Genotoxicity of coke-oven and urban air particulate matter in in vitro acellular assays coupled with 32P-postlabeling and HPLC analysis of DNA adducts

This study is an in vitro part of the ongoing biomarker studies with population from a polluted region of Northern Bohemia and coke-oven workers from Czech and Slovak Republics. The aim of this study is to compare DNA adduct forming ability of chemical compound classes from both the urban and coke-oven extractable organic mass (EOM) of airborne particles. The crude extracts were fractionated into seven fractions by acid-base partitioning and silica gel column chromatography. In in vitro acellular assays we used calf thymus DNA (CT DNA) with oxidative (+S9) and reductive activation mediated by xanthine oxidase (+XO) under anaerobic conditions. Both the butanol and nuclease P1 versions of 32P-postlabeling for detection of bulky aromatic and/or hydrophobic adducts were used. The results showed that the spectra of major DNA adducts resulting from both the in vitro assays are within the fractions similar for both the urban and coke-oven samples. The highest DNA adduct levels with S9-activation were detected for the neutral aromatic fraction, followed by slightly polar and acidic fractions for both samples. With XO-mediated metabolism, the highest DNA adduct levels were detected for both the acidic fractions. Assuming additivity of compound activities, then the acidic fraction, which in the urban sample comprises a major portion of EOM mass (28%), may contain the greatest activity in both in vitro assays (39 and 69%, +S9 and +XO, respectively). In contrast, the aromatic fraction constituting only 8% of total urban EOM mass may account for comparable activity (34%) with organic acids. The highest DNA adduct forming activity of the coke-oven sample accounts for the aromatic fraction (82 and 63%, +S9 and +XO, respectively) that also contains the greatest portion of the total EOM (48%). To characterize some of the specific DNA adducts formed, we coupled TLC on 20x20 cm plates with HPLC analysis of 32P-postlabeled adducts. In both S9-treated samples of the aromatic fraction, we tentatively identified DNA adducts presumably diolepoxide-derived from: 9-hydroxy-benzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide[+/-] (anti-BPDE), benzo[b,j,k]fluoranthenes (B[b]F, B[j]F, B[k]F), chrysene (CHRY), benz[a]-anthracene (B[a]A) and indeno[cd]pyrene (I[cd]P). These DNA adducts accounted for about 57% of total DNA adducts detected in both S9-treated samples of the aromatic fraction. DNA adducts of XO-treated samples were sensitive to nuclease P1 and HPLC profiles of the major adducts were markedly different from the major adducts of S9-treated samples. However, the combination of TLC and HPLC did not confirm the presence of DNA adducts derived from 1-nitropyrene (1 NP), 9-nitroanthracene (9 NA) and 3-nitrofluoranthene (3 NF) that were detected by GC-MS in the slightly polar fraction. We concluded that the chemical fractionation procedure facilitates the assessing of DNA adduct forming ability of different chemical compound classes. However, based on the results obtained with the whole extracts, it does not fulfil a task of the actual contribution of individual fractions within the activity of the whole extracts. Our results are the first in detecting of DNA adducts derived from urban air and coke-oven particulate matter.     

Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes

Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA adducts, genetic predisposition to cancer, and their interrelationships are under study in numerous laboratories. This report describes a modified 32P-postlabeling assay for the detection of polycyclic aromatic hydrocarbon-DNA adducts that uses immunoaffinity chromatography to enhance chemical specificity and quantitative reliability. The assay incorporates internal standards to determine direct molar ratios of adducts to unmodified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. High performance liquid chromatography is used to assure adequacy of DNA enzymatic digestion. The assay was validated using radiolabeled benzo(a)pyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby assessing all variables from enzymatic digestion to detection. Thirty-eight human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide-DNA adducts by immunoaffinity chromatography, high performance liquid chromatography, and synchronous fluorescence spectroscopy. A high correlation between the two assays was found (P = 0.006). The lung samples were then analyzed by the polymerase chain reaction for the presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione S-transferase mu (GST mu) genes. A positive association was identified for adduct levels and GST mu null genotypes (P = 0.038). No correlation was found between polycyclic aromatic hydrocarbon-adduct levels and CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related to adduct levels. Multivariate analysis indicated that only the GST mu genotype was associated with polycyclic aromatic hydrocarbon-DNA adduct levels. This work demonstrates that the 32P-postlabeling assay can be modified for chemically specific adduct detection and that it can be used in the assessment of potentially important genetic factors for cancer risk. The absence of a functional GST mu gene in humans is likely one such factor.     

Metabolic activation and carcinogen-DNA adduct detection in human larynx

Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.     

Changes in birth weight, birth length and head circumference of Hungarian children in the county Baranya between 1968 and 1979-1981

Wildland (forest) firefighters are exposed to a wide range of carcinogenic polycyclic aromatic hydrocarbons (PAH) in forest fire smoke. PAH undergo metabolic activation and can subsequently bind to DNA. In this study, we investigated the association between occupational and dietary PAH exposures and the formation of WBC PAH-DNA adducts in a population of wildland firefighters. An enzyme-linked immunosorbent assay using an antiserum elicited against benzo(a)pyrene-modified DNA was used to measure PAH-DNA adducts in WBC obtained from 47 California firefighters at two time points, early and late in the 1988 forest fire season. PAH-DNA adduct levels were not associated with cumulative hours of recent firefighting activity. However, firefighters who consumed charbroiled food within the previous week had elevated PAH-DNA adduct levels, which were related to frequency of charbroiled food intake. These findings suggest that dietary sources of PAH contribute to PAH-DNA adduct levels in peripheral WBC and should be evaluated when using this assay to assess occupational and environmental PAH exposure.     

Growth and cuticular synthesis in Ascaris suum larvae during development from third to fourth stage in vitro

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the adduct level ranged from 6.81 to 108.50 adducts/10(8) nucleotide. Thirty-three of 39 (85%) had detectable levels of BPDE-DNA adducts (> 1 adduct/10(9) nucleotide). Current heavy smokers (> 20 cigarettes/day) have significantly higher DRZ adduct levels compared to individuals smoking less than 20 cigarettes/day. By SFS combined with immunoaffinity column (IAC), 11 of 39 (28%) samples had detectable adduct levels, and 6 of 11 (55%) were detectable by SFS following purification of benzo[a]pyrene (BP)-tetrols by high pressure liquid chromatography (HPLC). Six of 33 (18%) samples were positive for BPDE-DNA adducts by both postlabeling and HPLC/SFS. No correlation was observed between the SFS and 32P-postlabeling assays for the detection of BPDE-DNA adducts. However, there was a good correlation between adduct levels detected by IAC/SFS and HPLC/SFS. We found a weak association between total PAH-DNA adduct levels in lung tissue and TP53 mutations.     

Tumors of the oral cavity, oropharynx and salivary glands. Role of TC and MRI in neoplasm staging

The 32P-postlabelling assay is one of the most sensitive methods for detection of DNA adducts induced by exposure to genotoxic chemicals. Under optimal conditions, detection limits of one adduct per 10(9)-10(10) nucleotides have been reported. This sensitivity now allows monitoring of occupational and even environmental exposure of humans to certain classes of chemicals, mainly polycyclic aromatic hydrocarbons (PAH). Despite its widespread use, 3P-postlabelling is still not a standardized method. Rigorous interlaboratory comparisons are scarce, and those that have been undertaken often show rather different results, both in relative and in absolute values, for the amounts of DNA adducts in the same samples. Furthermore, the optimization of many steps in the procedure has still not been given adequate attention. This paper deals with some technical aspects of detection of PAH-DNA adducts by 32P-postlabelling, in particular with assay calibration and adduct quantification. For this purpose, benzo[a]pyrene (BP)-modified DNA standards were prepared, the adduct contents of which were determined by use of an independent fluorometric method, viz. synchronous fluorescence spectrophotometry (SFS). These BP-DNA standards are processed along with the test samples throughout the entire 32P-postlabelling procedure, from the enzymic digestion up to and including the determination of radioactivity in adduct spots on the chromatogram. As such, these reference samples can be considered as external standards for inter-assay calibration. This method for adduct quantification was compared with the commonly used relative adduct labelling (RAL) and comparative dAMP labelling, which appeared to give rise to an underestimation of adduct levels. The method was applied in a biomonitoring study among workers in a carbon-electrode manufacturing plant, exposed to PAH. Although DNA adduct levels in peripheral blood lymphocytes of exposed workers, as determined by 32P-postlabelling, were not significantly different from those of controls, a significant difference was seen when smokers and non-smokers were compared.     

Ca2+-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.     

32P-postlabeling analysis of the DNA adducts of 6-fluorobenzo[a]pyrene and 6-methylbenzo[a]pyrene formed in vitro

Studies of benzo[a]pyrene (BP) and selected derivatives are part of the strategy to elucidate mechanisms of tumor initiation by polycyclic aromatic hydrocarbons. Substitution of BP at C-6 with fluorine to form 6-fluorobenzo[a]pyrene (6-FBP) or a methyl group to form 6-methylbenzo[a]pyrene (6-CH3BP) decreases tumorigenicity compared to BP. BP, 6-FBP, and 6-CH3BP formed adducts with DNA when (1) they were activated by 3-methylcholanthrene-induced rat liver microsomes, (2) they were activated by horseradish peroxidase (HRP), (3) their 7,8-dihydrodiols were activated by microsomes, or (4) the radical cation of BP, 6-FBP, or 6-CH3-BP was directly reacted with DNA. With microsomes, 6.5 mumol of [3H]6-FBP/mol of DNA-P and 10 mumol of [14C]6-CH3BP/mol of DNA-P were bound vs 15 mumol of [3H]BP. With microsomes, two major 6-FBP adducts and some minor adducts were obtained. One major adduct coincided with that from 6-FBP-7,8-dihydrodiol. With microsomes, the minor 6-FBP adducts coincided with the adducts obtained from 6-FBP radical cation plus DNA and the major adduct of HRP-activated 6-FBP. With microsomes, 6-CH3BP showed adducts similar to some formed with HRP and one from 6-CH3BP radical cation. 6-CH3BP-7,8-dihydrodiol produced a small amount of one adduct that did not coincide with any from 6-CH3BP. The adducts of 6-FBP appear to be formed mostly through the diolepoxide pathway, whereas those of 6-CH3BP appear to arise mostly via one-electron oxidation.     

The interaction between alcohol consumption and GSTM1 genotype on polycyclic aromatic hydrocarbon-DNA adduct levels in breast tissue

We investigated the association between alcohol consumption, GSTM1 genotype, and polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels in breast tissue. Women referred for breast surgery were enrolled prior to surgery, responded to an interview, and gave a blood sample. Women diagnosed with ductal carcinoma in situ and invasive ductal or lobular cancer were defined as cases, and women with benign conditions without atypia were defined as controls. Paraffin-embedded tumor and nontumor tissue from cases and benign tissue from controls were retrieved from the pathology samples. GSTM1 genotype status was determined by PCR using WBC DNA, and PAH-DNA adduct levels were measured in breast tissue using immunohistochemistry. In tumor and nontumor tissue from cases, the GSTM1-null genotype was associated with increased adduct levels among current alcohol consumers but not among nondrinkers. In nontumor tissue, the interaction between genotype and alcohol consumption was significant (P=0.02), but in tumor tissue, the interaction did not achieve statistical significance (P=0.10). In benign tissue from controls, there was no association between genotype and adducts, regardless of drinking status. Among subjects with the null genotype who drank alcohol, adduct levels were significantly higher in tumor and nontumor tissue from cases than in benign tissue from controls. These results indicate the presence of a novel gene-lifestyle interaction that influences PAH-DNA adduct levels in breast tissue from cases but not controls. This apparent difference in PAH metabolism in response to alcohol may be an important clue as to how alcohol influences breast cancer risk.     

Covalent DNA adducts formed in mouse epidermis by benzo[g]chrysene

The metabolic activation in mouse skin of benzo[g]chrysene (B[g]C), a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar, was investigated. Male Parkes mice were treated topically with 0.5 micromol B[g]C and DNA was isolated from the treated areas of skin at various times after treatment and analysed by 32P-post-labelling. Seven major adduct spots were detected, at a maximum level of 6.55 fmol adducts/microg DNA. Mouse skin treated with the PAH benzo[c]phenanthrene (B[c]Ph) gave a total of 0.24 fmol adducts/microg DNA. B[g]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 micromol of the optically pure putative proximate carcinogens, the (+)- and (-)-trans benzo[g]chrysene-11,12-dihydrodiols, led to the formation of adducts which comigrated on TLC and HPLC with those formed in B[g]C-treated mice, which suggested that the detected adducts were formed by the fjord region B[g]C-11,12-dihydrodiol-13,14-epoxides (B[g]CDEs). To test this, the four optically pure synthetic B[g]CDEs were reacted in vitro with DNA and the heteroco-polymers poly(dA x dT) and poly(dG x dC) and these samples 32P-postlabelled. Co-chromatography, on both TLC and HPLC, of in vitro and in vivo adducts indicated that B[g]C is activated in mouse skin through formation of the (-)-anti-(11R,12S,l3S,14R) and (+)-syn-(11S,12R,13S,14R) B[g]CDEs. (-)-anti-B[g]CDE formed five adducts with DNA, two of them with adenine and three with guanine bases. (+)-syn-B[g]CDE formed one adduct with each of these bases in DNA. The adenine adducts accounted for 64% of the total major adducts formed in B[g]C-treated mouse skin. The route of metabolic activation or B[g]C is similar to that reported for B[c]Ph, but the extent of activation to the fjord region diol-epoxides is significantly greater in the case of B[g]C, as demonstrated by the higher levels of adduct formation in vivo.     

Anti-benzo[a]pyrene diolepoxide--DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking

The level of (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39 coke oven workers exposed to polycyclic aromatic hydrocarbons (PAH) and 39 non-exposed persons (controls) were investigated, each of the groups consisting of smokers and non-smokers. The adduct level was measured by an improved HPLC/fluorescence method (Rojas, M., Alexandrov, K., van Schooten, F. J., Hillebrand, M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557-560, 1994) through the release of the corresponding benzo[a]pyrene (B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51% of coke oven workers exposed to PAH and in 18% of the non-exposed (control) subjects. The mean level of anti-BPDE-DNA adducts/10(8) nucleotides in coke oven workers (15.7 +/- 37.8) was approximately 8 times higher than in non-exposed subjects (2.0 +/- 8.7). The interindividual variation of adduct levels was approximately 100-fold in coke oven workers and approximately 50-fold in controls respectively. Smokers in the exposed group had 3.5 times more DNA adducts than non-smokers. With the exception of one non-smoker with very high adduct levels (52.8 adducts/10(8)), the control subjects showed the presence of barely detectable adducts in only 16% of the samples examined. The increased in vivo formation in some smokers and high variability of anti-BPDE-DNA adducts in coke oven workers suggests variations in genetically controlled activation/inactivation reactions of PAH metabolism.     

Concentration-dependent block of sodium current in guinea pig ventricular myocytes by a class III antiarrhythmic agent, MS-551

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.     

Lack of correlation between in vitro and in vivo replication of precisely defined benz-a-anthracene adducted DNAs

Like other polycyclic aromatic hydrocarbons, certain metabolites of benz[a]anthracene have been implicated as potent carcinogens. These effects are thought to be caused by the covalent binding of these species to nucleophilic groups on the bases of DNA. To address the molecular mechanisms by which these molecules induce mutations, this study employed oligonucleotides containing four site-specific N6 adenine-benz[a]anthracene diol epoxide adducts. Using a prokaryotic in vivo replication system, we have shown that both non-bay region anti-trans-benz[a]anthracene adducts are essentially nonmutagenic. In contrast, the bay region anti-trans-benz[a]anthracene lesions do induce point mutations at the adduct site. The mutagenic frequency of these bay region lesions is dependent on the stereochemistry about the adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated. The ability of the bacterial replication machinery to bypass the lesions does not correlate with the differences observed in their mutagenesis. While both non-bay region adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree. In vitro studies of the interactions of E. coli DNA polymerase III with these adducts have also been undertaken to further dissect the relationship between adduct structure and biological activity.     

Polycyclic aromatic hydrocarbon-DNA adducts in human placenta and modulation by CYP1A1 induction and genotype

This study investigated the relationship in human placenta between polycyclic aromatic hydrocabon (PAH)-DNA adduct levels and two biomarkers of cytochrome P4501A1 (CYP1A1): gene induction evidenced by CYP1A1 mRNA, and a genetic polymorphism, the CYP1A1 MspI RFLP. CYP1A1 codes for an inducible enzyme system that catalyzes the bioactivation of PAHs. Prior research found a high correlation in human lung tissue between CYP1A1 activity and DNA damage from PAHs. The CYP1A1 Mspi RFLP has been linked in some studies to risk of lung cancer. The relationships in human placenta between DNA damage, CYP1A1 activity and genotype have not been well characterized and may be relevant to risks from transplacental PAH exposure. The study cohort consisted of 70 newborns from Krakow, Poland, a city with elevated air pollution, and 90 newborns from nearby Limanowa, an area with lower air pollution but greater indoor coal use. Contrary to results seen previously in lung tissue, CYP1A1 mRNA was not significantly correlated with PAH-DNA adduct levels in the placenta. Smoking (self-reported maternal and infant plasma cotinine) was significantly associated with CYP1A1 mRNA levels (P < 0.01), but not with PAH-DNA adduct levels. Placental PAH-DNA adduct levels were significantly higher in infants with the CYP1A1 MspI restriction site compared with infants without the restriction site (P < 0.01), implicating a genetic factor in inter-individual variation in DNA damage in human placenta. Further studies are needed to determine the relevance of this finding to risk of transplacental carcinogenesis.     

Structure, conformations, and repair of DNA adducts from dibenzo[a, l]pyrene: 32P-postlabeling and fluorescence studies

The nature of stable DNA adducts derived from the very potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in the presence of rat liver microsomes in vitro and in mouse skin in vivo has been studied using 32P-postlabeling and laser-based fluorescence techniques. Analysis of DB[a,l]P-DNA adducts via 32P-postlabeling has been obtained by comparison of the adduct patterns to those obtained from reactions of synthetic (+/-)-anti-, (+)-anti-, (-)-anti-, and (+/-)-syn-DB[a,l]P-11,12-diol 13,14-epoxide (DB[a,l]PDE) with single nucleotides and calf thymus DNA. anti-DB[a,l]PDE-dA adducts derived from the (-)-enantiomer are the major adducts formed in calf thymus DNA and in mouse skin DNA. The ratio of deoxyadenosine to deoxyguanosine modification is approximately 2:1 in mouse skin exposed to DB[a,l]P; activation by rat liver microsomes leads to a similar profile of adducts but with two additional spots. The conformations of DB[a,l]P adducts in native DNA, as well as the possibility of conformation-dependent repair, have been explored by low-temperature fluorescence spectroscopy. These studies have been performed using polynucleotides and calf thymus DNA reacted in vitro with DB[a,l]PDE and native DNA from mouse epidermis exposed to DB[a, l]P. The results show that adducts are heterogeneous, possess different structures, and adopt different conformations. External, external but base-stacked and intercalated adduct conformations are observed in calf thymus DNA and in mouse skin DNA samples. Differences in adduct repair rates are also revealed; namely, the analysis of mouse skin DNA samples obtained at 24 and 48 h after exposure to DB[a,l]P clearly shows that external adducts are repaired more efficiently than intercalated adducts. These results, taken together with those for B[a]P-DNA adducts [Suh et al. (1995) Carcinogenesis 16, 2561-2569], indicate that the repair of DNA damage resulting from PAH diol epoxides is conformation-dependent.     

Peptide YY inhibition of rat gastric enterochromaffin-like cell function

The present study determined tumorigenicity, tumor classification and DNA damage induced in infant mice by benzo[a]pyrene (B[a]P) or Manufactured Gas Plant (MGP) residues after a single exposure. Male and female B6C3F1 mice were exposed to B[a]P or MGP residue from a single environmental site (MGP-4) and males were also exposed to MGP residue composite from seven different sites (MGP-M7). At 26, 39 and 52 weeks after exposure tumorigenesis was assessed in lung, forestomach and liver. Formation and persistence of DNA adducts were quantified by 32P-postlabeling. Exposure of males to B[a]P induced liver tumors in a dose and time dependent manner. MGP induced more advanced tumors than B[a]P. Only a single liver tumor was found in MGP-4 treated females. No forestomach and few pulmonary adenomas were induced in males or females. MGP-4, MGP-M7 or B[a]P induced DNA adducts in males and females. Adducts in liver, lung and forestomach peaked on different days and decreased at different rates. At 24 h post-exposure, no significant differences in initial DNA adduct levels occurred in males and females exposed to MGP-4 or B[a]P. Lack of DNA damage (adducted DNA) did not account for non-responsiveness of lung and forestomach in B6C3F1 genders as well as in liver in females. MGP tumorigenicity could not be accounted for solely by B[a]P content nor did it reflect additivity of B[a]P and other carcinogenic polycyclic aromatic hydrocarbons (PAHs) in MGP. Synergy among MGP-PAHs, presence of unidentified carcinogens and/or promoters in MGP may account for MGP potency. The B6C3F1 infant male model is a convenient and rapid assay for assessing MGP liver tumorigenicity and potency.     

Aromatic DNA adducts in foundry workers in relation to exposure, life style and CYP1A1 and glutathione transferase M1 genotype

Levels of aromatic DNA adducts in foundry workers and controls were followed at four annual samplings. During this time exposure to polycyclic aromatic hydrocarbons (PAH) decreased and the level of DNA adducts decreased accordingly. In the total group exposure was related to the level of adducts. Adduct levels correlated with urinary 1-hydroxypyrene (LOGU1OH), air benzo[a]pyrene, weekly working hours and daily cigarette consumption. In a multivariate model 1-hydroxypyrene had a consistent effect. Neither glutathione transferase M1 (GSTM1) nor cytochrome P450 1A1 (CYP1A1) genotypes had clear effects. Yet the individuals lacking GSTM1 had a stronger effect of LOGU1OH and some effect by other sources of PAH, such as charcoal broiled food, although all these variables were not significant in the multivariate model. The rare individuals with a CYP1A1 polymorphism MspI containing an amino acid change at isoleucine had an increased level of adducts. The results showed that the postlabelling method used was able to detect an increase in aromatic DNA adducts in leukocytes when exposure to benzo[a]pyrene in air was approximately 5 ng/m3. At such low levels smoking and charcoal broiled food may be important contributors to adducts.     

A study of multiple biomarkers in coke oven workers--a cross-sectional study in China

We conducted a cross-sectional molecular epidemiological study of coke oven workers exposed to the established carcinogen polycyclic aromatic hydrocarbons (PAHs) to evaluate the relationships between both traditional 'exposure markers' and a series of biomarkers, including urinary 1-hydroxypyrene as a marker of internal dose, leukocyte aromatic DNA adducts as markers of biologically effective dose, serum p53 protein as a response marker and genetic polymorphisms of cytochrome P4501A1 and glutathione S-transferase MI as susceptibility markers. Twenty-five male subjects each were randomly selected from the top, middle and bottom work areas of the oven, and the control plant. They were matched for age and smoking status. The mean levels of PAH exposure, monitored by stationary and personal samplers, and of worker urinary 1-hydroxypyrene differed significantly between the top, middle and bottom of the oven and control work areas. The highest stationary and personal PAH concentrations and 1-hydroxypyrene levels were demonstrated at the top work area. Good correlations were found between the stationary PAH levels, personal PAH levels and urinary 1-hydroxypyrene levels. No positive correlations were demonstrated between aromatic DNA adduct levels and current or cumulative PAH exposure dose. In the presence of genetic polymorphisms of cytochrome P4501A1, a positive correlation was demonstrated between aromatic DNA adducts and urinary 1-hydroxypyrene levels. There was also a significant correlation between serum p53 protein levels and the cumulated benzo[a]pyrene exposure dose. Although these biomarkers have certain limitations, they are applicable to cancer epidemiology, and may contribute to our understanding of the mechanisms of carcinogenesis.     

Reliability of the Agitated Behavior Scale

Polycyclic aromatic hydrocarbon-DNA adducts were evaluated in oral cells from 98 healthy volunteers by an immunohistochemical method using a specific antiserum against benzo(a)pyrene-DNA adducts revealed by the immunoperoxidase reaction. Mean adduct content, determined as relative staining intensity by absorbance image analyzer, was significantly higher in the cells from tobacco smokers compared with nonsmokers (330 +/- 98, n = 33 versus 286 +/- 83, n = 64, respectively) with a P = 0.013 obtained by two-sample t test with equal variances. We found that in the smoker group, the PAH-DNA adduct content increases with the number of cigarettes. Thus, the relative staining intensity was 305 +/- 105 in the group smoking 1-10 cigarettes/day (n = 16), 347 +/- 77 in the 11-20 group (n = 14), and 386 +/- 112 in the group smoking more than 20 cigarettes/day (n = 3; P = 0.03 by nonparametric test for trend). No significant association was detected between PAH-DNA adducts in oral cells and variables such as residential area, oral infections, alcohol or vitamin intake, grilled food consumption, and professional activity. This work confirms and extends previous data suggesting that this immunohistochemical method might be used as a valuable dosimeter of genotoxic damage in a carcinogen-exposed population, although further studies are needed to verify the applicability of the test in high-risk populations other than smokers.     

Inhibitory effects of naturally occurring coumarins on the metabolic activation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured mouse keratinocytes

Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic hydrocarbons.