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Carcinogen-DNA adducts, a marker of DNA damage, are capable of inducing mutations in oncogenes and tumor suppressor genes, resulting in carcinogenesis. We have shown previously that polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels in prostate cancer cases vary by cellular histology and that higher adduct levels are associated with biochemical recurrence. A nested case-control study was conducted in a historical cohort of 6692 men with histopathologically benign prostate specimens. PAH-DNA adduct levels were determined by immunohistochemistry in benign prostate specimens from 536 prostate cancer case-control pairs (59% White and 41% African American). We estimated the overall and race-stratified risk of subsequent prostate cancer associated with higher adduct levels. Prostate cancer risk for men with elevated adduct levels (defined as greater than control group median) was slightly increased [odds ratio (OR) = 1.28, 95% confidence interval (CI) = 0.98-1.67, P = 0.07]. After race stratification, elevated adduct levels were significantly associated with increased risk in African American men (OR = 1.56, CI = 1.00-2.44, *P = 0.05) but not White men (OR = 1.14, CI = 0.82-1.59, P = 0.45). Elevated PAH-DNA adduct levels were significantly associated with 60% increased risk of prostate cancer among cases diagnosed 1-4 years after cohort entry (OR = 1.60, CI = 1.07-2.41) with a greater risk observed in African Americans within the first 4 years of follow-up (OR = 4.71, CI = 1.97-11.26, ***P = 0.0005). Analyses stratified by age or tumor grade revealed no additional significant heterogeneity in risk. Increased prostate cancer risk associated with high PAH-DNA adduct levels in benign prostate was found only in African Americans; risk was greatest within 4 years of follow-up, possibly reflecting a carcinogenic process not yet histologically detectable.

Chromium(VI) [Cr(VI)] is a ubiquitous environmental and industrial contaminant. Cr(VI) exposure is strongly associated with a higher incidence of human lung cancer, but the mechanism of Cr(VI) carcinogenicity remains unclear. Cigarette smoking has been known as the prominent cause of lung cancer, and polycyclic aromatic hydrocarbons (PAHs), the major carcinogens in cigarette smoke, have been suggested as being responsible for the initiation and development of lung cancer. It has been reported that lung cancer from workers exposed to Cr(VI) has a high percentage of G to T transversion mutations in the non-transcribed strand of the p53 gene, a hallmark of PAH-induced mutation. Cr(VI) is a weak mutagen although it can induce a high percentage of G to T transversion mutations. These results raise the possibility that Cr(VI) may enhance PAH binding at the p53 gene in lung tissue. To test this possibility, we have determined the effect of Cr(VI) exposure on benzo[a]pyrene diol epoxides (BPDE)-DNA binding at total genomic DNA level and at the p53 gene in normal human lung fibroblast cells. We found that in lung cells Cr(VI) pre-exposure does not affect the BPDE-DNA binding at the total genomic DNA level or at exons 5, 6 and 9 of the p53 gene; however, it greatly enhances BPDE-DNA binding at exons 7 and 8 of the p53 gene, especially at mutational hotspots of lung cancer: codons 248, 273 and 282 of the p53 gene. No enhancement of BPDE-DNA binding in the p53 was observed when naked genomic DNA isolated from Cr(VI)-exposed cells was modified with BPDE in vitro. These results suggest that Cr(VI) exposure may enhance chromatin structure-dependent carcinogen-DNA binding. This effect may contribute to the synergism of Cr(VI) and BPDE on mutagenesis and cell transformation, and may also contribute to the higher incidence of lung cancer in Cr(VI)-exposed populations.

Murine class Alpha glutathione (GSH) transferase A1-1 (mGSTA1-1) is unique among mammalian Alpha class GSTs due to its exceptionally high catalytic activity toward (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated metabolite of an environmentally relevant carcinogen, benzo[a] pyrene (BP). However, the molecular basis for high catalytic activity of mGSTA1-1 toward (+)-anti-BPDE is not clear. In the present study, we demonstrate that an arginine residue at position 216, which is conserved in some but not all mammalian class Alpha GSTs, plays an important role in catalytic activity of mGSTA1-1 toward (+)-anti-BPDE and carcinogenic diol epoxides of other environmentally relevant polycyclic aromatic hydrocarbons (PAHs). The catalytic efficiency (k(cat)/K(m)) of mGSTA1-1 for the GSH conjugation of (+)-anti-BPDE (108/mM/s) was reduced by about 58% upon replacement of arginine 216 with alanine (R216A). This was mainly due to a significantly lower V(max) for the R216A mutant of mGSTA1-1 compared with wild-type mGSTA1-1. The R216A mutation also resulted in a statistically significant reduction (>70%) in specific activity of mGSTA1-1 toward racemic anti-diol epoxides of chrysene and benzo[c]phenanthrene (anti-CDE and anti-B[c]PDE, respectively). The catalytic activity of mGSTA2-2, which is a close structural homologue of mGSTA1-1, was also reduced upon R216A mutation. The results of the present study clearly indicate that an arginine residue at position 216 is critical for catalytic activity of mGSTA1-1 and mGSTA2-2 toward carcinogenic diol epoxide metabolites of various PAHs that are abundant in the environment and suspected human carcinogens.

While inhaled polycyclic aromatic hydrocarbons have long been suspected to induce lung cancer in humans, their dosimetry has not been fully elucidated. A key question is whether the critical exposure occurs during absorption in the lungs, or if toxicants in the systemic circulation contribute significantly to lung cancer risk. In particular, data are needed to determine how the physical properties of inhalants affect local dosimetry in the respiratory tract. Pyrene, a tobacco smoke component, was selected for study because it has physical properties between those of highly lipophilic benzo[a]pyrene and water-soluble nitrosamines. Aliquots of 5 ng of pyrene dissolved in a phospholipid/ saline suspension were instilled as a single-spray bolus in the posterior trachea of the dog just anterior to the carina. For 3 h after instillation, blood was repeatedly sampled from the azygous vein, which drains the mucosa around the point of instillation, and from both sides of the systemic circulation. At 3 h post-instillation, tissue samples were taken. Autoradiography was used to determine the depth distribution of pyrene in the tracheal mucosa. The concentration of pyrene-equivalent radioactivity in the azygous vein peaked 9 min after the instillation. At approximately 30 min after instillation, a rapid early clearance phase shifted into a distinctly slower second clearance phase. Rates of rapid clearance were, however, sufficiently slow to indicate diffusion-limited absorption of pyrene in the trachea. This finding was corroborated by high concentrations of pyrene in the epithelium as determined by autoradiography. High epithelial concentration of pyrene combined with a slow penetration into the circulating blood allowed substantial first-pass metabolic conversion of pyrene in the tracheal mucosa. A total of 13% of the instilled pyrene was retained in the tracheal mucosa 3.2 h after instillation; of this, 29% was parent compound, 52% was organic-extractable metabolites, 14% was water-soluble metabolites and 6% (approximately 1% of the instilled amount) was covalently bound to tracheal tissues. Results support the inference that lipophilic protoxicants, because of slow, diffusion-limited absorption, are more likely than water-soluble protoxicants to be bioactivated in the lining epithelium and, in turn, induce first-pass toxicity at the site of entry. In addition, limitations were identified in the use of systemically distributed biomarkers of PAHs, such as urinary hydroxypyrene levels, as indicators of the biologically effective dose in airway target cells.

Transplacental transfer of genotoxic material has been determined by measuring the polycyclic aromatic hydrocarbon-albumin adduct level in serum isolated from the mother and the umbilical cord using a competitive ELISA assay and the antibody (8E11) against benzo[a]pyrene (B[a]P) tetrols. Smoking women (median 5.54 fmol B[a]P equiv/microgram albumin; 21 cases) and non-smoking women living in rural areas (median 4.99; 30) had higher adduct levels than non-smoking women living in suburbia (median 4.09; 37), whereas non-smoking women living in the city of Aarhus had an intermediate level (median 4.82; 40). Exposure to passive smoking did not modify the adduct levels. When all non-smoking cases were combined, the transport time to/from the home became a major contributing factor to the adduct level. The median adduct level in umbilical cord blood was significantly lower than in maternal blood, the maternal/fetal ratio being approximately 1.3, and a positive association between the adduct levels in the mother and umbilical cord blood was observed. The frequency of the GSTM1 null genotype in the study population, females aged 19-44, was 55.4%, but the GSTM1 genotype did not significantly alter the serum albumin adduct level. This study indicates that the competitive ELISA to detect B[a]P bound to serum albumin is sensitive enough to detect differences in the burden of genotoxic compounds in non-occupational exposed individuals. The lower adduct level in people living in suburbia suggests that local production of incomplete combustion products, like vehicle exhaust or heat generation, is the major contributing factor to genotoxic compounds in the general environment.

Analysis of DNA adducts demands both high sensitivity and good resolution. A high-performance liquid chromatography method for 32P-postlabeled DNA adducts (32P-HPLC) was used to investigate DNA adduct formation from 38 polycyclic hydrocarbons and biphenyls in vitro. The 32P-HPLC method proved to be useful for separation, detection and characterization of DNA adducts from most of the substances. The in vitro method used to form the DNA adducts, with calf thymus DNA, nucleotide 3'-phosphates and metabolic activation through S-9 liver homogenate, gave poor quantitative reproducibility. However, the results showed that the 32P-HPLC method was suitable for characterizing DNA adducts from many substances. From 35 of the tested substances 365 DNA and nucleotide 3'-phosphate adducts were detected and characterized concerning retention times. Of the adducts, 171 were detected in DNA and 39 of them from five substances were characterized concerning target nucleotides. The retention time library built can be used in future analyses of DNA with complex patterns of DNA adducts.

Sixty-three male cigarette smokers were entered into a cross-sectional study to determine whether inverse associations existed between polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels and intake/serum levels of vitamin A, vitamin C and vitamin E. Associations between PAH-DNA adducts and intakes of carotene, as well as serum levels of beta-carotene, were also determined. Fasting blood samples were collected for assays of PAH-DNA adducts in circulating mononuclear cells, plasma cotinine and serum levels of vitamin A, beta-carotene, vitamin C and vitamin E. Since genetic deficiency in the detoxifying enzyme glutathione S-transferase M1 (GSTM1) has been associated with increased risk of lung cancer, GSTM1 genotype was also determined. Analysis of PAH-DNA adducts by competitive enzyme-linked immunosorbent assay (ELISA) indicated that 70% of the subjects had detectable adducts, with a mean of 4.38 adducts/10(8) nucleotides (range 1.00-24.1/10(8)). Pearson's method was utilized to determine whether any associations existed between the various host variables and PAH-DNA adducts. Previously, no significant associations were found between PAH-DNA adducts and cigarettes smoked/day, pack-years, daily/life-time tar exposures or plasma cotinine levels (Santella et al., Carcinogenesis, 13, 2041-2045, 1992). PAH-DNA adducts were inversely associated with serum cholesterol-adjusted vitamin E levels (r = -0.25, P < or = 0.05) and with smoking-adjusted vitamin C serum levels (r = -0.22, P < or = 0.09). Stratification by GSTM1 genotype indicated that these associations were limited to subjects with the null genotype. The relationship between adducts and serum cholesterol-adjusted vitamin E was significant in those of the null genotype (r = -0.38, P < or = 0.04), but not in those with the gene present (r = -0.12, P = 0.5). Similarly, for smoking-adjusted vitamin C, the relationship with adducts was stronger in subjects with the null genotype (r = -0.35, P < or = 0.06) than in those with GSTM1 present (r = -0.05, P = 0.77). These results are consistent with findings of prior epidemiological studies identifying significant inverse associations between anti-oxidant micronutrient status or GSTM1 genotype and the incidence of lung cancer. Additional studies should be conducted to confirm a possible role for vitamin E in PAH-DNA adduct formation and to explore further the possible roles of vitamin A, beta-carotene and vitamin C in modulating adduct formation and lung cancer risk.

Larynx cancer is strongly associated with tobacco smoking. The objective of this work was an analysis of aromatic DNA adducts in tumour and non-tumour larynx cells by means of the 32P-postlabelling method. Peripheral blood leukocytes were used as a reference tissue. The presence of aromatic DNA adducts was demonstrated in all the studied tissues obtained after surgery of larynx tumours. The highest level of DNA adducts was found in larynx tumour cells, followed by non-tumour larynx cells, which exceeded that found in leukocytes almost 2.5 times. Large interindividual differences were detected between subjects. The adduct level in tumour/non-tumour correlated only moderately. However a high correlation was found between the level of DNA adducts in larynx (tumour and non-tumour) cells and that in leukocytes.

Aromatic DNA adducts in total white blood cells, cytochrome P450 (CYP) class 1A1 and glutathione transferase (GST1) class mu genotypes and micronuclei in T- and B-lymphocytes were studied in 69 full-time chimney sweeps and 35 controls, all male subjects. The sweeps had a higher (22%) but statistically non-significant increase in the level of DNA adducts as compared to the controls when all individuals independent of genotype were compared. The non-inducible CYP1A1 genotype, m1/m1, lacking a MspI restriction site at the 3' end of the gene, was associated with significantly higher adduct levels in the sweeps. Among the 26 sweeps with the combined genotype m1/m1 and GST1(-), a statistically significant 60% increase in median adduct levels was observed as compared with those 14 control subjects with the corresponding genotype. Smoking also showed a significant effect on the level of adducts. The effect on DNA adducts by sweeping, smoking and genotype appeared to be additive and independent of each other. DNA adducts in sweeps were moderately but statistically significantly correlated with micronuclei in both T- and B-lymphocytes. The correlation between adduct levels and micronuclei was most marked in T-lymphocytes of individuals lacking the GST1 gene.

The 32P-postlabelling assay for analysis of DNA adducts of chemical carcinogens has been applied in a large number of experimental animal and human studies. Most human studies have dealt with occupational and environmental exposures to polycyclic aromatic hydrocarbons (PAHs). The postlabelling assay does not allow direct chemical identification, and most studies with this method have not been performed in a quantitative way. Very little is therefore known about the identity and absolute levels of adducts, which are important contributors to the process of risk identification and quantitation. In the present study it was, therefore, decided to test some parameters suspected to affect recoveries of adducts in the phosphorylation step of the assay. For this purpose 12 different PAHs were reacted individually and in a mixture with DNA in the presence of a rat liver S9 metabolizing system. Different concentrations of ATP, calcium chloride and polynucleotide kinase were tested using the nuclease P1 enhancement. We found that each factor contributed to adduct recovery and that optimal conditions could be defined. Diluting the modified DNA samples up to 1000 times had little influence on the recoveries of adducts. Comparing the nuclease P1 and the butanol extraction procedures for adduct purification showed that both methods gave similar patterns and levels of major adducts. The absolute recoveries in postlabelling, based on 3H-binding of radiolabelled compounds, were for most of the tested compounds relatively low. The fact that the nuclease P1 and the butanol extraction procedures gave similar recoveries points towards common factor(s) involved in the reduction of the recovered adduct levels. Based on the observed recoveries the conclusion can be drawn that when postlabelling related adducts in human samples the true total adduct levels can be considerably underestimated, even if optimal conditions are used.

Treatment of mouse skin with coal tar is known to initiate tumour formation, with the carcinogenic activity associated mainly with polycyclic aromatic hydrocarbons (PAHs). A sample of pharmaceutical coal tar was analysed by gas chromatography and 19 major PAHs were identified. 32P-postlabelling analysis was used to characterize those PAHs that are responsible for the DNA binding of coal tar and, by inference, its biological activity. PAHs were grouped according to their reported carcinogenic activities and applied as mixtures to mouse skin. Group A contained all of the 19 PAHs, group B seven PAHs for which there is sufficient evidence for carcinogenicity and group C 12 PAHs with only limited or inadequate evidence of carcinogenicity in experimental animals. 32P-Labelled DNA adducts formed by coal tar were resolved on TLC into a pattern of three discrete spots (2, 4 and 6) and four areas of diffuse radioactivity (1, 3, 5 and 7). By comparison of the pattern of adducts formed by coal tar with those formed by the synthetic mixtures it appeared that PAHs in group B formed coal tar-DNA adduct spots 4 and 6, and that adduct spot 2 was formed by PAHs in group C. Attempts to identify those PAHs responsible for the formation of coal tar-DNA adducts 4 and 6 were made by comparing the chromatographic mobilities of 32P-labelled coal tar-derived DNA adducts formed in mouse skin, using TLC and HPLC, with those formed by PAHs in group B. As benzo[ghi]perylene (B[ghi]P), a component of group C, has been demonstrated to exhibit significant DNA binding ability previously, the chromatographic mobility of coal tar-DNA adduct spot 2 was compared to that of the major DNA adducts formed by B[ghi]P in vivo and in vitro. It appeared that coal tar adduct spot 2 was the major adduct formed by B[ghi]P in vitro and that benzo[a]pyrene, benzo[b]fluoranthene, benzo [j]fluoranthene and benzo[k]fluoranthene contributed to the formation of adduct spot 6. None of the PAHs examined appeared to be responsible for the formation of adduct spot 4.

Studies on cigarette smoking related polycyclic aromatic hydrocarbon-DNA adducts in blood have produced conflicting results. To determine whether a subset of specific white blood cells is a useful marker for monitoring exposure to cigarette smoke, blood was obtained from 63 heavy smokers and 27 non-smokers. Adduct levels were determined by competitive enzyme-linked immunosorbent assay with a polyclonal antiserum recognizing benzo[a]pyrene and structurally related diolepoxide-DNA adducts. Analysis of the lymphocyte plus monocyte fraction from smokers indicated 70% had detectable adducts with a mean of 4.38 +/- 4.29 adducts/10(8) nucleotides, while in non-smokers the corresponding values were 22% and 1.35 +/- 0.78/10(8) (P < 0.001). Plasma cotinine levels differed significantly in smokers (286 +/- 90 micrograms/l) compared to non-smokers (4.4 +/- 3.3 micrograms/l) (P < 0.001). However, cotinine was not correlated with self-reported smoking history in these heavy smokers. Nor were DNA adducts in smokers correlated with cigarettes per day, pack-years and plasma cotinine, indicating large interindividual variation in DNA adduct formation. These data demonstrate lymphocytes plus monocytes from smokers have elevated levels of polycyclic aromatic hydrocarbon diolepoxide-DNA adduct levels compared to non-smokers.

It is well known that food restriction diminishes tumor formation, but mechanisms responsible are difficult to define because multiple physiological changes result from dietary alterations. Studies in this report were designed to focus specifically on the effects of food restriction on hepatic metabolism of polycyclic aromatic hydrocarbons following liver transplantation. By placing livers from food-restricted and untreated rats into naive controls, the effects of diet could be restricted to the liver. After a 15 min infusion of [3H]benzo[a]pyrene under these conditions, food restriction increased polar metabolites in liver (70 pmol/g) and blood (8 pmol/ml) compared to controls approximately 2-fold. Four hours after liver transplantation, levels of polar metabolites in blood were diminished by approximately 50% but were still approximately 2-fold higher in the food-restricted than in the control group. Lung, kidney, spleen, adrenal, ovary, colon, heart and brain also contained higher levels of polar metabolites in the food-restricted than in the control group. The more hydrophobic glucuronides and sulfate conjugates accounted for most of the elevation in polar metabolites in blood from the food-restricted group. In spite of the increase in circulating metabolites in blood of food-restricted animals, DNA binding in liver, lung and kidney was identical in tissues from control and food-restricted groups. In order to evaluate the hypothesis that food restriction stimulated the release of hepatic benzo[a]pyrene metabolites, a liver perfusion model was employed. Maximal rates of release of polar metabolites into the effluent perfusate were approximately 30 and approximately 45 nmol/g/h in livers of control and food-restricted rats respectively. Moreover, rates of metabolism of the model compound p-nitroanisole and glucuronidation of p-nitrophenol were also approximately 2-fold higher in livers from food-restricted than control rats. However, rates of monooxygenation were the same in microsomes prepared from livers of food-restricted or control animals. These results support the hypothesis that food restriction enhances the supply of cofactors which stimulate metabolism of polycyclic aromatic hydrocarbons. This detoxification process may be an important mechanism involved in the protective action of reduced food intake.

The Computer-Automated Structure Evaluation (CASE) Program, an expert system that automatically selects relevant descriptors for structure-activity relationships, has been used to analyze the binding of various ligands to the tetrachloro-dibenzo-p-dioxin (TCDD) receptor or Ah receptor. Two databases were analyzed. One database contained 136 polycyclic aromatic hydrocarbons (PAH), substituted dibenzo-p-dioxins, dibenzofurans and biphenyls whose binding affinities were measured by a sucrose density gradient technique. The other 87 compound database contained PAH, nitro-PAH, halo-PAH and N-heterocycles. Their binding affinities were measured by the electrofocusing assay. Within each training set significant correlations between the affinity for the TCDD receptor and relevant molecular fragments identified by the CASE program were observed. Among the halogenated aromatic hydrocarbons, fragments containing lateral halogens and a longitudinal hydrogen appeared important for TCDD receptor binding. The fragments of PAH and heterocyclic compounds that were most activating with respect to TCDD receptor binding were found to contain the classical 'bay' region and were in fact identical to the fragments found previously to be related to carcinogenicity. It was found that the activating fragments from PAH and heterocyclic compounds were different from those found within the halogenated compounds such as dibenzo-p-dioxins, dibenzofurans and biphenyls. One interpretation of the data is that two different recognition sites may be involved in Ah receptor binding.

Bay region diolepoxides of polynuclear aromatic hydrocarbons (PAHs) generally are more tumorigenic than their parent PAHs in newborn mice. This contrasts to the results obtained in mouse skin, in which the same diolepoxides are frequently less tumorigenic than their parents. In order to evaluate mechanism(s) responsible for this behavior we have investigated the binding of metabolites of [3H]benzo[a]pyrene (B[a]), [3H]5- and [3H6]6-methyl-chrysene (5-MeC and 6-MeC) and their corresponding dihydrodiols and bay region diolepoxides to pulmonary and hepatic DNA in male and female newborn mice and compared the results with their tumorigenic activities. Groups of 1 day old mice were treated with 0.4 or 4 nmol of the appropriate compounds in DMSO by i.p. injection. HPLC analysis of DNA hydrolysates obtained 24 h after treatment indicated that levels of diolepoxide-DNA adducts following treatment with (+-)-[3H]7,8-dihydroxy-7,8-dihydroB[a]P and (+-)-[3H]anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroB[a] P are 5- and 10-fold higher than those formed from [3H]B[a]P. The major products (70-80%) released upon enzymatic hydrolysis of DNA following treatment with [3H]B[a]P, [3H]5-MeC and [3H]6-MeC were unidentified polar compounds. Levels of these unknown products were lower and formation of diolepoxide--DNA adducts higher when test compounds were changed from parent PAHs to the corresponding dihydrodiols and diolepoxides. Comparison of these results with those of tumorigenesis studies indicates a correlation between formation of B[a]P-diolepoxide--DNA adducts and induction of tumors in newborn mouse lung, but not in liver. These observations are consistent with the high sensitivity of the newborn mouse lung towards the tumorigenic effects of bay region diolepoxides. Previous studies have demonstrated that 1R,2S-dihydroxy-3S,4R,epoxy, 1,2,3,4-tetrahydro-5-MeC (5-MeC-1R,2S-diol-3S,4R-epoxide) is a potent lung tumorigen while the corresponding diol-epoxide of 6-MeC had no effect in newborn mice. From the results of the present study, we estimate that at equimolar doses the formation of diolepoxide--DNA adducts from 5-MeC-1R,2S-diol-3S,4R-epoxide would be at least 20-fold greater than from the corresponding diolepoxide of 6-MeC in newborn mouse lung. Thus, the higher tumorigenic activity of 5-MeC-1R,2S-diol-3S,4R-epoxide compared to that of the corresponding diol epoxide of 6-MeC is partially due to its greater extent of DNA damage.

Two versions of the 32P-postlabeling assay (nuclease P1 and butanol extraction) enhance the detection limit of polycyclic aromatic hydrocarbon (PAH)-modified DNA. Previously published studies suggest that DNA adducts derived from N-substituted aryl compounds are poorly recovered in the nuclease P1 version. In this study, both versions were employed to ascertain whether the apparent differences in sensitivity could be used to select diagnostically for nitroaromatic-DNA adducts derived by treating calf thymus DNA with organic extracts isolated from four diesel and one gasoline vehicle emission particles. We enhanced the formation of nitrated-PAH-derived adducts through xanthine oxidase (XO)-catalyzed nitroreduction of nitrated-PAHs, constituents previously detected in the diesel emissions. Chromatographic mobilities of the XO-derived DNA adducts were compared to adducts detected in calf thymus DNA resulting from rat liver S9-mediated metabolism. All four diesel organic extracts treated with XO resulted in the formation of one major DNA adduct, chromatographically distinct from the multiple DNA adducts detected in the rat liver S9-treated incubations. This adduct was detectable with the butanol extraction but not the nuclease P1 version of the 32P-postlabeling assay and was chromatographically similar to DNA adducts formed following XO nitroreduction of 1-nitropyrene or ascorbic acid treatment of 1-nitro-8-nitroso-pyrene and 1-nitro-6-nitrosopyrene. In contrast, when S9 activation was used, multiple DNA adducts were detected along a diagonal zone of radioactivity and were radioactively labeled with equivalent efficiency irrespective of the assay version employed. The in vitro calf thymus DNA model described in this study enhances the diagnostic potential of the 32P-postlabeling assay through the selective formation of nuclease P1-sensitive N-substituted aryl-derived DNA adducts.

Engine lubricating oils are known to accumulate carcinogenic polycyclic aromatic hydrocarbons (PAHs) during engine running. Oils from nine petrol-powered and 11 diesel-powered vehicles, in addition to samples of unused oil, were analysed for PAH content and ability to form DNA adducts when applied topically to mouse skin. The levels of 19 PAHs, determined by GC, were in total, approximately 22 times higher in used oils from petrol engines than in oils from diesel engines. Male Parkes mice were treated with 50 microliters of oil daily for 4 days before they were killed and DNA isolated from skin and lung tissue. DNA samples were analysed by nuclease P1-enhanced 32P-postlabelling. Used oils from both diesel and petrol engines showed several adduct spots on PEI-cellulose plates at total adduct levels of up to 0.57 fmol/microgram DNA [approximately 60 times greater than in experiments with samples of unused oil in which adduct levels (0.01-0.02 fmol adducts/microgram DNA) were close to the limit of detection]. Higher adduct levels were generally formed by petrol engine oils than by diesel engine oils. Lung DNA contained similar total adduct levels to those in skin although the adduct maps were less complex. Total adduct levels correlated with extent of oil use in the engine, the total PAH concentration in oils and with the concentrations of certain individual PAHs present in the oils. An adduct spot that co-eluted with that of the major benzo[a]pyrene-DNA adduct accounted for 9-26% of the total adducts in skin DNA, and approximately 8% of the adducts in lung DNA, of mice treated with petrol engine oils. A major, and as yet unidentified, adduct spot comprised up to 30% of the total adducts in skin DNA, and up to 89% of the total adducts in lung DNA, of these animals.

The 4S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a cytoplasmic protein that binds PAHs with specificity and high affinity. We have used antisera for the PBP and unlabeled peroxidase anti-peroxidase immunohistochemistry to demonstrate its possible localization in cell types known to have xenobiotic metabolizing capabilities. Cellular sites of the PBP in liver, lung and kidney of C57BL/6 and DBA/2 mice were probed. The PBP was visualized in hepatocytes throughout the liver lobule and was not preferentially located in either centrilobular or periportal areas. However, cellular heterogeneity with respect to PBP content was clearly evident in the hepatocyte population. The positive reactivity correlated with substantial levels of benzo[a]pyrene (B[a]P) binding in liver cytosol. In the lung, the PBP was found in the bronchiolar epithelium and the alveolar septa, and was localized in ciliated and non-ciliated Clara and alveolar type II cells as well as in alveolar macrophages. In the kidney, the glomeruli and epithelia of proximal and distal convoluted tubules and collecting ducts were labeled. Staining for the PBP was greatest in the apical region of the pyramid and was localized in the epithelial lining of the collecting ducts. Relatively lower levels of the PBP were detected in the lung and kidney than in the liver. Staining was localized in the cytoplasmic compartment of cells in all tissues examined. Similar immunoreactivities were exhibited in the tissues of both C57BL/6 and DBA/2 mice. Treatment with beta-naphthoflavone (beta NF) altered neither the intensity nor pattern of immunostaining. Furthermore, treatment with beta NF or isosafrole has no effect on the Kd and Bmax of B[a]P binding to liver cytosolic PBP. The results of our experiments demonstrate localization of the PBP to sites of active physiological response to PAH exposure.

The effect of environmental pollution on DNA adducts in humans was analysed in a highly industrialized area of Poland. Coded samples of white blood cell DNA were analysed by 32P-postlabelling and immunoassay from three populations: coke workers, exposed occupationally to high levels of polycyclic aromatic hydrocarbons (PAHs); residents of the towns around cokeries (local controls); and residents from rural Poland (countryside controls). Local controls exhibited adduct levels and patterns similar to those of coke workers, while the levels in rural controls were 2-3 times lower. The results, based on coded samples and two different assays, suggest that environmental pollution is likely to contribute to the adduct levels in local controls. Furthermore, the results show that the levels of aromatic adducts in white blood cell DNA do not linearily relate to ambient air levels of PAHs but other sources such as food may be important contributors.

The effect of ingesting charcoal-broiled (CB) beef on polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels in nucleated peripheral white blood cells (WBCs) was examined in four healthy, non-smoking males who consumed an average of 280 g CB beef daily for 7 days. PAH-DNA adducts were quantitated by enzyme-linked immunosorbent assay. During the week of CB beef consumption, two individuals exhibited a 3- to 6-fold increase in PAH-DNA adducts above baseline levels observed during the month prior to CB beef consumption. In contrast, PAH-DNA adduct levels in the two other subjects did not increase. Thus, dietary sources of PAH can contribute to the PAH-DNA adduct load in peripheral WBCs in some individuals.

Occupational exposure to polycyclic aromatic hydrocarbons (PAH) in an aluminum smelter has been characterised simultaneously by three different techniques. Air samples were collected by personal sampling and analysed by capillary gas chromatography. Urine samples of exposed workers and a control group, smokers and non-smokers, were collected after shift and analysed for PAH and PAH-metabolites by reversed metabolism and high performance liquid chromatography using fluorescence detection. Blood samples were collected from the same persons and the frequency of sister chromatid exchange (SCE) in the lymphocytes determined. The results showed that workers are exposed to very high concentrations of PAH. This high exposure is not reflected in significantly different values of excreted PAH metabolites or in SCE when comparing exposed to non-exposed workers. However, differences between smokers and non-smokers are easily identified both in exposed and non-exposed workers.

The role of metabolic activation of carcinogens in fetal tissue as a determinant of sensitivity in transplacental carcinogenesis was investigated in a pharmacogenetic experiment utilizing backcrosses of C57BL/6 (AhbAhb, responsive to induction of aromatic hydrocarbon metabolism) and DBA/2 (AhdAhd, non-responsive) mice. Responsive (C57BL/6 X DBA/2)F1 and non-responsive DBA mothers, all carrying both responsive (AhbAhd) and non-responsive (AhdAhd) fetuses, were given i.p. doses of the carcinogen 3-methylcholanthrene (MC) ranging from 5 to 175 mg/kg on gestation day 17. At 10 months of age the metabolic phenotype of each offspring was determined, and correlated with number of lung and liver tumors. Both male and female AhbAhd (responsive) offspring in most dose groups presented a consistent two- to three-fold higher incidence of lung tumors than did non-responsive AhdAhd littermates. The difference held for offspring of both (C57BL/6 X DBA)F1 and DBA mothers and it was of statistical significance for one or both sexes at most dosage levels. Hepatocellular tumors were also significantly more frequent in responsive male AhbAhd progeny of (C57BL/6 X DBA/2)F1 mothers than in non-responsive AhdAhd littermates. Progeny of the DBA mothers exhibited significantly more liver and lung tumors than did those of the (C57BL/6 X DBA/2)F1 mothers receiving the same dose. These results suggest that in this model system both maternal and fetal genotype for responsiveness to induction of aromatic hydrocarbon metabolism are important factors modulating fetal carcinogenic risk.

Five nitrated polycyclic aromatic hydrocarbons, synthesized from benzo[a]pyrene, fluoranthene, pyrene, and chrysene induced dose-dependent transformation of Syrian hamster embryo cells. Benzo[a]pyrene, a known carcinogen, induced transformation while the other parental compounds, which are non-carcinogens, were not effective. The transforming potential of the nitro derivatives varied from compound to compound; on a molar basis, 1,8-dinitropyrene was the most effective nitrated hydrocarbon followed in order by 3-nitrofluoranthene, 1-nitropyrene, 6-nitrochrysene, and 6-nitrobenzo[a]pyrene. The ability to obtain dose-dependent transformation frequencies indicates that hamster cell transformation represents a responsive model for elucidating the mechanism of action of nitrated carcinogens. Because of their ubiquitous distribution and their ability to induce morphological transformation in mammalian cells, nitrated polycyclic aromatic hydrocarbons must be considered as potential carcinogens.

High prevalences of idiopathic hepatic lesions, including neoplasms (e.g., hepatocellular carcinomas, cholangiocellular carcinomas) (27%, 20 of 75 fish) and foci of cellular alteration (putative 'preneoplastic' lesions) (44%, 33 of 75 fish), were found in English sole (Parophrys vetulus) exposed to creosote-contaminated sediments in Eagle Harbor, Puget Sound, WA. Sediments from the contaminated region of the harbor contained particularly high concentrations of aromatic hydrocarbons (e.g., benzo[a]pyrene and benz[a]anthracene), and a variety of nitrogen-containing aromatic compounds (e.g., carbazole and acridine). The composition of the aromatic compounds was characteristic of creosote. Dramatically lower concentrations of aromatic compounds were found in sediments from a reference site in which the bottom-dwelling fish examined were free of detectable neoplastic or 'preneoplastic' hepatic lesions. Food organisms in the stomachs of the English sole from Eagle Harbor contained substantially higher concentrations of aromatic hydrocarbons than comparable organisms from the reference site. The concentrations of individual aromatic hydrocarbons in muscle and liver from the Eagle Harbor fish were low; however, high concentrations of metabolites of aromatic compounds were present in the bile. The findings strongly suggest an association between exposure to creosote and the prevalence of hepatic lesions, including neoplasms, in the bottom-dwelling fish, and furthermore support the putative role of aromatic hydrocarbons in liver carcinogenesis in fish.

The metabolism of benzo[a]pyrene, aflatoxin B1, N-nitrosodimethylamine, N-nitrosoethylmethylamine, and N-nitrosopyrrolidine has been studied in cultures of normal human and rat urinary bladder epithelial cells. The cultures were incubated with radioactively labeled carcinogens for 24 h, and the metabolism was assayed by binding of reactive metabolites to DNa and by the release of metabolites into the medium. Only slight variation in binding level of benzo[a]pyrene to DNa among the three human bladder cell lines was seen, the level of binding being higher than to rat DNA. The major benzo[a]pyrene-DNA adduct (80%) in human bladder cells eluted prior to the adducts formed by reaction of 7,8-dihydroxy-9,10-epoxy - 7,8,9,10-tetrahydrobenz[a]pyrene with guanine by high pressure liquid chromatography, but has yet to be identified. The benzo[a]pyrene-DNA adducts were quickly removed and only about 10% of the radioactivity remained associated with human bladder DNA 72 h post-treatment with benzo[a]pyrene. The 7,8- and 9,10-diols of benzo[a]pyrene were the major organo-soluble metabolites formed by both rat and human bladder cells. The primary benzo[a]pyrene metabolites were conjugated to a minor extent only. The highest level of modification of DNA was seen in the case of N-nitrosodimethylamine. N-Nitrosopyrrolidine was oxidized in both the alpha-and beta-position by all three cell lines, the oxidation at the alpha-position being predominant. No binding to DNA was detectable with N-nitrosoethylmethylamine, although this compound was metabolized as measured by the formation of CO2 and aldehydes. These results add the urinary bladder to the list of human organs which have been shown to metabolize chemical carcinogens into electropositive metabolites. However, qualitative differences exist between the data from bladder cells and those from other human organs.

DNA from normal human bone marrow mononuclear and non-mononuclear cells was analysed by 32P-post-labelling for the presence of aromatic adducts. Ten out of 10 individuals showed the presence of adducts at levels of 1-9 adducts per 10(8) nucleotides that were not detected in four samples of human foetal bone marrow. Inter-individual variations in the patterns of these presumed aromatic adducts were observed. Similar adducts were also present in the DNA of peripheral white blood cells of both smokers and non-smokers, although at lower levels than in bone marrow. The data suggest that the adducts result from environmental exposure to an as-yet-unidentified genotoxic agent or agents.

The effect of trans-stilbene oxide (TSO) induction on the microsome-catalyzed binding of polycyclic aromatic hydrocarbon metabolites to DNA was investigated using two rodent species (Sprague-Dawley rat and C57BL/6N or NMRI Swiss mouse) and 2 different binding substrates (benzo[a]anthracene). It was determined that TSO exerts 2 separate effects on polycyclic aromatic hydrocarbons - it increases the rate of oxidation at the K-region of the molecule due to its induction of specific monooxygenases, and it increases the rate of deactivation of epoxide intermediates by induction of microsomal epoxide hydrolase activity. The importance of these individual effects were determined by inducing monooxygenase activity with BP, altering region specificity and inducing epoxide hydrolase (EH) activity with TSO, assessing the combined inductive effects of TSO and BP, inhibiting EH with 1,1,1-trichloropropene oxide, or increasing its activity by the addition of pure enzyme. This study shows that these effects are similar for both substrates examined, and that the effect of TSO on the binding to DNA of highly carcinogenic bay-region diol-epoxides is multi-faceted, due to its multiple inductive effects.

The tumorigenic and mutagenic effects induced by polycyclic aromatic diol epoxides are widely believed to involve the covalent binding of these electrophilic metabolites of polycyclic aromatic hydrocarbons to DNA. Here the relationships between the formation of non-covalent intercalation complexes with DNA, the DNA-catalyzed reaction rates, the formation of tetraols (which constitutes a detoxification pathway) and the level of covalent binding to DNA, are examined critically within the context of available experimental results. When the rates of reaction of the diol epoxide molecules at DNA binding sites dominate over those in the external aqueous solution, the level of covalent binding is independent of the intercalative non-covalent binding (association constant K, M-1) which precedes the covalent binding reaction. However, under conditions in which DNA does not catalyze or enhance the reaction rate of diol epoxide molecules (e.g. at high salt concentrations), or when there are efficient competitive reaction pathways in the solution external to the DNA, the level of covalent binding to DNA is predicted to be directly proportional to K. Since these latter reaction conditions are expected to prevail in a cellular environment, the ability of a given diol epoxide derivative to form intercalative non-covalent complexes with DNA prior to covalent binding should be an important factor in determining the biological activities of these compounds. The experimental conditions which are suitable for testing the relationships between non-covalent intercalative complex formation and covalent binding to DNA in vitro are described.

The physiological state of pregnancy confers significant resistance to polycyclic aromatic hydrocarbon (PAH)-induced mammary tumorigenesis. We have tested the abilities of primary mammary cells from pregnant and virgin rats cultured on collagen-coated plates to metabolize PAHs and convert these carcinogens to mutagenic derivatives. Using a cell-mediated mutagenesis assay, we found that mammary epithelial cells from pregnant rats produced half the levels of mutagenic 7,12-dimethylbenz[a]anthracene (DMBA) metabolites formed by cells from virgin rats. Pregnant-derived mammary cells also showed consistently lower levels of metabolism of DMBA and benzo[a]pyrene (B[a]P) than cells from virgin rats. H.p.l.c. analysis of B[a]P metabolism by these cell populations indicated no significant qualitative differences in the extracellular and intracellular metabolites formed. We have previously shown that mammary cells from rat strains exhibiting significant differences in susceptibility to PAH-induced tumors have equivalent qualitative and quantitative abilities to metabolize PAH carcinogens. Our current data suggest that modifications in mammary tumor susceptibility found in various physiological states, unlike genetically determined differences, may be related in part to an altered ability to activate chemical carcinogens within the mammary gland.

Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity.

Dependence of polycyclic aromatic hydrocarbon (PAH)-induced mutagenicity on the bay region of the molecule and on the activating cytochrome P-450 enzyme was studied. Eleven PAHs with and six without a bay region were activated by postmitochondrial supernatants from control and 3-methylcholanthrene (MC)-pretreated C57BL/6 mice and from control, MC- and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-pretreated DBA/2 mice and from control and MC-pretreated Sprague-Dawley and Lewis rats. S-9 fractions from MC- or TCDD-treated animals induced more mutagenicity with PAHs with a bay region compared with S-9 fractions from control animals or MC-treated D2 mice. Mutagenicities of PAHs without a bay region were largely independent of the source of activating enzyme. There were three exceptions, namely benzo[e]pyrene, phenanthrene and perylene (each possessing a bay region), which were not mutagenic. These studies support the notion that the Ah-locus-controlled induction of cytochrome P1-450 activating PAHs into reactive intermediates at the bay region of the hydrocarbon molecule is of prime importance in the mutagenicity of PAHs. Qualitative correspondence to carcinogenicity is also apparent.

Singlet delta oxygen (1O2) was generated at atmospheric pressure using heterogeneous gas-phase photosensitization. Polycyclic aromatic hydrocarbons (PAH), known constituents of the atmosphere, were found to be efficient sensitizers in the production of singlet delta oxygen under simulated environmental conditions. A novel mechanism involving singlet delta oxygen is proposed in which PAH on particle surfaces are self-activated to form direct-acting mutagens.

The metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and benz[a]anthracene (BA) by epithelial cell aggregates and fibroblasts in culture has been investigated using mammary tissue obtained from female Wistar rats. The results show that: (a) both types of mammary cells metabolise all three hydrocarbons into ether- and water-soluble derivatives; (b) the patterns of metabolites produced by epithelial cells and fibroblasts are similar but that fibroblasts form more of the water-soluble materials; (c) the major dihydrodiols formed are the 8,9-dihydrodiols of BA and DMBA and the 7,8- and 9,10-dihydrodiols of BP; (d) all three hydrocarbons are metabolise to form products that bind covalently to protein but only the potent carcinogens BP and DMBA are metabolised to form derivatives that react covalently with DNA; and (e) the chromatographic profiles of the hydrocarbon-deoxyribonucleoside adducts formed in epithelial cells treated with either BP or DMBA are similar to those obtained in analogous experiment with fibroblasts.

A keratinocyte cell-mediated mutation assay was used to examine the importance of cellular proximity to the transfer of mutagenic and cytotoxic polycyclic aromatic hydrocarbon (PAH) metabolites between cells. The PAHs 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (BP) were both mutagenic and cytotoxic to V-79 fibroblasts co-cultured on the surface of a keratinocyte feeder layer. However, neither PAH was mutagenic to the V-79 cells when the V-79 cells were co-incubated with the cultured keratinocytes under conditions in which the two cell types were separated by 0.5-1.5 mm but continually shared a common medium. Similarly, DMBA cytotoxicity was eliminated and BP-dependent cytotoxicity was greatly reduced in co-incubation protocols. These results suggest that cells must either be in contact or very close to facilitate the transfer of mutagenic PAH metabolites between cells.

With the help of results on the metabolism and the carcinogenic activities of polycyclic aromatic hydrocarbons (PAH), the comprehensive metabolic process in vivo is discussed. It is thought that the carcinogenic activity exhibited by a PAH is determined by the competition between the carcinogenesis and detoxification in which it participates. It is suggested that the essential agent of carcinogenesis should be, as a rule, the highest delocalization energy (beta unit) of the carbonium ion at the aromatic angular ring (A region), which is obtained by the pertubational molecular orbit (PMO) method. Since there are no essential distinctions in the molecular geometry and delocalization energy states between two carbonium ions of the aromatic angular ring and of the bay region, the A region can be looked upon as the extended bay region. On the basis of discussion of the overall metabolism, evaluation of the detoxification efficacy of each kind of the competing carcinogenic factors, including the biological factor B and three structural factors of the PAH molecule: K, A and L, was made. After making necessary approximation, K = 0.228, A = 0.5, L = 1.22 and B = 0.7 are obtained. It can be seen from these values that the L region plays the most important role in detoxification processes, and the K region plays the least important role. The effect of biological factor B is approximately the sum of the K region and the A region. This paper suggests the concept of a carcinogenic constant. For the PAHs with the same number of aromatic rings (N), C is a constant. The curve of function C = f(N), including the extensional line, is an isosceles triangle. The author suggests that it should be called the 'Pyramid Rule'. The final form of the quantitative equation is log R = C[delta E3deloc/(0.7 + 0.228nk + 0.5na + 1.22n1)]. The values for 50 PAHs which had been tested by animal experiments were calculated. Of these, 92% of the PAHs are in agreement with experiments on carcinogenic activities.