs of cigarettes per day (Hartge et al., 1987 and P. Hartge, personal commu- nication). The daily dose of ABP was estimated based on a published value of 2.4 ng ABP per American cigarette (Patrianakos and Hoffman, 1979; Vineis, 1992). The smoking-related DNA adduct levels are from three different studies in which adducts have been measured in human bladder by different methods (Cuzick et al., 1990; Talaska et al., 1991; Lin et al., 1994). 3.2. Epidemiological studies of smoking-related bladder cancer As an initial approach, expected yearly increases in bladder tumor incidence for Caucasian males in the United States (from Table 2 in Silverman et al., 1992) were summed between ages 20 and 70 and corrected so that individuals with cancer did not remain in the cohort. Thus calculated, the cumulative bladder tumor incidence at age 70 was estimated to be 4.4%. A more accurate, but similar value of 4.82% was calculated (P. Hartge, personal communi- cation) from the most recent SEER cancer statistics (Kosary et al., 1995). Among Caucasian men aged 65-84 in the United States the relative risk of blad- der cancer upon smoking 1-2 packs of cigarettes per day was determined to be 2.13 (95%CI 1.74-2.62) and the population attributable risk for all smoking was 40% (P. Hartge, personal communication). Cal- culated from the population attributable risk (Kosary et al., 1995; P. Hartge, personal communication), the lifetime absolute risk (bladder tumor incidence) asso- ciated with this level of smoking was 2.56%, ob- tained by subtracting a background of 2.26% for non-smokers from the 4.82% value for 1-2 packs/ day smokers. 3.3. DNA adduct quantities in human bladder The measurement of human DNA adducts has often been compromised by a lack of specificity in the methods employed; however, this analysis has been made credible partly because consistent--quanti- tative data a~e available for the dG-C8-ABP in hu- man bladder biopsies in two studies. The adduct levels used in this analysis are from two studies that will be described below (Talaska et al., 1991; Lin et al., 1994). Values from the Talaska and Lin studies are almost identical, and are similar to those reported by Cuzick et al. (1990) for human bladder DNA adducts determined only by 32 P-postlabeling. In the Cuzick study, eight smokers, average age 69 years, had a mean adduct level of 0.103 fmol//~g DNA and 11 non- and ex-smokers, average age 72 years. had a mean adduct level of 0.058 fmol/l~g DNA. By subtraction, the smoking-related adducts were 0.045 fmol/l~g DNA. A more-specific approach was reported by Talaska et al. (1991), who analyzed DNA from human bladder biopsies of 13 smokers (average age 68 years) and 29 non-smokers (average age 69 years) by 32 P-postlabeling. The advantage of this study was that a value was obtained for one adduct spot, found by co-chromatography on HPLC to co-elute with an authentic dG-C8-ABP standard; this adduct appeared to constitute about 20% of the total smoking-related adducts. The average smoking-related butanol-extractable dG-C8-ABP, with values for non-smokers subtracted, was 0.036 fmol/~g DNA. Finally in a third study, (Lin et al., 1994), negative ion gas chromatography/mass spec- trometry was used to measure the dG-C8-ABP adduct in eight urinary bladder mucosa specimens from individuals with unknown smoking habits; the mean for these samples was 0.037 fmol/~g DNA. Be- the Talaska study provided the most extensive cause subject information coupled with good adduct identi- fication, the value of 0.036 fmol/izg DNA was used for the subsequent analysis. 3.4. Extrapolation for correlation between dose, DNA adducts and tumor incidence in bladders of male Caucasian smokers In constructing this analysis, linearity with dose was assumed for tumorigenicity and DNA adduct levels. This approach was considered reasonable r~ since the relative risk of urinary bladder cancer is o linearly related to the extent of cigarette consump- ~r~ tion (Mommsen and Aagaard, 1983). In addition, r.o proliferative histologic changes in the human bladder co are proportional to the extent of cigarette consump- on tion (Auerbach and Garfinkel, 1989). There is no real knowledge of the low end of the human dose-

iM.C. Poirier, F.A. Beland/Mutation Research 376 (1997) 177-184 181 Table 2 Extrapolation for correlation between dose. dG-C8-ABP, and tumor incidence in bladders of mate human smokers ~ABP dose Smoking-related dG-C8-ABP Smoking-related bladder tumors _| (l~g/kg b.wt./day) (fmol/~g DNA) (%) ~bserved 0.0013 a 0.036 b 2.56% ]~ Extrapolated e 0.0254 . 0.703 50% .~ a 75 kg male smoking 2 packs/day with 2.4 ng of ABP/cigarette. b From Talaska et al. (1991). • / c Smoking-related bladder tumor incidence for Caucasian males in the United States aged 65-84 years who smoked 1-5 packs of cigarettes | per day (P. Hartge, personal communication and Kosary et al., 1995). • ,t Based on linearity for all parameters. / response curv~ for DNA adduct formation in urinary linear extrapolation, the adducts/dose averaged 27.7. bladder; however, at very low dose levels in the Therefore, under steady-state dosing conditions, the • 1 mouse (7-14 ppm of ABP in drinking water) adduct observed level of smoking-related dG-C8-ABP in .~ levels appear to be linear with dose. Table 2 presents human bladder epithelium, expressed as a function of the results of proportional extrapolation to 50% tu- daily ABP intake, is about 3500-fold higher than .~ . mor incidence, based on the previously-discussed similar data for mice. | human values for dose and adducts at the observed The analysis in Table 3 makes no attempt to ~ smoking-related bladder tumor incidence of 2.56%. correct for inter-species dosage comparisons. Using the method of Freireich et al. (1966) to correct for l~ surface area differences between men and mice, • 4. Mouse-human comparison for DNA adduct whatever dose is given to a mouse is divided by 12 • formation as a function of chronic ABP intake to estimate the human equivalent. Therefore, at 2.6% d tumors, the mouse dose of 6,300 ~g/kg b.wt./day, | 4.1. The comparison which was associated with 55.5 fmol adducts/~g -- DNA, would be equivalent to a human dose of 525 • Further analysis of the data in Tables 1 and 2 is Izg/kg b.wt./day. If humans formed adducts at the i shown in Table 3. For the mouse, the data for 2.6% same efficiency as mice, the adducts associated with tumors and 50% tumors were obtained from the a dose of 525 I~g/kg b.wt./day would be 4.0 curves of dose vs. tumors and dose vs. adducts fmol//~g DNA. However, given the ratio of ~ published previously (Poirier and Beland, 1994). In adducts/dose observed in humans, a dose of 525 i the mouse, the ratio of adducts/dose, which suggests /~g/kg b.wt./day would be expected to produce efficiency of adduct formation, was similar at both adduct levels of 14,542 fmol/l~g DNA. Therefore, ~I tumor incidences, and averaged 0.0075. For the hu- even with an inter-species correction for surface I man, where the data at 50% tumors were obtained by area, the ratio of ABP-induced DNA adduct levels to I~ Table 3 i~ Mouse-human comparison at 2.6% and 50% bladder tumor incidence, using data from Table I and Table 2 Species Tumor incidence ABP dose dG-C8-ABP in bladder Adducts/dose l~ (~g/kg b.wt./day) (fmol/p.g DNA) ~ Mouse 2.6% 6300 55.5 0.0088 "~ 50% 22400 140 0.0062 ~ Human 2.56% 0.0013 0.036 27.7 I 0.0254 0.703 27.

182 M. C. Poirier, F.A. Beland / Mutation Research 376 (1997) 177-184 ABP intake in the human bladder appears to be approximately 3500-fold higher than similar data for the mouse. This comparison further demonstrates that at ei- ther tumor incidence (2.6% or 50%) the dG-C8-ABP levels in mouse bladder were at least 100-fold higher than those observed in human bladder. This suggests that factors other than dG-C8-ABP formation con- tribute significantly to the smoking-related bladder tumor burden. Such factors undoubtedly include DNA adducts of other carcinogens, the influence of promoting agents, and synergistic effects produced by combinations of chemicals. 4.2. The caveats and considerations In this comparison we have analyzed data from mice given ABP in the drinking water and humans receiving ABP by inhalation. Both situations have in common that the target tissue is the same, and is distal from the intake site. In addition, there is no compelling evidence that different metabolic mecha- nisms come into play when aromatic amines enter circulation by these different routes (Kadlubar and Guengerich, 1992; Bois et al., 1995). However, the comparative efficiencies of dose absorption in lung and gastrointestinal tract have not been accounted for, and therefore constitute a possible source of difference. In addition, it has been estimated that the concentration of ABP in sidestream smoke is 10-fold higher than in mainstream smoke (Patrianakos and Hoffman, 1979), suggesting that the actual dose to a smoker might be higher than the 2.4 ng per cigarette contained in mainstream smoke. The quantitative uncertainties in the 32 P-postlabel- ing assay are a possible source of error. However, by choosing a study in which the dG-C8-ABP adduct spot was identified by HPLC, at least the identity of adduct is clear. Also, the quantity reported by Ta- laska et al. (1991) appears to be sound by compari- son with the very similar numbers obtained using GC-MS (Lin et al., 1994). It is possible that these values are not completely representative, since the number of individuals was small, and further adduct studies of human bladder biopsies using different techniques are clearly warranted. In mouse liver (Poirier et al., 1995), adduct values determined by radioimmunoassay and 32p-postlabeling were very comparable, indicating that values for bladder deter- mined only by 32p-postlabeling, and used in this analysis, should also be valid. Another possible source of error is the assumption of linearity between dose and tumors, and dose and adducts for the human smokers. However, there is strong evidence in a study by Mommsen and Aa- gaard (1983) that the relative risk of developing bladder cancer for men increases linearly with the lifetime consumption of cigarettes. Also, a study by Auerbach and Garfinkel (1989) has shown that pre- neoplastic morphoiogic changes in human bladder epithelium, including atypical nuclei and hyperpla- sia, increase as a function of daily cigarette con- sumption. In addition, the weight of evidence from studies of AAF and ABP in mice suggests that the assumption of linearity at very low doses is reason- able. One important consequence of this analysis is the suggestion that human bladder cancer is higher than would be expected by just comparing ABP adducts in both species. Clearly there are other smoking-re- lated DNA adducts that have been observed bv 32P-postlabeling. Talaska et al. (1991) found that levels of total smoking-related butanol-extractable adducts were about 5-fold higher than the observed dG-C8-ABP levels; absolute quantitation cannot be determined given the unknown efficiency of uniden- tified adduct phosphorylation in 3ZP-postlabeling. However, the mutagenic efficiencies of some of these unknown adducts may vary and the bladder epithelial replication rate may not be the same for the two species. In addition, large interindividual differences in metabolism are known to exist (Kadlubar and Guengerich, 1992) and an attempt to model this using parameters from the dog predicted at least a 1000-fold interindividual variability in humans. Synergistic effects of carcinogens and pro- moting agents may enhance the tumor incidence more than would be otherwise observed if exposure were only to one carcinogen or one promoter. Future analyses may be able to incorporate some of these presently-unknown variables. 5. Conclusions Bound by the conditions and caveats stated here. the analysis has produced two major predictions. | |. |_ | |

i i M. C. Poirier, F.A. Beland / Mutation Research 376 (19971 177-184 First, that as a function of daily ABP intake, humans chronically exposed to ABP through cigarette smoke have about 3500-fold more dG-C8-ABP adducts in bladder epithelium than mice given ABP daily in the drinking water. The implication is that human may perform the biotransformation of ABP more effi- ciently than mice. Second, at any bladder tumor incidence between 2.6% and 50%, mouse bladder DNA contained much higher adduct levels than hu- bladder DNA. At 2.6% there was a 1400-fold man difference and at 50% there was a 170-fold differ- ence. This suggests that factors other than ABP-DNA adducts, contribute substantially to smoking-related bladder cancerin humans. The overall implication is that the dG-CS-ABP adduct alone contributes less to tumor formation in the human, under conditions of chronic smoking exposure, than it does in the mouse, given chronic dosing on a controlled experimental protocol. A number of factors may produce these inter- species differences. Mice have a 2-year lifespan and a very rapid metabolic rate. Humans live for more than 70 years and have a slower overall metabolism. It is possible that this allows time for mutations to accumulate in many different critical genes. In addi- tion, the longer life span allows time for exposure to other chemical carcinogens, as well as inflammatory and promoting agents that may act in concert to accelerate tumorigenesis. Again, the potential influ- ence of other smoking-related toxicities to exert syn- ergistic tumorigenic effects should not be underesti- mated. As more extensive chronic dosing studies are performed in rodents, and human epidemiologic and DNA adduct data become available, this type of analysis may be possible for other classes of chemi- carcinogens, as aflatoxins, heterocyclic cal such amines and polycyclic aromatic hydrocarbons, and may be useful for the application of human DNA adduct information within the framework of human cancer risk assessment. Acknowledgements 183 The authors wish to extend thanks to Drs. Patricia i Hartge, Fred Kadlubar, Nathaniel Rothman and Stu- art Yuspa critical reading of the manuscript and discussions of the analysis. In addition, we are greatly indebted to Dr. Hartge for calculating the lifetime human bladder cancer incidence from her own data and recent SEER statistics. The editorial assistance of Margaret Taylor is much appreciated. References Auerbach, O. and L. Garfinkel (1989) Histologic changes in the urinary bladder in relation to cigarette smoking and use of artificial sweeteners, Cancer, 64, 983-987. Bois, F.E., Krowech, G. and L. Zeise (1995) Modeling human interindividual variability in metabolism and risk: the example of 4-aminobiphenyl, Risk Anal,, 15, 205-213. Clayson, D.B. (1981) Specific aromatic amines as occupational bladder carcinogens, Natl. Cancer Inst. Monogr., 58, 15-19. Cohen, S.M. and L.B. Ellwein (1990) Proliferative and genotoxic cellular effects in 2-acetylaminofluorene bladder and liver carcinogenesis: biological modeling of the ED01 study, Toxi- col. Appl. Pharmacol,, 104, 79-93. Cuzick, J., M.N. Routledge, D. Jenkins and R.C. Garner (1990) DNA adducts in different tissuesof smokers and non-smokers, Int. J. Cancer, 45, 673-678. Freireich, E.J., Gehan, E.A., Rail, D.P., Schmidt, L.H. and H.E. Skipper (1966) Quantitative comparison of toxicity of anti- cancer agents in mouse, rat, dog, monkey and man, Cancer Chemothe. Rep., 50, 219-244. Hartge, P., D. Silverman, R. Hoover, C. Schairer. R. Altman, D. Austin, K. Cantor, M. Child, C. Key, L.D. Marrett, T.J. Mason, J.W. Meigs, M.H. Myers, A. Narayana, J.W. Sullivan, G.M. Swanson, D. Thomas and D. West (1987) Changing cigarette habits and bladder cancer risk: a case-control study, J. Natl. Cancer Inst., 78, 1119-1125. Kadlubar, F.E and F.P. Guengerich (1992) Inducibility of human cytochromes P-450 primarily involved in the activation of chemical carcinogens, Chemosphere, 25, 201-204. Kosary, C.L., L.A.G. Ries, B.A. Miller, B.F. Hankey, A. Harras and B.K. Edwards (Eds.) (1995) SEER Cancer Statistics Re- view, 1973-1992: Tables and Graphs, National Cancer Insti- tute, NIH Pub. No. 96-2789, Bethesda, MD. Lin, D, J.O. Lay, M.S. Bryant, C. Malaveille. M. FrieSen, H. Bartsch, N.P. Lang and F.F. Kadlubar (1994) Analysis of 4-aminobiphenyl-DNA in human urinary bladder and lung by alkaline hydrolysis and negative ion gas chromatography/mass spectrometry, Environ. Health Perspect., 102 (Suppl. 6), 11- 16. Mommsen, S. and J. Aagaard (1983) Tobacco as a risk factor in bladder cancer, Carcinogenesis, 4, 335-338. Patrianakos, C. and D. Hoffman (1979) Chemical studies of tobacco smoke, J. Anal. Toxicol., 3, 150-154. Poirier, M.C., N.F. Fullerton, T. Kinouchi, B.A. Smith and F.A. Beland ~t991) Comparison between DNA adduct formation and tumorigenesis in livers and bladders of mice chronically fed 2-acetylaminofluorene, Carcinogenesis, 12. 895-900.

184 M.C. Poirier, F.A. Beland / Mutation Research 376 (1997) 177-184 Poider, M.C., N.F. Fullerton, B.A. Smith and F.A. Beland (1995) DNA adduct formation and tumorigenesis in mice during the chronic administration of 4-aminobiphenyl at multiple dose levels, Carcinogenesis, 16, 2917-2921. Polder, M.C. and F.A. Beland (1992) DNA adduct measurenients and tumor incidence during chronic carcinogen exposure in animal models: implications for DNA adduct-based human cancer risk assessment, Chem. Res. Toxicol., 5, 749-755. P0irier, M.C. and F.A. Beland (1994) DNA adduct measurements and tumor incidence during chronic carcinogen exposure in rodents, Environ. Health Perspect., 102 (Suppl 6), 161-165. Sehiefersteia, G.L, N.A. Littlefield, D.W. Gaylor, W.G. Sheldon and G.T. Burger (1985) Carcinogenesis of 4-aminobiphenyl in BALB/cStCrlfC3Hf/Nctr mice, Eur. J. Cancer Clin. Oncol., 21, 865-873. $ilverman, D.T., P. Hartge, A.S. Morrison and S.S. Devesa (1992) Epidemiology of bladder cancer, Hematol. Oncol. Clin. North. Am., 6, 1-30. Staffa, J.A. and M.A. Mehlman (1979) Innovations in cancer risk assessment (ED0f) study, J: Environ. Pathol. Toxicol., 3. 1-246. Talaska, G., A.Z. A1-Juburi and F.F. Kadlubar (1991) Smoking related carcinogen-DNA adducts in biopsy samples of human urinary bladder: identification of N-(deoxyguanosin-8-yl)-4- aminobiphenyl as a major adduct, Proc. Natl. Acad. Sci. USA. 88, 5350-5354-. Vineis, P. (1992) Epidemiological models of carcinogenesis: the example of bladder cancer, Cancer Epidemiol. Biol. Prey.. 1. 149-153. I I