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Individual susceptibility to the toxic effects of cigarette smoke may be modified by inherited variability in carcinogen metabolism. The purpose of the present study was to investigate pancreatic cancer risk associated with cigarette smoking and 33 variants within carcinogen metabolism genes and examine whether these variants modify the association between smoking and pancreatic cancer. A population-based study was conducted with 455 pancreatic cancer cases and 893 controls. Epidemiological and smoking data were collected from questionnaires and variants were genotyped by mass spectrometry. Age- and sex-adjusted odds ratio (ASOR) and multivariate-adjusted odds ratio (MVOR) estimates were obtained using multivariate logistic regression, and interactions between each variant and smoking were investigated. Current smoker status [MVOR = 2.29, 95% confidence interval (95% CI): 1.62, 3.22], 10–27 pack-years (MVOR = 1.57, 95% CI: 1.13, 2.18), >27 pack-years (MVOR = 1.77, 95% CI: 1.27, 2.46) and longer durations of smoking (19–32 years: MVOR = 1.46, 95% CI: 1.05, 2.05; >32 years: MVOR = 1.78, 95% CI: 1.30, 2.45) were associated with increased pancreatic cancer risk. CYP1B1-4390-GG (ASOR = 0.36, 95% CI: 0.15, 0.86) and Uridine 5'-diphospho glucuronosyltransferase 1 family, polypeptide A7-622-CT (ASOR = 0.77, 95% CI: 0.60, 0.99) were associated with reduced risk. N-acetyltransferase 1-640-GT/GG (ASOR = 1.75, 95% CI: 1.00, 3.05), GSTM1 (rs737497)-GG (ASOR = 1.41, 95% CI: 1.02, 1.95), GSTM1 gene deletion (ASOR = 4.89, 95% CI: 3.52, 6.79) and glutathione S-transferase theta-1 gene deletion (ASOR = 4.41, 95% CI: 2.67, 7.29) were associated with increased risk. Significant interactions were observed between pack-years and EPHX1-415 (P = 0.04) and smoking status and N-acetyltransferase 2-857 (P = 0.03). Variants of carcinogen metabolism genes are independently associated with pancreatic cancer risk and may modify the risk posed by smoking.

Measurement of human urinary carcinogen metabolites is a practical approach for obtaining important information about tobacco and cancer. This review presents currently available methods and evaluates their utility. Carcinogens and their metabolites and related compounds that have been quantified in the urine of smokers or non-smokers exposed to environmental tobacco smoke (ETS) include trans,trans-muconic acid (tt-MA) and S-phenylmercapturic acid (metabolites of benzene), 1- and 2-naphthol, hydroxyphenanthrenes and phenanthrene dihydrodiols, 1-hydroxypyrene (1-HOP), metabolites of benzo[a]pyrene, aromatic amines and heterocyclic aromatic amines, N-nitrosoproline, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (NNAL and NNAL-Gluc), 8-oxodeoxyguanosine, thioethers, mercapturic acids, and alkyladenines. Nitrosamines and their metabolites have also been quantified in the urine of smokeless tobacco users. The utility of these assays to provide information about carcinogen dose, delineation of exposed vs. non-exposed individuals, and carcinogen metabolism in humans is discussed. NNAL and NNAL-Gluc are exceptionally useful biomarkers because they are derived from a carcinogen- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- that is specific to tobacco products. The NNAL assay has high sensitivity and specificity, which are particularly important for studies on ETS exposure. Other useful assays that have been widely applied involve quantitation of 1-HOP and tt-MA. Urinary carcinogen metabolite biomarkers will be critical components of future studies on tobacco and human cancer, particularly with respect to new tobacco products and strategies for harm reduction, the role of metabolic polymorphisms in cancer, and further evaluation of human carcinogen exposure from ETS.

The transformation of chemicals is important in carcinogenesis, both in bioactivation and detoxification. Major advances in the past 20 years include appreciation of the migration of reactive electrophiles, the ability of Phase II conjugating enzymes to activate chemicals, understanding of the human enzymes, the realization that DNA modification can result from endogenous chemicals, and the demonstration that cancers can result from the metabolism of chemicals to non-covalently bound products. Pathways of transformation in which major insight was gained during the past 20 years include nitropolycyclic hydrocarbons, polycyclic hydrocarbons and their diols, vinyl halides and dihaloalkanes. Advances in analytical methods and recombinant DNA technology contributed greatly to the study of metabolism of chemical carcinogens. Major advances have been made in the assignment of roles of individual enzymes in reactions. The knowledge developed in this field has contributed to growth in the areas of chemoprevention, molecular epidemiology and species comparisons of risk. Some of the areas in which future development relevant to carcinogen metabolism is expected involve pathways of transformation of certain chemicals, regulation of genes coding for many of the enzymes under consideration and genomics.

Following exposure of mice to the arylamine carcinogen 2-aminofluorene, DNA-carcinogen adducts can be found in the target tissues liver and bladder, and also in circulating leukocytes. Evidence is presented here that mouse mononuclear leukocytes (MNL) are capable of metabolizing 2-aminofluorene to DNA-binding metabolites which give rise to the adducts found in the MNL. Both lymphocytes and monocytes were able to acetylate arylamines during 18 h of culture. The degree of acetylation was determined by the N-acetyltransferase genotype of the mice as shown through use of acetylator congenic strains which differ only in the Nat-2 gene. Cultured MNL from rapid acetylator mice (C57BL/6J and A.B6-Natr) produced about twice as much N-acetylaminofluorene from 2-aminofluorene and 6- to 8-fold as much N-acetyl-p-aminobenzoic acid from p-aminobenzoic acid as cells from slow acetylator mice (B6.A-Nat(s) and A/J). Other differences in arylamine metabolism by MNL in culture were observed and shown to be due to genetic factors, currently unidentified, other than N-acetyltransferase. DNA adduct formation following incubation of MNL with the arylamine carcinogen 2-aminofluorene was related to both acetylation capacity and to other genetic metabolic factors in the mouse genome. MNL from rapid acetylator mice with the C57BL/6J background (B6) had 3-fold the DNA adduct levels of cells from the corresponding slow acetylator congenic (B6.A-Nat(s)). Similarly, MNL from rapid acetylator mice with the A/J background (A.B6-Natr) had twice the DNA adduct levels of those from their corresponding slow congenic (A). Adduct levels in MNL from C57BL/6J were nearly the same as those of MNL from A/J, again indicating the involvement of loci other than acetylation in DNA adduct formation. The finding of genetically dependent arylamine carcinogen metabolism and DNA adduct formation in cultured MNL suggests the possibility of using cultured MNL for assessing individual susceptibility to arylamine-induced DNA damage.

A large number of chemical carcinogens require metabolic activation before they are biologically active. The metabolism of a few of these compounds has been investigated in cultured human tissues and cells and the metabolism has been compared with the metabolism in organs from experimental animals in which the compounds induce cancer. Generally, only quantitative differences could be observed between animal and human tissues. The development of new methods to detect carcinogen-DNA adducts makes it feasible to study and compare the metabolism in human and animal tissues of an increasing number of potential human carcinogens. Furthermore, construction of cell lines, expressing the human forms of the carcinogen metabolizing enzymes, by biotechnology provides a new model to study the metabolism and to monitor for genetic markers in the same cells.

The effects of diabetes on the liver microsomal monooxygenase enzymes and carcinogen metabolism have been studied in rats. Treatment with streptozotocin causes a marked enhancement in microsomal N-nitrosodimethylamine (NDMA) demethylase activity. The enhancement is due mainly to the induction of a high affinity NDMA demethylase (Km, approximately 0.05 mM) which is accompanied by the induction of a protein species with mol. wt. of 50,000. The treatment also induces aniline hydroxylase whose activity is in parallel with NDMA demethylase. Streptozotocin-induced diabetes also increases the metabolism of N-nitrosomethylethylamine but not that of N-nitrosomethylaniline or N-nitrosomethylbenzylamine. On the other hand, diabetes decreases the metabolism of benzo[a]pyrene, benzphetamine, and ethylmorphine. The result suggest that diabetes causes an alteration of the composition of cytochrome P-450 isozymes; the forms efficient in metabolizing NDMA are increased while certain other forms of cytochrome P-450 are decreased.

Chemical carcinogens are generally activated enzymatically to electrophiles that form covalently bound carcinogen-DNA adducts. Detoxifying enzymes are competing with the activating enzymes for these procarcinogenic chemical substrates. Wide person-to-person variations in these two types of enzymatic activities are found. Repair rates of DNA damage caused by carcinogens also vary among individuals. These interindividual differences in the metabolism of chemical carcinogens and repair rates of carcinogen-induced DNA damage reflect acquired and inherited host factors that may influence an individual's risk for development of cancer.

Extrapolation from rodent genotoxicity data to humans is complicated by variables such as interspecies differences in carcinogen metabolism and DNA repair. A xenograft system containing human bronchial epithelial cells was used to assess the induction of unscheduled DNA synthesis (UDS) by carcinogens and to compare the response with that of rat tracheal epithelium. Cells from human bronchus were grown in explant culture, inoculated into de-epithelialized rat tracheas and implanted subcutaneously into nude mice. Within six weeks, a differentiated mucociliary epithelium lined the xenografted tracheas. Fresh rat tracheas and human xenografts were cut into rings and incubated in media containing [3H]thymidine and either the direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 3-1000 microM), or a carcinogen requiring metabolic activation, 4-nitroquinoline-1-oxide (4-NQO, 3-100 microM). Tissues were then fixed, sectioned, processed for autoradiography and the number of nuclear grains (NG) determined for 100 epithelial cells lining the trachea in each section. A time- and concentration-dependent increase in NG was observed in both human xenografts and rat tracheas after treatment with MNNG or 4-NQO, indicating induction of UDS by these agents. The UDS response to MNNG in the human xenografts was similar to that observed in the rat tracheas, whereas the response to 4-NQO was greater in rat tracheas. These studies indicate that the human xenograft system should have applications for the study of carcinogen-induced damage in normal bronchial epithelial cells.

The metabolism of chemical carcinogens was investigated in liver preparations from 28 captive woodchucks (Marmota monax). Of these, 23 were naturally infected with the woodchuck hepatitis virus (WHV), and eight also had primary hepatocellular carcinoma (PHC). Twenty-nine parameters were investigated in liver subcellular fractions, including cross-reactivity with HBsAg, and biochemical parameters, such as gamma-glutamyl transpeptidase, cytochrome P-450 and microsomal monooxygenases (aryl hydrocarbon hydroxylase, ethoxycoumarin and ethoxyresorufin deethylases, aminopyrine and dimethylnitrosamine demethylases, and testosterone 7 alpha-, 16 alpha- and 6 beta-hydroxylases), uridine 5'-diphosphoglucuronosyl transferase, GSH and related enzymes (peroxidase, reductase and S-transferase), as well as other cytosolic enzyme activities (glucose 6-phosphate and 6-phosphogluconate dehydrogenases, NADPH- and NADH-dependent diaphorases, and DT diaphorase). In addition, liver preparations were used in order to quantify the metabolic activation into bacterial mutagens of five procarcinogens (aflatoxin B1, the pyrolysis products Trp-P-2 and MeIQ, 2-aminofluorene and dimethylnitrosamine) and the decrease of potency of three direct-acting mutagens (sodium dichromate, ICR 191 and 4-nitroquinoline 1-oxide). WHV infection produced a significant stimulation of carcinogen metabolism, as shown by the simultaneous change in detoxification parameters (GSH depletion) and activation indices (enhancement of microsomal monooxygenases and of procarcinogen activation into mutagenic metabolites). There were no significant differences between WHV-positive samples from animals without PHC and the noncancerous tissue of PHC-bearing animals, whereas a decrease of both activation and detoxification indices was recorded in the tumorous tissue. There was a considerable interindividual variability among WHV carriers, which was tentatively ascribed to genetic factors. Pregnancy was the only known factor influencing the results in WHV carriers. However, even by excluding pregnant animals, the effects on carcinogen metabolism produced by WHV infection were still statistically significant. These results, together with previous data obtained in humans, revealed that metabolic factors may play a role in the synergism between viral hepatitis and chemical hepatocarcinogens in the etiopathogenesis of PHC.

Four inbred strains of mice (C3H/HeJ, C57BL/6J, AKR/J, and DBA/2J) were injected s.c. with 150 micrograms of dibenz[a,h]anthracene and followed for 9 months. Strains C3H/HeJ and C57BL/6J were most susceptible to dibenz[a,h]anthracene-induced carcinogenesis with 80% and 53% incidence of tumors, respectively. Strains AKR/J and DBA/2J were much less sensitive with only one tumor observed out of a total of 60 treated mice. Dibenz[a,h]anthracene was shown to induce hepatic aryl hydrocarbon hydroxylase activity in the two sensitive strains, C3H/HeJ and C57BL/6J, but not in the two resistant strains, AKR/J and DBA/2J. When 3-methylcholanthrene-treated liver microsomes from the four strains were studied for dibenz[a,h]anthracene metabolism in vitro, the two sensitive strains not only demonstrated a 3- to 4-fold greater overall rate of metabolism than the two resistant strains, but also showed a quantitative shift with a greater percentage of the total metabolites being the 3,4-diol of dibenz[a,h]anthracene. This diol is the presumed precursor to the apparent ultimate carcinogen, dibenz[a,h]anthracene 3,4-diol-1,2-epoxide.

Polychlorinated biphenyls (PCBs) have previously been shown to be inhibitors of carcinogenesis in trout. The mechanism of this inhibition was investigated by studying the effects of PCBs on aflatoxin B1 (AFB1) distribution, metabolism and DNA adduct formation, both in vivo and in vitro. A 24 h distribution study of injected tritiated AFB1 showed more radioactivity in blood, liver and bile in fish fed PCBs, but less in residual carcass. The metabolites of AFB1 found in vivo in blood plasma and liver homogenates were shifted by PCB pretreatment towards greater production of the polar metabolite aflatoxin M1 (AFM1) and glucuronide conjugates. The major metabolite in bile of PCB fish was the glucuronide of aflatoxicol M1 (AFL-M1), which was enhanced 15-fold over controls. Levels of aflatoxicol (AFL) glucuronide, the major conjugate in controls, were unaltered by PCBs. The pattern of AFB1 metabolism in isolated hepatocytes from PCB-prefed fish was consistent with in vivo metabolism. AFB1--DNA adduct formation in a 1 h assay was similar in hepatocytes from PCB-fed and control fish. However, the total rate of AFB1 metabolism was significantly elevated in hepatocytes from PCB-fed fish such that the degree of AFB1--DNA adduct formed per unit AFB1 metabolized was 42% lower than control. Similarly, adduct formation in vivo during the first 24 h post-AFB1 injection in PCB fish was not significantly different from controls. However, over a longer 21 day period, adduct levels in PCB fish were only 48-69% of controls (P less than 0.005, analysis of variance), once peak adduct formation was reached. Thus, initial rates of adduct formation may be misleading in the absence of further information on rates of carcinogen metabolism in vitro and/or pharmacokinetics of peak adduct formation in vivo. These results indicate that PCB inhibition of AFB1 carcinogenesis in trout involves dramatic initial changes in carcinogen distribution, metabolism and elimination which, over time, results in a net reduction of DNA adduct formation.

Immunohistochemical investigation of liver tissue 48 h after single doses of the hepatocarcinogens diethylnitrosamine (DEN), dimethylnitrosamine, aflatoxin B1 and methylazoxymethanol acetate revealed the generation of a population of single glutathione S-transferase placental form (GST-P) positive hepatocytes. Yield was carcinogen dose dependent. Although the mixed function enzyme inducers sodium phenobarbital (PB), methylcholanthrene (Mech), polychlorinated biphenyls (PCB) and isosafrole (IS) did not in themselves induce comparable single cell lesions, their application prior to DEN caused significant alteration in the resultant numbers of GST-P positive hepatocytes observed. While PB and IS were associated with decreased yield, Mech and PCB, in contrast, brought about an increase. The results gained from investigation of the effects of 3-aminobenzamide administration and partial hepatectomy carried out at different times after carcinogen treatment also pointed to an 'initiated' character for the lesions and suggest that GST-P may be a useful marker for analyzing factors relevant to the initiation stage of hepatocarcinogenesis. Thus the interplay between carcinogen metabolism, DNA adduct formation and repair, toxicity and proliferation may be assessable in terms of numbers of enzyme-altered solitary hepatocytes.