Hemoglobin Adducts, DNA Adducts, and Urinary ... - ACS Publications

Chapter 17

Hemoglobin Adducts, DNA Adducts, and Urinary Metabolites of Tobacco-Specific Nitrosamines As Biochemical Markers of Their Uptake and Metabolic Activation in Humans

Downloaded by CORNELL UNIV on October 29, 2016 | http://pubs.acs.org Publication Date: March 28, 1994 | doi: 10.1021/bk-1994-0553.ch017

Stephen S. Hecht, Neil Trushin, and Steven G. Carmella Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, Valhalla, NY 10595 Methods have been developed for quantitation in humans of hemoglobin and DNA adducts resulting from metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), two carcinogenic tobacco specific nitrosamines, and for quantitation in human urine of two NNK metabolites, 4-(methylnitrosamino)-1-(3 -pyridyl)-1-butanol (NNAL) and its glucuronide. The hemoglobin and DNA adducts are formed by α-hydroxylation of NNK and NNN, and release 4-hydroxy-1-(3pyridyl)-1-butanone (HPB) upon mild hydrolysis. The released HPB is derivatized and analyzed by gas chromatography-mass spectrometry. Hemoglobin adduct levels are elevated above background in 15-20% of smokers and in most snuff-dippers. DNA adduct levels are higher in lung tissue from smokers than non-smokers. Diastereomeric NNAL glucuronides have been characterized as major urinary metabolites of NNK in the patas monkey. This led to the development of a gas chro matography-Thermal Energy Analysis method for detection of NNAL and its glucuronides in smokers' urine. These NNK metabolites have been detected in all smokers but not in non-smokers. The results of this research are providing new insights on the metabolic activation and detoxification of carcinogenic nitrosamines in humans. Tobacco alkaloids are nitrosated during the curing and processing of tobacco, produc ing a group of nitrosamines called tobacco-specific nitrosamines (1,2). Two of these compounds, 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N'-nitroso nornicotine (NNN), have well documented carcinogenic effects in laboratory animals. NNK is a potent pulmonary carcinogen, inducing predominantly adenocarcinoma in mice, rats, and hamsters independent of the route of administration (7-5). Extensive dose response studies in rats have demonstrated that the total doses of NNK required to produce lung tumors are similar to the total doses to which life-long smokers would be exposed, based on the amounts of NNK in mainstream cigarette smoke (6). NNK and its metabolite NNAL also induce pancreatic tumors in rats (4). They are the only tobacco smoke constituents known to induce acinar and ductal tumors of the pancreas in laboratory animals. NNN, which produces esophageal tumors in rats, is the most 0097-6156/94/0553-0211$08.00/0 © 1994 American Chemical Society

Loeppky and Michejda; Nitrosamines and Related N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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prevalent esophageal carcinogen in tobacco smoke (7,2). A mixture of NNK and NNN has been shown to cause oral cavity tumors in rats (7). These carcinogenicity data are discussed in more detail in the chapters by Hoffmann et al, and Murphy. Based on these data, we have proposed that NNK and/or NNN are possible causative agents for cancers of the lung, pancreas, esophagus, and oral cavity observed in humans who use tobacco products (8). In this chapter, we review our work on the development and application of methods to assess human uptake and metabolic activation of these nitrosamines. Our goal is to understand their mechanisms of metabolic activation and detoxification in humans. NNK and NNN are suitable substrates for achieving this goal because human exposure to these compounds is extensive. Our hypothesis is that the probability of tumor development in an exposed person will be at least partially determined by that individual's ability to metabolically activate or detoxify these nitrosamines. Two types of approaches will be discussed. In one, we are quantifying hemoglobin adducts and DNA adducts of NNK and NNN in order to provide an estimate of the dose of metabolically activated substrate which reaches cells. In the other, we are assessing levels of urinary metabolites of NNK to obtain an estimate of its uptake, and eventually, a profile of its metabolic activation and detoxification. Hemoglobin and DNA Adducts of NNK and NNN. Figure 1 summarizes pathways of NNK and NNN metabolism, based on studies carried out in laboratory animals (7,2,9, 70). The important reactions leading to hemoglobin and DNA adduct formation are the α-hydroxylation pathways (11-14). α-Methylene hydroxylation of NNK gives intermediate 9, which spontaneously decomposes to methane diazohydroxide (16). This electrophile alkylates DNA and hemoglobin. α-Methyl hydroxylation of NNK produces intermediate 10, which upon loss of formaldehyde generates the pyridyloxobutane diazohydroxide 18. This diazohydroxide reacts with aspartate or glutamate in hemoglobin, with the formation of ester adducts 22 (75). Hydrolysis of these adducts with mild base releases 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB, 26). DNA adducts are also produced by 18. Acid hydrolysis of these adducts gives HPB. The formation of HPB-releasing hemoglobin and DNA adducts by NNK in rats has been investigated in some detail (72-20. The pathway leading to pyridyloxobutylation of globin and DNA also occurs in animals treated with NNN, via α-hydroxylation at the 2-position to 12. a-Hydroxylation of NNN at the 5'-position is a known metabolic pathway but DNA and globin adducts arising in this way have not been characterized. Quantitation of hemoglobin or DNA adducts of NNK and NNN will provide data on the extents to which these metabolic activation pathways may occur in humans. The use of hemoglobin adducts as dosimeters of carcinogen exposure was suggested by Ehrenberg and co-workers (27). Several groups have developed methodology which can now be applied to assess human exposure to, and/or metabolic activation of, a number of carcinogens including ethylene oxide, aromatic amines, and polynuclear aromatic hydrocarbons (22). Advantages of hemoglobin adducts as dosimeters include the relatively long lifetime of the erythrocyte in humans (approximately 120 days), which permits integration of dose over a somewhat extended period, and the relative ease with which ample quantities of hemoglobin can be obtained. Disadvantages include the probable lack of relevance of the hemoglobin adducts to the carcinogenic process, and the necessity to establish a predictable relationship between hemoglobin adduct levels and the biologically relevant DNA adducts. A more direct approach is the quantitation of DNA adducts, which has been attempted in numerous studies using techniques such as immunoassays, P-postlabelling, fluorescence spectroscopy, and gas chromatography-mass spectrometry (GC-MS)(23). The clear advantage of this approach is the relevance of some DNA adducts to carcinogenesis. However, DNA is difficult to obtainfrompotential target 32



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tissues in quantities sufficient for analysis and the interpretation of adduct measurements can be confounded by repair and other mechanisms of removal. In spite of some of these limitations, quantitation of hemoglobin or DNA adducts can potentially provide important information on human exposure to important DNA damaging intermediates produced by carcinogen metabolism. We have chosen HPB, released by hydrolysis of hemoglobin or DNA, as a dosimeter of NNK and NNN metabolic activation. HPB releasing hemoglobin and DNA adducts can only be formedfromNNK, its metabolite NNAL, and NNN, as far as we are aware. Thus, detection of these adducts can be traced specifically to these compounds or certainly to tobacco-derived exposures. In contrast, methylation of hemoglobin and DNA by NNK would not be a specific dosimeter, because methylation has many sources, including endogenous ones, leading to high backgrounds and potentially confounding interpretation of the data. Both the methylation and pyridyloxobutylation pathways of NNKmetabolism have biological significance with respect to its carcinogenic activity; pyridyloxobutylation is essential for NNN carcinogenicity (24). It should be noted that hemoglobin and DNA adducts, such as those releasing HPB, give a measure of both carcinogen uptake and metabolic activation in an individual. This measurement of "internal dose" is distinctfrombiomarkers of overall exposure to tobacco smoke, such as urinary cotinine. The methodology employed to quantify HPB, released by base hydrolysis of human hemoglobin, has been described (25,26). In this method, an HPB-enriched fraction is prepared by a series of partition steps. The HPB is derivatized as its pentafluorobenzoate and, after an HPLC cleanup step, the derivative is separated and detected by capillary column gas chromatography-negative ion chemical ionization mass spectrometry, with selected ion monitoring (GC-NICI-MS-SIM). Deuterated HPB is used as an internal standard. The sensitivity of this method is excellent, with a detection limit of approximately 0.1 fmol HPB-pentafluorobenzoate. A trace obtained from a smoker's hemoglobin is presented in Figure 2. Data obtainedfromseveral completed or ongoing studies are presented in Figure 3 (24-26). In non-smokers, we have generally not observed levels of HPB releasing hemoglobin adducts which are significantly above background levels of the method (approximately 200 fmol/g Hb). This is consistent with the fact that NNK and NNN are tobacco-specific nitrosamines. In three cases, however, elevated levels were observed suggesting exposure of these individuals to environmental tobacco smoke. This requires further investigation. In smokers, we have detected HPB-releasing hemoglobin adducts above background in approximately 15-20% of the individuals tested. Hemoglobin adduct levels have not been found to correlate with plasma cotinine or numbers of cigarettes smoked. Thus, the elevated levels may relate to more efficient metabolic activation of NNK or NNN in these smokers than in the others. Limited studies carried out to date on snuff-dippers indicate that these individuals have generally higher levels of HPB releasing adducts than do smokers, possibly due in part to higher levels of exposure to NNK and NNN. Hemoglobin adduct levels in F344 rats treated chronically with NNK, and determined by the same GC-MS method, are also illustrated in Figure 3. Most of the hemoglobin adduct values observed in humans fall within the same range as those quantified in rats that had been exposed to expected human doses of NNK and NNN, based on the amounts of these nitrosamines in tobacco products. However, some are well above this range. The factors governing higher hemoglobin adduct levels in some individuals require further research. Based on these data, and on our experience with this assay, we can conclude that the pathway illustrated in Figure 1, leading to pyridyloxobutylation of hemoglobin via the intermediate 18 (or a related electrophile), exists in humans exposed to NNK and NNN. Our studies to date have demonstrated that the published GC-NICI-MSSIM methodology is accurate and reproducible, giving a reliable quantitation of HPB in samples of base treated hemoglobin. However, there are many questions to be


NTTROSAMINES AND RELATED ΛΓ-NITROSO COMPOUNDS


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Figure IB. Further metabolism of the NNK metabolite 4-(methylnitrosamino)pyridyl)-l-butanol (NNAL).


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NITROSAMINES AND RELATEDtf-NITROSOCOMPOUNDS


HPB-pentafluorobenzoate

22.0

22.2

ι

1

22.4

22.6

1

1

1

1

22.8 23.0 23.2 23.4 retention time (min)

1

1

1

23.6

23.8

24.0

Figure 2. GC-NICI-MS-SIM chromatograms obtained upon analysis of a smokers' hemoglobin; A) SIM at m/z 359 (molecular ion of HPBpentafluorobenzoate and B) SIM at m/z 361 (molecular ion of internal standard, [4,4-D ]HPB-pentafluorobenzoate. 2

Non 2000 r Smoker

Smoker

Snuff Dipper

1750 -

F344 Rats 1916 ± 1 5 3

NNK 5yg/kgl.p. 5k weekly, S wks

1500 1250 1000 750 500 •s-329 fe-163

n=37

n=100

7 n=35

517 ± 3 . 2 η Approximate (Ιμ9 / kg) I range of estimated I exposures to 247 ± 5.9 -I NNK and NNN

n=3

Figure 3. Data obtained upon analysis for released HPBfromhemoglobin of nonsmokers, smokers, snuff-dippers, and F-344 rats treated with NNK. Data are from ongoing studies conducted in our laboratory by S.E. Murphy, P.G. Foiles, and S. Akerkar, as well asfrompublished studies (ref. 25).



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answered. The mechanism of adduct formation in hemoglobin will be investigated to better define the origin and significance of the adducts. The kinetics of formation and removal of the adduct in humans will be determined to supplement what is already known in rats. The relationship between hemoglobin adduct levels and DNA adduct levels in a given individual will be assessed in future studies. In rats, a predictable but non-linear relationship between hemoglobin adducts and DNA adducts in lung and liver has been observed in studies to date (77). Datafromthese studies will facilitate interpretation of adduct levels determined in humans and may provide insights on the parameters which lead to high adduct levels in only certain individuals. Properly designed epidemiologic studies of tobacco use and cancer induction, which incorpo rate HPB releasing hemoglobin adduct data, may provide insight on the possible relationship of adduct levels to the probability of cancer development. The methodology for determination of HPB-releasing DNA adducts is similar to that employed for hemoglobin adducts, except that thefirststep requires acid hydrolysis to release HPB (27). This method has not been applied as widely as the hemoglobin assay in studies to date, principally because DNA is more difficult to obtain in the required quantities. In one study, DNA adduct levels rangingfrom3-49 fmol/mg DNA were found in smokers' periperal lung and trachea, obtained at immedi ate autopsy. The amounts of HPB-releasing DNA adducts were higher than in nonsmokers (27). As in rats, levels of HPB-releasing adducts were higher in DNA than in hemoglobin, when expressed per weight of macromolecule (77). These studies support the hypothesis that NNK or NNN are metabolically activated to intermediates which pyridyloxobutylate DNA in human lung. Metabolism of NNK in the Patas Monkey Although the metabolism of NNK has been well characterized in rodents, limited data are available in primates {28,29 ). Since one of our goals is to identify and quantify metabolites of NNK in human urine, we initiated a study of NNK in the patas monkey. These studies were carried out in collaboration with Lucy M. Anderson and Jerry M. Rice of the National Cancer Institute. Female monkeys were given i.v. injections of 0.1 μg/kg-4.9 mg/kg [5- H]NNK, labelled with tritium at the 5-position of the pyridine ring. Blood and urine were collected and analyzed by HPLC. The time course of NNK and its metabolites in serum is illustrated in Figure 4. NNK disappeared rapidlyfromserum, while NNAL and its glucuronides persisted for longer times. The results for NNK and NNAL are consistent with previous observations in baboons. An important observation was the rapid and extensive formation of keto acid 21 and hydroxy acid 28. These are products of α-hydroxylation which entail the formation of adducts, as discussed above. HPLC analysis of serum and urine demonstrated the presence of a major peak which did not coelute with any of the known metabolites of NNK. Hydrolysis experi ments with β-glucuronidase indicated that this metabolite was a glucuronide of NNAL. However, it had a different chromatographic retention time from NNAL-Gluc that had been previously characterized in rat and mouse urine (arbitrarily assigned as NNAL-Gluc(I)-Figure 1-since the absolute configuration is unknown)(J0). This peak was collected and its H - and C-NMR spectra were determined. They were quite similar to those of NNAL-Gluc(I) indicating that the new metabolite was the diastereomer, NNAL-Gluc(II). This was confirmed by HPLC isolation of NNALGluc(I) and (II) from monkey urine, followed by hydrolysis to the enantiomeric NNAJL(I) and NNAL(II), which were converted to the corresponding diastereomeric carbamates by reaction with R-(+)-a-methylbenzyl isocyanate. Significantly, the levels of NNAL-Gluc(I) and (II) in monkey urine accounted for up to 23% of the urinary metabolites after a dose of 0.1 μg/kg NNK, approximately equivalent to a smokers' exposure to NNK. These results encouraged us to analyze human urine for NNALGluc. s

3

1

13


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Metabolites of NNK in Human Urine The analytical method which we developed for analysis of NNAL and NNAL-Gluc in human urine is summarized in Figure 5. Fraction 1 contained unconjugated NNAL. The aqueous portion of the urine was treated with β-glucuronidase, and the released NNAL was further purified, silylated, and analyzed by GC-TEA. Tracesfrom5 smokers and 5 non-smokers are illustrated in Figure 6. In each case, the peak marked with the asterisk, which corresponds in retention time to the trimethylsilyl ether of NNAL, was detected in Fractions 1 and 2 from smokers. Of the seven non-smokers examined, only small amounts of NNAL-Gluc were detected in one; all other nonsmoker urines were negative. The following evidence supports the identity of NNAL and its glucuronides in smokers' urine: (1) the GC retention time of silylated NNAL was identical to that of a standard but well resolvedfromthat of silylated 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL), which was added to samples to determine silylation efficiency (see Figure 6); (2) the material which was collected from HPLC, prior to silylation, had the same retention time as NNAL; and (3) the GC retention time of NNAL from these samples was the same as that of a standard, in analyses that were performed without silylation. Some experiments were carried out without β-glucuronidase, or with sulfatase instead of β-glucuronidase, or with β-gluc uronidase in the presence of saccharic acid 1,4-lactone, an inhibitor of β-glucuronidase activity. No NNAL was detected in Fraction 2 of these samples. Other experiments, involving the addition of nitrite or monitor amines to urine samples, demonstrated that artefactual formation of NNAL and NNAL-Gluc under our conditions was minimal. The results of analyses of urine from 11 smokers are summarized in Table I. Six samples were also analyzed for NNK, by collecting the appropriatefractionfrom HPLC, reducing with NaCNBH , and analyzing as above. NNK was not detected. 3

Table I. NNAL-Gluc and NNAL in Smokers' Urine Cigarettes/ NNAL (itg/24 h) Subject Sex Day free total glucuronide 1 M 36 2.4 0.46 2.9 2 F 29 3.0 0.37 3.4 3 F 20 3.5 0.35 3.9 4b M 25 0.92 0.43 1.4 5 F 20 2.1 0.69 2.8 6b F 29 2.1 1.0 3.1 7 F 25 2.3 0.23 2.5 8 F 25 0.31 0.70 1.0 9b F 12 0.76 0.87 1.6 10b F 20 1.8 0.29 2.1 11 F 15 1.5 0.28 1.8 Expressed as NNAL equivalents. For conversion to μg NNAL-Gluc, multiply by 1.85 Levels of NNK in mainstream smoke of the cigarettes used by these volunteers were available. These were multiplied by cigarettes per day to obtain estimated daily dose of NNK, as follows (in /xg) #4, 3.4; #6, 4.7; #9, 1.6; #10, 2.7 a

These data clearly demonstrate the presence in smokers' urine of NNAL and NNAL-Gluc. Their amounts are in the range expected based on the levels of NNK in the mainstream smoke of cigarettes used by four of the subjects. These data confirm the uptake of NNK by smokers in quantities which are comparable to those which induce lung tumors in laboratory animals, as previously estimated based on NNK levels in mainstream cigarette smoke (6). It is possible that some of the NNAL and


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hydroxy acid

3

Figure 4. Time course of [5- H]NNK and selected metabolites in patas monkey serum after i.v. injection of [5- H]NNK. Hydroxy acid and keto acid are compounds 28 and 2 i of Figure 1. Open and closed symbols represent experiments with two different monkeys. 3

24h urine + Ammonium Sulfamate 1. pH7 2. [5- 3 H]NWAL-Gluc (il) 3. EtOAc

Fraction 1

H20

EtOAc 1. [5- 3 H]NNAL 2. HPLC 3. Silylation

1. β-Glucuronldase 2. p H 2 3 . EtOAc

GC-TEA H20

EtOAc

1. p H 7 2. C H 2 C I 2

Fraction 2

CH2CI2

H20

1. HPLC 2. Silylation GC-TEA

Figure 5. Scheme for analysis of NNAL and NNAL-Gluc in human urine.


NITROSAMINES AND RELATED JV-NTTROSO COMPOUNDS

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Smokers


1

I

2

À

LU 0

5 10 15 20

0 5 10 15 20

0 5 10 15 20

0 5 10 15 20

0

0 5 10 15 20

UL 0 5 10 15 20

Non-Smokers 6

ILL 0

5 10 15 20

5 10 15 20

5 10 15 20

Time (min)

Figure 6. GC-TEA traces of Fraction 2 (see method in Figure 5)fromsmokers (1-5) and non-smokers (6-10). The peaks eluting at approximately 10 and 17 min in all traces are internal standards, nitrosoguvacoline (injection standard) and isoNNAL (silylation standard). The peak marked with the asterisk is the trimethylsilyl ether of NNAL.


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NNAL-Gluc in urine may arise from NNAL in cigarette smoke, but its presence in smoke has not been reported. The presence of NNAL and NNAL-Gluc in smokers* urine, but apparent absence of NNK, is consistent with the studies described above which showed that NNAL-Gluc(I) and (II) were major constituents of patas monkey urine, in contrast to NNK (10). The ratios of NNAL-Gluc to NNAL in human urine are potentially interesting. They varyfrom0.44 to 10. Most smokers had ratios greater than 1, indicating preferential excretion of NNAL as its glucuronide conjugate. It is possible that smokers who conjugate NNAL poorly may α-hydroxylate it more extensively, potentially leading to higher levels of hemoglobin and DNA adducts.


Perspectives Although our knowledge of carcinogen metabolism and mechanisms of carcinogenesis in laboratory animals has greatly increased over the past four decades, our understanding of these processes in humans has lagged behind. The development of reliable techniques to quantify carcinogen metabolic activation, detoxification, and macromolecular binding in humans should lead to advances in understanding causes of human cancer. The techniques of molecular biology have led to the detection of mutations in critical cellular genes associated with cancer such as ras and p53 (37,52). However, the origin of these changes with respect to particular carcinogen exposures is still speculative. Human exposure to tobacco-specific nitrosamines through use of tobacco products is widespread in spite of the known hazards of smoking. The methodology reported here has already produced new insights on their metabolic activation and detoxification in humans. The detection of elevated levels of tobacco-specific nitrosamine hemoglobin adducts in only 15-20% of smokers was a surprising finding although it should be noted that further refinements in the GC-MS assay may lead to inclusion of a greater percentage of subjects above background levels. Nevertheless, the reasons for these relatively elevated adduct levels need to be determined since they may indicate potential susceptibility factors. While the interpretation of data obtained from analysis of hemoglobin adducts and DNA adducts is somewhat complex because of multiple factors which may influence their formation and removalfromcells, interpretation of urinary metabolite data is more straightforward and can be useful with respect to determination of overall uptake and individual profiles of metabolic activation and detoxification reactions. The two metabolites analyzed here can arise onlyfromNNK or NNAL, while other metabolites of NNK and NNN may also be formedfromnicotine. Nevertheless, stereochemical differences in the formation of some of these metabolitesfromNNK and NNN versusfromnicotine may allow us to confidently assign their origin. By combining datafromadduct determinations with thosefromanalysis of urinary metabolites, we expect to achieve a more complete understanding of NNK and NNN metabolic activation and detoxification in humans. Literature Cited 1. 2. 3. 4. 5.

Hoffmann, D; and Hecht, SS. Cancer Res., 45, 935(1985). Hecht, SS; and Hoffmann, D. Carcinogenesis, 9, 875(1988). Belinsky, SA; Foley, JF; White, CM; Anderson, MW; and Maronpot, RR. Cancer Res., 50, 3772(1990). Rivenson, A; Hoffmann, D; Prokopczyk, B; Amin, S; and Hecht, SS. Cancer Res., 48, 6912(1988). Lijinsky, W; Thomas, BJ; and Kovatch, RM. Jpn. J. Cancer Res., 82, 980(1991).


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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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Hecht, SS; and Hoffmann, D. In: "The Origins of Human Cancer: A Comprehensive Review", Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1991, 745. Hecht, SS; Rivenson, A; Braley, J; DiBello, J; Adams, JD; and Hoffmann, D. Cancer Res., 46, 4162(1986). Hecht, SS; and Hoffmann, D. Cancer Surv., 8, 273(1989). Hecht, SS; Castonguay, A; Rivenson, A; Mu, B; and Hoffmann, D. J. Environ. Health Sci. CI, CI 1, 1(1983). Hecht, SS; Trushin, N; Reid-Quinn, C; Burak, E; Jones, AB; Southers, J; Gombar, C; Carmella, SG; Anderson, LM; and Rice, JM. Carcinogenesis, in press (1992). Hecht, SS; Trushin, N; Castonguay, A; and Rivenson, A. Cancer Res., 46, 498(1986). Carmella, SG; and Hecht, SS. Cancer Res., 47, 2626(1987). Hecht, SS; Spratt, TE; and Trushin, N. Carcinogenesis, 9, 161(1988). Hecht, SS; and Trushin, N. Carcinogenesis, 9, 1665(1988). Carmella, SG; Kagan, SS; and Hecht, SS. Chem. Res. Toxicol., 5, 76(1992). Spratt, TE; Trushin, N; Lin, D; and Hecht, SS. Chem. Res. Toxicol., 2, 169(1989). Murphy, SE; Palomino, A; Hecht, SS; and Hoffmann, D. Cancer Res., 50, 5446(1990). Carmella, SG; Kagan, SS; Spratt, TE; and Hecht, SS. Cancer Res., 50, 5453(1990). Peterson, LA; Carmella, SG; and Hecht, SS. Carcinogenesis, 11, 1329(1990). Peterson, LA; Mathew, R; Murphy, SE; Trushin, N; and Hecht, SS. Carcinogenesis, 12, 2069(1991). Ehrenberg, L; and Osterman-Golkar, S. Teratog., Carcinog., Mutag., 1, 105(1976). Skipper, PL; and Tannenbaum, SR. Carcinogenesis, 11, 507(1990). Santella, RM. Environ. Carcino. & Ecotox. Revs., C9(1), 57(1991). Hecht, SS; Carmella, SG; Foiles, PG; Murphy, SE; and Peterson, LA. Environ. Health Perspect., 99, in press(1992). Carmella, SG; Kagan, SS; Kagan, M; Foiles, PG; Palladino, G; Quart, AM; Quart, E; and Hecht, SS. Cancer Res., 50, 5438(1990). Hecht, SS; Carmella, SG; and Murphy, SE. Methods in Enzymology, in press(1992). Foiles, PG; Akerkar, SA; Carmella, SG; Kagan, M; Stoner, GD; Resau, JH; and Hecht, SS. Chem. Res. Toxicol., 4, 364(1991). Castonguay, A; Tjälve, H; Trushin, N; d'Argy, R; and Sperber, G. Carcinogenesis, 6, 1543(1985). Adams, JD; LaVoie, EJ; O'Mara-Adams, KJ; Hoffmann, D; Carey, KD; and Marshall, MV. Cancer Lett., 28, 195(1985). Morse, MA; Eklind, KI; Toussaint, M; Amin, SG; and Chung, F-L. Carcinogenesis, 11, 1819(1990). Bos, JL. Cancer Res., 49, 4682(1989). Hollstein, M; Sidransky, D; Vogelstein, B; and Harris, CC. Science, 253, 49(1991).

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