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Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke constituent, is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [(15)N(5)]-(±)-anti-DB[a,l]PDE-N(6)-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 μg of digested DNA as the matrix. Two adducts were detected and identified as (-)-anti-cis and (-)-anti-trans-DB[a,l]PDE-dA in the oral tissues of mice following the direct application of DB[a,l]P (240 nmol per day, for 2 days) into the oral cavity, indicating that DB[a,l]P is predominantly metabolized into (-)-anti-DB[a,l]PDE in this target organ. We also compared the formation and removal of adducts as a function of time, following the direct application of DB[a,l]P (24 nmol, 3 times per week for 5 weeks) into the oral cavity of mice. Adducts were quantified at 48 h, 1, 2, and 4 weeks after the last dose. Maximal levels of adducts occurred at 48 h, followed by a gradual decrease. The levels (fmol/μg DNA) of (-)-anti-trans adducts (4.03 ± 0.27 to 1.77 ± 0.25) are significantly higher than (-)-anti-cis-DB[a,l]PDE-dA adduct (1.63 ± 0.42 to 0.72 ± 0.04) at each time point (p < 0.005). The results presented here indicate that the formation and persistence of (-)-anti-DB[a,l]PDE-dA adducts may, in part, contribute to the initiation of DB[a,l]P-induced oral carcinogenesis.

Hexavalent chromium is a known inducer of DNA-protein cross-links (DPCs) that contribute to repression of inducible genes and genotoxicity of this metal. Lymphocytic DPCs have also shown potential utility as biomarkers of human exposure to Cr(VI). Here, we examined the mechanism of DPC formation by Cr(VI) and the impact of its main cellular reducers. In vitro reactions of Cr(VI) with one-electron reducing thiols (glutathione and cysteine) or two-electron donating ascorbate were all efficient at DPC production, indicating a dispensable role of Cr(V). No Cr(VI) reducer was able to generate DPC in the presence of Cr(III)-chelating EDTA or phosphate. A critical role of Cr(III) in DNA-protein linkages was further confirmed by dissociation of Cr(VI)-induced DPC by phosphate. EDTA was very inefficient in DPC dissociation, indicating its poor suitability for testing of Cr(III)-mediated bridging and reversal of complex DPC. Reactions containing only one Cr-modified component (protein or DNA) showed that Cr(III)-DNA adduction was the initial step in DPC formation. Cross-linking proceeded slowly after the rapid formation of Cr-DNA adducts, indicating that protein conjugation was the rate-limiting step in DPC generation. Experiments with depletion of glutathione and restoration of ascorbate levels in human lung A549 cells showed that high cellular reducing capacity promotes DPC yield. Overall, our data provide evidence for a three-step cross-linking mechanism involving (i) reduction of Cr(VI) to Cr(III), (ii) Cr(III)-DNA binding, and (iii) protein capture by DNA-bound Cr(III) generating protein-Cr(III)-DNA cross-links.

Polychlorinated biphenyls (PCBs) are toxic industrial chemicals, complete carcinogens, and efficacious tumor promoters. However, the mechanism(s) of PCB-mediated carcinogenicity remains largely undefined. One likely pathway by which these agents may play a role in carcinogenesis is the generation of oxidative DNA damage by redox cycling of dihydroxylated PCB metabolites. We have now employed a new (32)P-postlabeling system to examine novel oxidative DNA lesions induced by Cu(2+)-mediated activation of PCB metabolites. (32)P postlabeling of DNA incubated with various PCB metabolites resulted in over a dozen novel polar oxidative DNA adducts that were chromatographically similar for all active agents. The most potent metabolites tested were the hydroquinones (hydroxyl groups arranged para to each other), yielding polar oxidative adduct levels ranging from 55 to 142 adducts/10(6) nucleotides. PCB catechols, or ortho-dihydroxy metabolites, were up to 40% less active than their corresponding hydroquinone congeners, whereas monohydroxylated and quinone metabolites did not produce detectable oxidative damage over that of vehicle. With the exception of 2,4,5-Cl-2',5'-dihydroxybiphenyl, this oxidative DNA damage seemed to be inversely related to chlorine content: no chlorine approximately mono->di->trichlorinated metabolites. Importantly, copper, but not iron, was essential for activation of the PCB metabolites to these polar oxidative DNA adducts, because in its absence or in the presence of the Cu(+)-specific scavenger bathocuproine, no adducts were detected. Intervention studies with known reactive oxygen species (ROS) modifiers suggested that H(2)O(2), singlet oxygen, hydroxyl radical, and superoxide may also be involved in this PCB-mediated oxidative DNA damage. These data indicate a mechanistic role for several ROS, in addition to copper, in PCB-induced DNA damage and provide further support for oxidative DNA damage in PCB-mediated carcinogenesis.

Polycyclic aromatic hydrocarbons (PAHs) are a class of widespread environmental carcinogens. Most of our knowledge of their mechanisms of metabolic activation to DNA-binding "ultimate carcinogenic" metabolites has come from analysis of the DNA interaction products formed by these highly reactive intermediates. Studies of their role in forming DNA-binding intermediates identical to those formed in vivo from the PAH itself have also allowed identification of the particular cytochrome P450 enzymes involved in activating various structural classes of carcinogenic PAHs. It has been established that PAHs, after metabolic activation in vivo, are capable of inducing mutations in oncogenes and, by inducing multiple mutations, may result in tumors. PAHs also cause changes in cellular gap-junction communication similar to those caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Thus, PAHs may also act through a promotional mechanism in addition to serving as tumor initiators. Previous studies on these mechanisms are described and summarized.

The N-1-(2,3,4-trihydroxybutyl)adenine (N-1-THB-Ade) adducts induced by 1,3-butadiene (BD) were analysed from lymphocytes of 15 workers occupationally exposed to BD and 11 controls by (32)P-post-labelling using HPLC with radioactivity detection. The difference in the adduct levels between the BD-exposed workers (4.5 +/- 7.7 adducts/10(9) nucleotides) and the controls (0.8 +/- 1.2 adducts/10(9) nucleotides) was statistically significant (Wilcoxon rank sum test, P = 0.038). This study shows for the first time BD-induced DNA adducts in humans and suggests that N-1-THB-Ade adducts may be used to biomonitor human exposure to BD.

The effect of solvent polarity and lipophilicity on DNA adduct formation by polycyclic aromatic hydrocarbons in skin and lung has been studied in CD1 mice exposed cutaneously in vivo to benzo(a)pyrene ( approximately 0.01-7.0 microg/animal) in either tetrahydrofuran or n-dodecane. The nature and amounts of DNA adducts, measured as 7R,8S, 9R-trihydroxy-10S-(N(2)-deoxyguanosyl-3'-phosphate)-7,8,9, 10-tetrahydrobenzo(a)pyrene, in relation to exposure dose and treatment regime was determined by (32)P-postlabelling. In skin DNA there was a linear relationship between exposure dose and adduct formation with both solvents, though the amount of adduct formed was significantly lower from treatment with benzo(a)pyrene in n-dodecane than in tetrahydrofuran. The amounts of adducts measured in skin DNA ranged from 67 amol adducts/microg DNA at the lowest exposure dose of benzo(a)pyrene in n-dodecane to 3.5 fmol adducts/microg DNA (1 adduct in 5 x 10(7) nucleotides to 1 adduct in 9 x 10(5) nucleotides) at the highest dose. In tetrahydrofuran the corresponding levels were 89 amol adducts/microg DNA (1 adduct in 3 x 10(7) nucleotides) to 16.9 fmol adducts/microg DNA (1 adduct in 2 x 10(5) nucleotides). DNA adducts could not be detected in lung tissue following cutaneous treatment of animals with benzo(a)pyrene in n-dodecane. Cutaneous treatment of animals with benzo(a)pyrene in tetrahydrofuran, however, resulted in adducts in lung DNA at a level of 88 amol/microg DNA from exposures only at the highest dose (6.72 microg/animal). The difference in octanol-water partition coefficient, log P(ow) between n-dodecane compared to tetrahydrofuran is considered to be the most likely reason for the reduction in the bioavailability of benzo(a)pyrene and/or its metabolites and hence the degree of genotoxicity in tissues. The results suggest that other paraffinic hydrocarbon solvents may moderate the genotoxicity of polycyclic aromatic hydrocarbons in vivo. The assessment of the genotoxicity in vivo of mixtures of compounds should be carried out on complete mixtures of substances of interest in order to take account of these possible antagonistic or synergistic effects.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.

Incubation of the pentachlorophenol (PCP) metabolites, tetrachloro-p-benzoquinone (chloranil, TCpBQ), tetrachloro-p-hydroquinone (TCpHQ) and tetrachloro-p-benzoquinone (TCoBQ) with V79 Chinese hamster cells led to a significant enhancement of the amount of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA. With PCP itself and the metabolite tetrachloro-o-hydroquinone (TCoHQ) no distinct induction of this lesion could be observed. The average yields of 8-OH-dG were about 2-2.5 times above background levels. In addition, TCpBQ and TCpHQ were able to generate DNA single-strand breaks, while PCP, TCoHQ and TCoBQ failed to induce this lesion. All incubations were performed for 1 h without exogenous metabolic activation and concentrations were 25 microM of the respective agent. It is concluded that these metabolites may contribute to the carcinogenicity of PCP observed in mice, by generating reactive oxygen species (ROS) through their redox cycling properties.

Syrian golden hamsters are much more susceptible than Wistar rats to the induction of tracheal tumors by benzo(a)pyrene (BP). In order to investigate whether this difference is reflected in the pattern of DNA-adduct induction and removal, tracheas from either species were isolated and exposed to BP (5 micrograms/ml) in organ culture. At various time-points BP-DNA adducts in the epithelial cells were quantified by 32P-postlabeling; unscheduled DNA synthesis (UDS) was determined by [3H]thymidine incorporation. In an induction-repair experiment tracheas were exposed to BP for 2 days, and cultured for another 4 days without BP. After 2 days of exposure total BP-DNA adduct levels were 10 times higher in hamster compared to rat tracheas. In hamster tracheas one major adduct was formed (95%), vs. the adduct between (+)-anti-BP-diolepoxide and deoxyguanosine (BPDE-N2dG). In rat tracheas BPDE-N2dG comprised about 60% of the total adduct level. During exposure to BP the adduct level in hamster trachea increased to 36 +/- 19 adducts/10(6) nucleotides (add/10(6) n) on day 2. Two days after removal of BP the BP-DNA adduct level had decreased to 60% of that on day 2; there was no further decrease in the BP-DNA adduct level. UDS increased during exposure to BP and decreased after removal of BP. In rats, removal of BP did not lead to a decrease in the BP-DNA adduct level, which agreed with the observed absence of UDS. In a second experiment tracheas were exposed to BP continuously for 15 days. In hamster tracheas the total BP-DNA adduct level increased from 11 +/- 0.7 add/10(6) n after 1 day of exposure to 105 +/- 2 add/10(6) n after 15 days; also UDS increased with increasing exposure until day 11. In rat tracheas no progressive increase in the BP-DNA adduct level was seen. It was concluded that the difference in trachea tumor susceptibility between hamsters and rats exposed to BP correlates with the difference between the 2 species in BP-DNA adduct kinetics in the trachea epithelial cells.

Benzo[a]pyrene (B[a]P) injected intramuscularly into the base of the arms of cuttlefish was released continuously from the injection site and removed from the organism. Only a portion of the compound accumulated in the body. Twenty-four hr after its injection, 75% of B[a]P applied in olive oil was removed from the cuttlefish, and 1.2% was found in the body outside the head, the site of injection. If the carcinogen was dissolved in dimethylformamide, the removal of B[a]P was slower, so that only 18% of the injected B[a]P was removed from the organism and 0.36% accumulated in the body outside the head 24 hr after injection. The high level of B[a]P in gills and hemolymph 4 hr after injection and the kinetics of the decrease of its concentration with time indicate that these two organs could be involved in the excretion of B[a]P from the body. The B[a]P/DNA adducts characteristic for vertebrates could not be demonstrated in gills, skin, brain, hepatopancreas, and lymphocytes of the cuttlefish 24 hr after injection of B[a]P. The dose of the carcinogen injected into the cuttlefish was 2-4 times higher than the dose resulting in the formation of a high level of B[a]P/DNA adducts in the vertebrates. A different metabolism of B[a]P in the tissue of cephalopods, compared to vertebrates, could be less favorable to the process leading to malignant transformation and could explain the absence from the literature of reports of tumors in cephalopods.

Detection of carcinogen-DNA adducts in DNA from exfoliated urothelial cells from animals and humans exposed to potential environmental carcinogens is described. In an animal model, 4-aminobiphenyl-DNA adducts were detected, and the shape of the dose-response curve was related to the levels of 4-aminobiphenyl-hemoglobin adducts. In a human study, five distinct adducts were two to nine times higher in smokers than in nonsmokers. The association of four adduct measures with smoking was corroborated by significant correlations with levels of 4-aminobiphenyl-hemoglobin adducts, type and number of cigarettes smoked, and/or urinary mutagenicity. One adduct seemed chromatographically similar to N-(deoxyguanosin-8-yl)-4-aminobiphenyl. This adduct showed the strongest correlation with 4-aminobiphenyl-hemoglobin adduct levels. These data suggest that noninvasive techniques can be applied to the study of carcinogen-DNA adducts in the target organ of humans at risk for urinary bladder cancer.

N-Heterocyclic aromatics (NHA) are widely occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. NHA are found in significant amounts in tobacco condensates, synthetic fuels, gasoline engine exhaust, and effluents from the heating of coal. Dibenz[a,j]acridine (DBA) is an example of NHA. The potency of many carcinogenic compounds is related, at least in part, to the efficiency of their biological activation. We undertook studies to determine which initial metabolites of DBA lead to the formation of high levels of carcinogen-DNA adducts in vivo. DBA and its metabolites, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD), trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD), and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were applied to the skin of mice. DNA was isolated using enzyme-solvent extraction method. DNA was 32P-postlabeled under conditions of limiting [32P]ATP. In skin, DBA produced two distinct adducts. The same two adducts were seen when DBA-3,4-DHD was applied. In addition the total adduct level elicited by DBA-3,4-DHD was higher than that of parent compound. Two adducts were seen when DBA-5,6DHD was applied, but these were very different from adducts seen with DBA. These results suggested that activation of DBA to DNA-binding compounds in skin includes initial formation of DBA-3,4-DHD. The data support development of biomarkers for the exposure and effect of this compound, and also suggest that specific metabolic susceptibility markers might be able to predict populations at increased risk.

The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8-dihydro-2-nitrofluoranthene, trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The activated metabolite, N-hydroxy-2-aminofluoranthene, was prepared in situ and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8-substituted deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed at a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.

The proportion and amount of benzo[a]pyrene (B[a]P) that binds to DNA through the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] increases with time of exposure to B[a]P in cell cultures derived from a number of species. Pretreatment of primary rat hepatocyte cultures for 12 h with 1 microgram B[a]P/ml medium increased the subsequent metabolism of [3H]B[a]P by 47% and [3H]B[a]P-DNA binding by 53% compared with acetone-pretreated hepatocytes. The amount of (+)-anti-BPDE bound to DNA in the B[a]P-pretreated hepatocytes increased 175%. B[a]P pretreatment also increased DNA-binding 2-fold in hepatocytes treated with [3H]7,8-dihydroxy-7,8-dihydro-B[a]P but had no effect on DNA binding in cells treated with anti-B[a]P-7,8-diol-9,10-epoxide. Western blotting showed that cytochrome P450IA1, which was not detectable prior to B[a]P treatment, was selectively increased by B[a]P treatment. A monoclonal antibody that specifically inhibits cytochrome P450IA1 reduced the binding of B[a]P to DNA by greater than 90% in microsomal preparations from B[a]P-pretreated hepatocytes. These results indicate that the time-dependent increase in the formation of (+)-anti-BPDE-DNA adducts results from an increase in the amount and proportion of B[a]P metabolized to this ultimate carcinogen by P450IA1 that is induced by the B[a]P treatment. The importance of P450IA1 induction by the B[a]P for its activation to this ultimate carcinogenic metabolite suggests that long-term exposure of cells to B[a]P could result in activation of a higher proportion of the B[a]P to the carcinogenic (+)-anti-BPDE.

Certain finfish species living in chemically polluted environments exhibit a high incidence of gastrointestinal tract tumors. Carnivorous fish in such environments are likely to consume invertebrates which contain chemical procarcinogens and the invertebrate biotransformation products of these compounds. The retention in tissues, extent of DNA adduct formation in liver and intestine, and metabolite composition of bile was investigated in southern flounder following gavage with pure [3H]- or [14C]benzo[a]pyrene (BaP), pure [14C]benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8D), or hepatopancreas from spiny lobsters previously dosed with [3H]- or [14C]BaP (Metab.HP). Metab.HP contained mainly polar conjugates of BaP diols, triols and tetraols. BaP-7,8D was retained in fish tissues and bile at 24 h to a greater extent (33.6% of the dose), than either BaP (19.00%) or Metab.HP (6.6%). Hepatic and intestinal DNA isolated from all dosed fish contained covalently bound radioactivity, but exposure to BaP-7,8D or BaP resulted in significantly higher binding in both tissues than exposure to Metab.HP. Hepatic DNA from BaP and BaP-7,8D-dosed flounder contained 0.24 +/- 0.07 and 0.33 +/- 0.06 pmol BaP equivalents/mg DNA respectively (mean +/- S.E.), while hepatic DNA isolated from Metab.HP-dosed flounder contained 0.006 +/- 0.002 pmol BaP equivalents/mg DNA. Binding of radioactivity to intestinal DNA was significantly higher than to hepatic DNA for flounder dosed with Metab.HP (0.026 +/- 0.003) or with BaP (0.76 +/- 0.27) but not for flounder dosed with BaP-7,8D (0.44 +/- 0.09). These studies show that dietary BaP, and metabolites likely to be present in invertebrates, can be absorbed by the southern flounder and form DNA adducts in target organs.

Specific and sensitive methods are now available to detect DNA and protein adducts induced by a variety of carcinogens. These measurements of individual exposures are now starting to be used in cancer epidemiological studies. In this paper the requirements and the problems in implementation of the studies are discussed, as well as aspects of interpretation of the results in relation to assessments of past exposure and biological relevance to the disease process.

To assess the utility of DNA adducts as biomarkers of exposure to carcinogens in an industrial population, a pilot study of roofers occupationally exposed to a mixture of polycyclic aromatic hydrocarbons was conducted. DNA was isolated from peripheral white blood cells of roofers and non-occupationally exposed subjects matched for age, sex and smoking status. Occupational exposures to anthracene, fluoranthene, pyrene, benzanthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene and benzo[k]fluoranthene were assessed by personal breathing zone air sampling and skin wipes. Exposures to benzo[a]pyrene in air of exposed subjects ranged from 0.60 microgram/m3 to 1.39 micrograms/m3, and exposures to total polycyclic aromatic hydrocarbon (the sum of eight hydrocarbons) ranged from 6.0 micrograms/m3 to 13.8 micrograms/m3 on the day before blood collection. In the biomarker studies 10 of 12 roofers, but only 2 of 12 comparison subjects, had detectable levels of aromatic DNA adducts by 32P-postlabelling assay (p less than 0.01). The two non-roofers with detectable adducts had levels at or near the detection limit of 2 adducts per 10(9) nucleotides. In two roofer samples which were studied in a mixing experiment, the major adduct spots did not co-migrate with the guanosine N2 adduct of benzo[a]pyrene diol epoxide. These results suggest that the 32P-postlabelling assay may be useful for monitoring exposures to complex mixtures of aromatic hydrocarbons in industrial populations.

The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole (DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolites of DBC was investigated in liver and skin of female CD-1 mice. After topical application to the skin of 37 mumol/kg of DBC or the phenolic metabolites, DNA adducts were measured by a 32P-post-labeling assay employing carrier-free [gamma-32P]ATP and ATP-deficient conditions. In liver, DBC produced four major and several minor chromatographically distinct adducts of as yet undetermined chemical structure. The adduct pattern elicited by 3-OH-DBC was qualitatively similar to the DBC adduct pattern, while this was not the case for 2-OH-DBC and 4-OH-DBC. On the basis of co-chromatography experiments under various conditions, the DBC and 3-OH-DBC adducts appeared identical, and the total of adduction elicited by these compounds in liver was substantial. Similar results were observed when DBC or 3-OH-DBC were administered i.p. As a major difference between the two compounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-fold lower than the corresponding DBC adduct after i.p. and topical dosing, respectively. In skin, DBC produced two major adduct fractions after topical application, one of which could be chromatographically resolved into three subcomponents. Prominent adducts produced in skin DNA by each of the three metabolites were different from those elicited by DBC, and the level of adduction by the metabolites was significantly lower than that by DBC. Comparison of the skin and liver DBC-DNA adduct patterns after topical application of DBC showed that only one of the four major chromatographically resolved skin adducts corresponded to a major liver adduct (no. 3), and that total adduction in liver was 13.5-fold higher than in skin. These results suggested that activation of DBC to DNA-binding compounds in liver occurs through at least two pathways with 3-OH-DBC being a proximate carcinogen involved in the formation of most of the adducts; 3-OH-DBC and the other two phenolic metabolites investigated play a minor role, if any, in the formation of DBC-DNA adducts in skin; metabolic activation of DBC to DNA-binding compounds in liver and skin appears to follow pathways that are different in terms of both the chemical nature and the amount of the adducts formed; and DBC and 3-OH-DBC exhibit a strong preference for liver versus skin DNA.

Antibodies raised against the bovine serum albumin conjugates of N-(guanosin-8-yl)-N-2-acetylaminofluorene (Guo-8-AAF), the imidazole ring-opened form of N-(guanosin-8-yl)-2-aminofluorene (roGuo-8-AF) and the methylated bovine serum albumin complex of DNA modified with (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) have been employed in a highly sensitive enzyme-linked immunosorbent assay (ELISA) to determine their affinity for DNA modified with the corresponding carcinogens at various levels of modification. All antibodies recognized highly modified DNA more efficiently than DNA of low modification. This property, which may be common to all antibodies raised against carcinogen-DNA adducts, has to be taken into account when these antibodies are used to quantify carcinogen-DNA adducts in biological samples. Appropriate DNA preparations of low modification have to be used as reference compounds in immunoassays to allow reliable quantification of adduct levels in DNA from animals and human cells.

Covalent adduction of DNA by chemical agents is commonly thought to be an essential part of the initiation of chemical carcinogenesis. Until recently, assays of DNA damage by covalent binding of chemicals have been restricted mostly to substances that are available in radiolabeled form, which excludes many environmental compounds with carcinogenic potential. In this paper, the binding of non-radioactive 7H-dibenzo[c,g]carbazole (DBC), a known environmental carcinogen, to DNA in female CD-1 mice after s.c. injection of 44 mumol/kg of the compound has been investigated using a 32P-postlabeling assay. DBC showed strong hepatic specificity with a mean total level of 107 adducts per 10(7) nucleotides at 24 h, while much lower levels of binding were seen in kidney, lung, spleen, skin and brain with 4.3, 2.1, 1.3, 0.4 and 0.04 adducts, respectively, per 10(7) nucleotides. Proportions of individual DBC adducts also varied considerably between tissues. The degree of hepatic preference displayed by DBC is not seen with other polycyclic aromatic carcinogens such as benzo[a]pyrene and 2-acetylaminofluorene. The DNA-binding data, together with other hepatotoxic effects of the compound, may be causally related to the known hepatocarcinogenicity of DBC.

1-Nitropyrene (1-NP), a ubiquitous environmental pollutant, is a mammalian mutagen and causes cancer in animals. The ability of the lung, liver and kidney to form 1-NP-DNA adducts was determined in adult male B6C3F1 mice following a single intratracheal instillation of 1-NP. 1-NP-DNA adducts were isolated and characterized in mouse lung, liver and kidney by HPLC analysis of the enzymatically digested DNA. Multiple DNA adducts were present in lung, liver and kidney at 1 day after administration. One of the major adducts in lung (20% of the total eluted radioactivity) coeluted with the synthetic marker, N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-dG-AP). This adduct (10% of total eluted radioactivity) and others were still present in the lung at 28 days after administration of 1-NP. One of the adducts in liver and kidney DNA digests also coeluted with C8-dG-AP. Treatment of the adducts with 0.3 M NaOH resulting in earlier eluting peaks containing radioactivity, indicative of an imidazole ring-opening adduct. A portion of the original peak of radioactivity that coeluted with C8-dG-AP and other adducts, however, was not affected by 0.3 M NaOH. Thus, the chromatographic properties and chemical behavior of the adducts formed in vivo suggest that one of the adducts in the lung is C8-dG-AP which is formed by nitroreduction of 1-NP. Other adducts may be formed via ring-oxidation followed in some instances by nitroreduction. These data indicate that DNA adducts of 1-NP metabolites may be formed in the lung (a primary site for inhaled particles), liver and kidney following inhalation of airborne particles containing 1-NP.

The 32P-postlabelling assay is a recently developed analytical tool for the detection and measurement of nucleic acid (DNA and RNA) adducts formed by covalent binding of identified or unidentified electrophiles. The detection limit of the assay for many adducts is as low as 0.3 amol adduct/microgram DNA ( = one adduct/10(10) DNA nucleotides, or one adduct per mammalian genome). As presented here, the method can be applied to DNA alterations elicited by (i) complex mixtures of genotoxicants (e.g., cigarette smoke, occupational exposures), (ii) oestrogens (i.e., hormones that cause DNA damage via the formation of unidentified electrophiles), and (iii) DNA-reactive chemicals that may be formed metabolically in animal tissues without known exposure and give rise to adduct-like DNA alterations (termed I compounds).

The metabolic activation of polycyclic aromatic hydrocarbons (PAH), for example benzo[a]pyrene, leads to the formation of carcinogen-macromolecular adducts. Methods that make it possible to detect low levels of these adducts in human peripheral blood samples should be useful in the dosimetry of human exposure to carcinogens. We demonstrated previously the usefulness of enzyme immunoassays and of synchronous fluorescence spectroscopy (SFS) for detecting and characterizing low levels of PAH-macromolecular adducts present in synthetic adduct mixtures. These methods have now been refined and applied to the analysis of samples of peripheral blood collected from occupationally exposed individuals (coke-oven workers) and from people attending smoking cessation clinics. The results of both immunoassays and SFS show the presence of benzo[a]pyrene diol epoxide (BPDE)-DNA, BPDE-haemoglobin and other putative PAH-macromolecular adducts in peripheral blood samples from certain individuals.

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.

Transplacental exposure of fetuses to carcinogens is known to induce tumors in the offspring, often with a high incidence and short latency. While covalent adduction of DNA appears to be essential for tumor initiation, little is known about the binding of carcinogens to the DNA of fetal tissues. A sensitive 32P-postlabeling method enabled us to study the binding of the environmental carcinogens safrole (600 mumol/kg p.o.), 4-aminobiphenyl (800 mumol/kg), and benzo(a)pyrene (200 mumol/kg) to the DNA of various maternal and fetal tissues after administration of test carcinogens to pregnant ICR mice on day 18 of gestation. The results show that these carcinogens bound to the DNA of maternal and fetal liver, lung, kidney, heart, brain, intestine, skin, maternal uterus, and placenta, with organ-specific quantitative and qualitative differences. It was possible for the first time to analyze DNA adduct patterns in minute amounts of tissue, for example those available from fetal heart. The covalent binding index (mumol adducted nucleotides per mol of DNA nucleotides/mumol carcinogen administered per g body weight) 24 h after safrole treatment was estimated for the different organs and ranged from 0.1 to 247 and 0.1 to 5.8 for maternal and fetal DNA, respectively. Covalent binding index values of 0.2 to 13 and 0.1 to 0.3 for maternal and fetal DNA, respectively, were found for 4-aminobiphenyl. Benzo(a)pyrene treatment yielded covalent binding index values of 0.6 to 6.5 and 0.3 to 0.7 for maternal and fetal DNA, respectively. In both maternal and fetal tissues, safrole exhibited preferential binding to liver DNA. 4-Aminobiphenyl bound preferentially to DNA of maternal liver and kidney but showed no preference among fetal tissues. Benzo(a)pyrene exhibited weak tissue preference in both maternal and fetal organs. For all of the compounds studied, the fetal adduct levels were generally lower than the corresponding maternal adduct levels, especially when the level of maternal adduction was high. The major finding was that several carcinogens of diverse structure or their metabolites readily crossed the placenta and gave rise to DNA adducts in fetal organs. The resulting DNA damage in rapidly proliferating tissues may play a critical role in transplacental carcinogenesis.