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PubMed1982

Metabolism of benzo[a]pyrene by fish cells in culture.

Benzo[a]pyrene (BaP) metabolism was studied in cell lines derived from rainbow trout (RTG-2), bluegill fry (BF-2), and fathead minnow (FHM). Confluent cultures were exposed to 3H-BaP (0.5 nmol/ml), and, after various exposure times, metabolites were extracted from the media with an organic solvent and analyzed by high-pressure liquid chromatography. BF-2 and RTG-2 cells converted 63% of the BaP to water-soluble metabolites within 24 h, while FHM cells converted only 12%. BF-2 and RTG-2 cells metabolized more than 90% of the BaP by 48 h, while only 67% of the BaP was converted to water-soluble metabolites by FHM cells after 96 h. The major organic-solvent-extractable metabolites in all three cell lines were 9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene and unidentified polar metabolites. Of the water-soluble metabolites formed by BF-2, FHM, and RTG-2 cells, 67, 42, and 19%, respectively, were converted to ethyl-acetate-extractable metabolites by treatment with beta-glucuronidase. All three cell lines formed a glucuronide of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol); in BF-2 and FHM cells, the 7,8-diol represented almost half of the metabolites released by beta-glucuronidase treatment. Thus, cell lines derived from three widely distributed species of freshwater fish have the capacity to metabolize BaP to a form that is a proximate carcinogen in rodents and to produce a water-soluble conjugate of this metabolite.

Animals↗
PubMed1979

Generation and characterization of condensation aerosols of vanadium pentoxide and pyrene.

Generation and characterization of submicron aerosols of V2O5 and pyrene, two materials with very different physicochemical properties, were studied. Vaporization-condensation methods were used to generate the aerosols. The effects of various experimental conditions on particle size and aerosol mass concentration were investigated. Also, chemical stability of the aerosols and the source materials with respect to temperature was determined. Experimental results show that there are significant differences in the nature of pyrene and V2O5 aerosols. These methods and experimental conditions can be easily adapted for generation of heterogeneous condensation aerosols of pyrene and V2O5.

Aerosols↗
PubMed2005

2'-bis-pyrene modified oligonucleotides: sensitive fluorescent probes of nucleic acids structure.

A new type of fluorescent nucleic acid probes, 2-bis-pyrene-modified oligonucleotides, is described. Preparation of these conjugates involves attachment of two pyrene moieties to the 2'-phosphate group introduced into any position within a sequence by solid-phase phosphoramidite synthesis. Good hybridization properties of the 2'-bis-pyrene probes, their nuclease resistance and sensitivity of fluorescence to the type of complementary nucleic acid have been demonstrated.

DNA↗
PubMed1981

Expression of keratin and vimentin intermediate filaments in rabbit bladder epithelial cells at different stages of benzo[a]pyrene-induced neoplastic progression.

Rabbit bladder epithelium, grown on collagen gels and exposed to the chemical carcinogen benzo[a]pyrene, produced nontumorigenic altered foci as well as tumorigenic epithelial cell lines during 120-180 d in culture. Immunofluorescence studies revealed extensive keratin filaments in both primary epithelial cells and benzo[a]pyrene-induced altered epithelial foci but showed no detectable vimentin filaments. The absence of vimentin expression in these cells was confirmed by two-dimensional gel electrophoresis. In contrast, immunofluorescence staining of the cloned benzo[a]pyrene-transformed rabbit bladder epithelial cell line, RBC-1, revealed a reduction in filamentous keratin concomitant with the expression of vimentin filaments. The epithelial nature of this cell line was established by the observation that cells injected into nude mice formed well-differentiated adenocarcinomas. Frozen sections of such tumors showed strong staining with antikeratins antibodies, but no detectable staining with antivimentin antibodies. These results demonstrated a differential expression of intermediate filament type in cells at different stages of neoplastic progression and in cells maintained in different growth environments. It is apparent that the expression of intermediate filaments throughout neoplastic progression is best studied by use of an in vivo model system in parallel with culture studies.

Animals↗
PubMed1989

Catechol-induced alterations in metabolic activation and binding of enantiomeric and racemic 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes to DNA in mouse skin.

Catechol (1,2-dihydroxybenzene) is a potent co-carcinogen with benzo[a]pyrene (BaP) and with (+/-)-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene (BaP-7,8-diol) in mouse skin. The effects of catechol on the metabolic activation of (+)- and (-)-[3H]BaP-7,8-diols and on epidermal DNA adduct formation of racemic and enantiomeric [3H]BaP-7,8-diols were examined by applying the tritiated diols to mouse skin. The major metabolite of the (+)-[3H]BaP-7,8-diol was the hydrolysis product of (-)-[3H]-7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE). This suggests that a peroxyl radical-mediated pathway is predominantly responsible for the epoxidation of this diol. Formation of (-)-anti[3H]BPDE from (+)-[3H]BaP-7,8-diol was greater than that of (+)-anti-BPDE from (-)-[3H]BaP-7,8-diol. Co-application of catechol with [3H]BaP-7,8-diols inhibited epoxidation of the (+) enantiomer to a greater extent than that of the (-) enantiomer. Catechol decreased the total DNA-binding and the formation of the major adduct with (+)-[3H]BaP-7,8-diol metabolites but catechol had no significant effect on the binding and formation of (+)-anti-[3H]BPDE-deoxyguanosine, the major DNA adduct derived from (-)-[3H]BaP-7,8-diol. Co-administration of catechol with (+/-)-[3H]BaP-7,8-diol increased the ratio of (-)- to (+)-[3H]BaP-7,8-diol derived major DNA adducts in mouse skin suggesting that catechol selectively inhibits certain pathways of metabolic activation of (+/-)-[3H]BaP-7,8-diol. Thus, catechol modifies the tumorigenic activity of (+/-)-BaP-7,8-diol either by alteration of the relative proportion of various hydrocarbon:DNA adducts or by a totally different as yet unexplored mechanisms.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

The site of substitution of the methyl group in the bioalkylation of benzo[a]pyrene.

The major product of the S-adenosyl-L-methionine methyltransferase dependent substitution reaction (bioalkylation) of benzo[a]pyrene in preparations of rat liver cytosol was isolated by solvent extraction and high performance liquid chromatography and its structure determined by GC/MS and NMR. The results indicate that the methyl group is introduced at the center of highest chemical reactivity (or carbon atom at which it is easiest to localize pi electrons) in the anthracene nucleus, giving rise to 6-methylbenzo[a]pyrene. These data, unequivocally demonstrate the site of substitution of the methyl group in the bioalkylation of benzo[a]pyrene.

Alkylation↗
PubMed1990

Systematic modulation by human diet levels of dietary fibre and beef on metabolism and disposition of benzo[a]pyrene in the gastrointestinal tract of Fischer F344 rats.

Groups of male Fischer F344 rats isocalorically consuming cooked, low-fat human diets were given magnetic polyethyleneimine (PEI) microcapsules and [14C]benzo[a]pyrene (B[a]P) by gavage in order to determine the effects of 3-fold changes in levels of dietary fibre non-starch polysaccharide (NSP) and beef protein during gastrointestinal transit on microcapsule trapping and associated parameters. Total 14C trapped by microcapsules during 70 h was decreased by fibre and increased by beef with significant effects (P = 0.03) for dietary mass ratio beef/fibre and compared to controls eating chow. Total B[a]P excreted in faeces and the ration for faeces/urine were increased by fibre and not by beef; the distribution of B[a]P metabolites between liquid and solids of faeces was increased by both factors but there was a net decrease by fibre when allowing for its influence on faecal mass. The distribution of binding between microcapsules and faecal solids was increased significantly by beef, but fibre had no effect when mass-normalized. B[a]P metabolites were extracted from microcapsules by methanol-ammonia and HPLC assay showed mainly benzo[a]pyrene-3,6-dione (B[a]P 3,6-dione), benzo[a]pyrene-1,6-dione (B[a]P 1,6-dione), an unidentified metabolite and substances consistent with tetraols; the beef (P = 0.02) and beef/fibre ratio (P = 0.01) significantly increased B[a]P-1,6-dione trapped and released. In vitro, B[a]P 3,6-semidione (dione reduced form) was bound by PEI microcapsules; the extent of extraction/hydrolysis was much lower than for B[a]P e,6-dione or B[a]P 7,8-diol 9.10-epoxide. Urinary excretion of B[a]P metabolites was decreased by fibre but not by beef. Nearly all the above parameters were different in a control group consuming rat chow. Taken together, these results are consistent with the following conclusions; (i) dietary fibre NSP overall decreases the availability of B[a]P metabolites to contact microcapsules and intestinal mucosa through static bulking rather than absorption; (ii) beef protein increases binding to microcapsules, and enhances free radical activation of B[a]P to its 6-yl radical form; (iii) a systematic set of concordant relationships for B[a]P disposition were found between faecal solids, faecal liquids, microcapsules and urine; and (iv) rat chow produced effects quite unrepresentative of the range for human diets prepared to be in the norm for human use. Endogenous UV-absorbing substances trapped by microcapsules or excreted in urine were also altered by dietary beef levels and were different from those of chow-consuming animals. It is generally concluded that human diets can and should be used in studying carcinogen metabolism and disposition.

Animals↗
PubMed1991

Fluorescence detection of benzo[a]pyrene--DNA adducts in human lung.

Improved techniques are described for the specific identification of benzo[a]pyrene-diolepoxide (BPDE)--DNA adducts in human tissues. Immunoaffinity chromatography, synchronous fluorescence spectroscopy and second-derivative synchronous fluorescence spectroscopy have previously been used to detect BPDE-DNA adducts in human placenta. Here we report how these methods, together with HPLC and the generation of complete fluorescence excitation--emission matrices, have been used to identify unequivocally BPDE-DNA adducts in samples of human lung. BPDE nucleotide adducts were isolated with immunoaffinity chromatography columns bearing antibodies raised against the (+/-)anti-7,8-diol-9,10-epoxide-deoxyguanosine adduct of benzo[a]pyrene. These adducts were hydrolyzed to tetrahydrotetrols and the hydrolysis products subjected to HPLC. The major product isolated by HPLC, benzo[a]-pyrene-7,10/8,9-tetrahydrotetrol, was determined by fluorescence spectroscopy. Using this method, levels of BPDE-DNA adducts in the range of 1-40 in 10(8) nucleotides were measured in 6 out of 25 samples, with a lower detection limit of one adduct in 10(8) nucleotides. The data may also indicate that adduct levels show regional variation in different parts of the same lung.

Adolescent↗
PubMed1992

NADPH-supported and arachidonic acid-supported metabolism of the enantiomers of trans-7,8-dihydrobenzo[a]pyrene-7,8-diol by human liver microsomal samples.

Using a new sensitive reverse-phase HPLC assay relying on UV detection at 344 nm, the capacity of 18 human liver microsomal samples to support NADPH-dependent, cytochrome P450-mediated oxidation and arachidonic acid-dependent oxidation of the enantiomers of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-DHD) was determined. The (-)-7R,8R-enantiomer, the preferred substrate of cytochrome P450, formed 94% diolepoxide 2 (anti-isomer; 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene) measured as derived alcohols, and the (+)-7S,8S-enantiomer formed 67% diolepoxide 1 (syn-isomer; 7S,8R-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). Arachidonic acid-supported oxidations gave approximately 70% diolepoxide 2 from each enantiomer. The involvement of different sets of cytochrome P450 isozymes was supported by incubations in the presence of alpha-naphthoflavone (alpha-NF) (50 microM) and correlation studies. In the absence of alpha-NF, a positive correlation was found between the metabolism of the (-)-enantiomer but not the (+)-isomer of B[a]P-7,8,-DHD and the relative content of P450IA2. In the presence of alpha-NF, the P450IIIA3/4 content correlated positively with the metabolism of both the (+)-enantiomer and the (-)-enantiomer. Gestodene (100 microM) inhibited the alpha-NF-stimulated metabolism, confirming the involvement of cytochrome P450IIIA3/4. No difference was found between the extent of arachidonic acid-supported, peroxyl radical-mediated metabolism of the (+)- and (-)-enantiomers of B[a]P-7,8-DHD. The metabolism was almost completely abolished by 2 microM butylatedhydroxyanisole and 100 microM nordihydroguaiaretic acid, confirming the free radical nature of the reaction.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1994

Validation of a new fluorometric assay for benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells: comparisons with 32P-postlabeling and ELISA.

A new fluorometric assay was validated for quantification of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts in white blood cells (WBC) from humans exposed to polycyclic aromatic hydrocarbons (PAH). This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene derived from acid hydrolysis of BPDE-DNA, and can measure 1 BPDE adduct per 10(8) unmodified nucleotides. The quantity of WBC DNA required depends on the modification level and varies between 5 and 500 micrograms. The assay was applied to seven WBC DNA samples from lung cancer patients, six of whom were heavy smokers, and to three WBC DNA samples from healthy subjects employed in an aluminum production plant. High levels of BPDE-DNA adducts, ranging from 62 to 533 adducts/10(8) nucleotides were found in six out of seven DNA samples from the lung cancer patients. In WBC DNA from healthy persons BPDE-DNA adducts were detected only in two non-smokers, but at a much lower level than in lung cancer patients (4-10 adducts/10(8) nucleotides). Using coded WBC DNA samples, BPDE-DNA adduct levels measured by fluorometry of the B[a]P-tetrols, were compared with the results obtained by 32P-postlabeling (nuclease P1 enrichment) and ELISA measurements. A good correlation and proportionality was found between the levels of BPDE-DNA adducts measured by fluorometry and 32P-postlabeling (r = 0.95, P < 0.001, n = 8). The correlation between fluorometry and ELISA was much lower and not significant (r = 0.61, P = 0.1, n = 6). Moreover, the ELISA grossly overestimated BPDE-DNA adduct levels measured by the other two methods. The results demonstrate that the highly sensitive and specific fluorometric assay is suitable for measuring BPDE-DNA adducts in WBC from humans exposed to benzo[a]pyrene.

Benzo(a)pyrene↗
PubMed1994

Cyclopenta[cd]pyrene-induced tumorigenicity, Ki-ras codon 12 mutations and DNA adducts in strain A/J mouse lung.

Cyclopenta[cd]pyrene (CPP) is a ubiquitous cyclopenta-fused polycyclic aromatic hydrocarbon. CPP is highly genotoxic in bacterial and mammalian systems inducing gene mutations, sister chromatid exchanges and morphological transformation. CPP is a mouse skin carcinogen, a mouse skin tumor initiator and induces pulmonary tumors in newborn mice. We have examined the tumorigenic activity of CPP in strain A/J mice, have determined the formation and persistence of CPP-induced DNA adducts in lung tissue, and analyzed the mutational spectrum in the Ki-ras oncogene from CPP-induced tumors. CPP dissolved in tricaprylin was administered by i.p. injection to male A/J mice (20 mice/dose) at 0, 10, 50, 100 and 200 mg/kg. Animals were killed 8 months later and the lungs removed, fixed, and surface adenomas enumerated. CPP proved to be highly tumorigenic in A/J mice in terms of inducing lung adenomas. The observed tumor multiplicities (lung adenomas/mouse) were: 97.7 +/- 28.7 at 200 mg/kg, 32.8 +/- 15.4 at 100 mg/kg, 4.63 +/- 2.11 at 50 mg/kg and 0.58 +/- 0.82 at 10 mg/kg. Tricaprylin-treated controls produced 0.60 +/- 0.58 lung adenomas/mouse. Groups of mice treated under the same dosing conditions as those in the tumor studies were killed 1, 3, 7, 14 and 21 days after treatment. The lungs were removed, and the DNA was subjected to DNA adduct analysis by the 32P-postlabeling method. Total CPP-DNA adducts in mouse lung peaked at day 3 with 5870 amol CPP adducts/micrograms DNA after a single dose of 200 mg/kg. DNA adduct levels decreased to 1800 amol CPP adducts/micrograms DNA at day 21. Qualitative DNA adduct analysis revealed four major adducts and one minor adduct. Co-chromatography of the lung DNA from CPP-treated mice with calf thymus DNA treated with CPP-3,4-oxide indicated that all DNA adducts were oxide derived and comparison with CPP-3,4-oxide-treated polydeoxyguanylic acid suggests that almost all of these adducts are CPP-3,4-oxide-2'-deoxyguanosine adducts. Ki-ras codon 12 mutation analysis of the DNA from tumors taken from the 100 and 200 mg/kg CPP dose groups demonstrated the following patterns: GGT-->CGT (50%); GGT-->GTT (15%); GGT-->TGT (25%); GGT-->GAT (10%). We conclude that CPP is highly tumorigenic in the A/J mouse lung adenoma model, being five times more active than benzo[a]pyrene. This is unlike the result of CPP as a mouse skin tumorigen or tumor initiator in which CPP is considerably less potent than benzo[a]pyrene.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoma↗
PubMed1994

Mutagenic selectivity at the HPRT locus in V-79 cells: comparison of mutations caused by bay-region benzo[a]pyrene 7,8-diol-9,-10-epoxide enantiomers with high and low carcinogenic activity.

Earlier studies from our laboratories characterized the mutation profile of the optically active (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-BPDE--the ultimate carcinogenic metabolite of benzo[a]pyrene] in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene of Chinese hamster V-79 cells. In the present study, we evaluated the mutation profile of (-)-7S,8R-dihydroxy-9R, 10S-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-BPDE-a weakly carcinogenic or inactive enantiomer] and compared its mutation profile with that of (+)-BPDE. In both diol epoxide enantiomers, the benzylic 7-hydroxy group and epoxide oxygen are trans. The mutation frequency for V-79 cells treated with DMSO vehicle or with a low, non-cytotoxic dose (0.5 microM) or a high cytotoxic dose (2.0 microM) of (-)-BPDE was 1, 25 or 185 8-azaguanine-resistant colonies/10(5) survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the HPRT gene was amplified by the polymerase chain reaction and sequenced. Altogether, 92 (-)-BPDE-induced mutant clones were examined. At both doses, base substitutions were the most prevalent mutations observed (present in approximately 7% of the mutant clones), followed by exon deletions (present in approximately 22% of the mutant clones) and frame shift mutations (present in approximately 6% of the mutant clones) in the cDNAs analyzed. At the high cytotoxic dose, 5 out of 36 base substitutions occurred at AT base pairs (14%) and 31 at GC base pairs (86%). At the low, non-cytotoxic dose, 7 out of 34 base substitutions were at AT base pairs (21%) and 27 were at GC base pairs (79%). Although there was a trend towards an increase in the proportion of mutations at AT base pairs when the dose of (-)-BPDE was decreased, this trend was not statistically significant. The data also indicated no dose-dependent differences in the kinds of base substitutions or exon deletions in cDNAs induced by (-)-BPDE. Ninety-one per cent of the (-)-BPDE-induced mutations that occurred at guanine were on the non-transcribed strand of DNA and 9% were on the transcribed strand. In contrast to these results, 50% of the (-)-BPDE-induced mutations that occurred at adenine were on the transcribed strand and 50% on the non-transcribed strand.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Tumorigenicity in newborn mice of fjord region and other sterically hindered diol epoxides of benzo[g]chrysene, dibenzo[a,l]pyrene (dibenzo[def,p]chrysene), 4H-cyclopenta[def]chrysene and fluoranthene.

Diol epoxides of benzo[g]chrysene, dibenzo[a,l]pyrene (dibenzo[def,p]chrysene), 4H-cyclopenta[def]chrysene and fluoranthene were tested for tumorigenicity in newborn mice. The compounds tested were racemic trans-11,12-dihydroxy-anti-13,14-epoxy-11,12,13, 14-tetrahydrobenzo[g]-chrysene (BgCDE), trans-11, 12-dihydroxy-anti-13,14-epoxy-11,12,13,14-tetrahydrodibenzo [a,l]pyrene (DB[a,l]PDE), trans-1,2-dihydroxy-anti-3, 3a-epoxy,1,2,3,3a-tetrahydro-4H-cyclopenta[def]chrysene (C[def]C-1,3a-DE), trans-6,7-dihydroxy-anti-8,9-epoxy-10b,1, 2,3-tetrahydrofluoranthene (FDE). BgCDE and DB[a,l]PDE are fjord region diol epoxides and their tumorigenic activities were compared to those of trans-3,4-dihydroxy-anti-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPDE), a fjord region diol epoxide with known high tumorigenicity and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]-pyrene (BPDE), a highly tumorigenic bay region diol epoxide. The protocol called for testing of each compound at a total dose of 25 nmol per mouse, administered on days 1, 7 and 15 of life, with killing at age 35 weeks. BgCDE had similar activity as BcPDE for induction of lung tumors and was more active than BcPDE for induction of liver tumors in male mice. Both compounds were significantly more tumorigenic than BPDE. DB[a,l]PDE was highly toxic. All mice died within 1 week of the first dose. It was then tested in a second study using total doses of 5 and 1 nmol per mouse. Only the first dose of the intended 5 nmol total dose was given due to toxicity. The full course of doses with a total of 1 nmol per mouse was administered; DB[a,l]PDE induced a significant incidence and multiplicity of lung tumors and, in male mice, liver tumors at both doses. These results demonstrate that fjord diol epoxides are highly active tumorigens in newborn mice, with activity greater than that of the most active unsubstituted bay region diol epoxide, BPDE. C[def]C-1-3a-DE and C[def]-6-9-DE were compared to trans-1,2-dihydroxy-anti-3, 4-epoxy-1,2,3,4-tetrahydrochrysene (CDE), at a total dose of 500 nmol per mouse. FDE was also tested at this dose. The most active compound among the chrysene derivatives was C[def]C-1-3a-DE, followed by C[def]C-6-9-DE and CDE. C[def]C-1-3a-DE has a sterically constrained bay region, in which the benzylic carbon of the tri-substituted epoxide ring is part of a fused ring system. This feature is also present in FDE, which and considerable tumorigenic activity, greater than that of CDE in lung and greater than any of the chrysene derivatives in liver.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Influences of chronic cholangiohepatitis and cholestasis on hepatic metabolism of benzo[a]pyrene in white suckers (Catostomus commersoni) from industrially polluted areas of Lake Ontario.

White suckers from polluted regions of western Lake Ontario have an increased prevalence of cholangiocellular and hepatocellular and hepatocellular neoplasms associated with an idiopathic chronic cholangiohepatitis. We examined the hypothesis that bile duct obstructions and cholestasis in these fish might increase the susceptibility of liver to administered benzo[a]pyrene (B[a]P). Cytosolic glutathione S-transferase (GST) activity (CDNB) was reduced in obstructed liver to 45% of activity in adjacent unobstructed liver. At micromolar concentrations, chenodeoxycholic acid, deoxycholic acid, bilirubin and haematin each inhibited GST activity of hepatic cytosolic and S-hexylglutatione-affinity-purified GST preparations from unobstructed liver. Liver cytosol and affinity-purified hepatic GSTs from normal white sucker liver reduced DNA binding of 3H-benzo[a]-pyrene-7,8-diol-9,10-epoxide (3H-BPDE) after preincubation in vitro in the presence of 5 mM GSH. Under these conditions, cytosol from adjacent unobstructed liver had a moderately stronger protective activity against DNA binding by BPDE (16.4 +/- 1.3 pmol BPDE/mg DNA) than did cytosol from obstructed liver (20.6 +/- 1.6 pmol BPDE/mg DNA). Suckers with obstructed livers identified by laparotomy were orally administered 3H-benzo[a]pyrene (3H-B[a]P) (0.2 mmol/kg) or unlabelled B[a]P (2.0 mg/kg) and the level of B[a]P macromolecular binding was analyzed in liver tissue by liquid scintillation counting and by immunohistochemistry with antibodies to BPDE-DNA adducts. Covalent binding of 3H-B[a]P to hepatic protein was 30% less in adjacent unobstructed liver compared to obstructed liver; however, there was no significant difference in the levels of 3H-B[a]P bound to DNA in the obstructed lobes compared with non-obstructed adjacent liver. These studies demonstrate that some endogenous non-substrate ligands that accumulate during cholestasis can reduce hepatic GST activity in white suckers. While these changes are insufficient to influence total 3H-B[a]P-DNA adducts in obstructed liver, the preferential localization of BPDE-DNA adducts in GST-deficient hyperplastic biliary tracts suggests that cholangiohepatitis might increase susceptibility to cholangiolar neoplasia in fish exposed to genotoxic polycyclic aromatic hydrocarbons.

Animals↗
PubMed1996

Hemoglobin adducts of benzo[a]pyrene diolepoxide in newspaper vendors: association with traffic exhaust.

Benzo[a]pyrene diol epoxide adducts with hemoglobin (Hb) were measured to detect human exposure to environmental benzo[a]pyrene from traffic exhaust. Benzo[a]pyrene tetrahydrotetrols (BPTs) released from Hb after acid hydrolysis were quantitated by gas chromatography-mass spectrometry after immunoaffinity chromatography. Fifty three newspaper vendors were enrolled. The median adduct concentration was 0.3 fmol BPTs/mg Hb in high density traffic-exposed vendors and < or = 0.1 fmol BPTs/mg Hb in those exposed to low density traffic; the difference was not significant (P = 0.09). Among non-smokers, adducts were detectable in 60% of high exposure subjects (median 0.3 fmol BPTs/mg Hb) and in 28% of those with low exposure (median < or = 0.1 fmol/mg Hb). This difference was significant (P = 0.02). In low exposure smokers the median of adducts was 0.26 fmol BPTs/mg Hb, while in low exposure non-smokers it was < or = 0.1 fmol BPTs/mg Hb (P = 0.08, not significant). Adduct concentration was no different for low and high density traffic-exposed smokers (P = 0.82). The data indicate a significant difference in adduct concentration related to traffic exhaust exposure among non-smokers.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system.

Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogen for benzo[a]pyrene (BP). We have demonstrated previously that the interaction between sulfite, the physiological form of sulfur dioxide, and (+/-) -7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenic form of BP, results in an enhanced mutagenic effect in Salmonella typhimurium strains TA98 and TA100. We report here that this same co-mutagenic effect of sulfite occurs in a mammalian cell line. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE, a concentration on the linear portion of the dose-response, resulted in a four-fold increase in mutations at the hprt locus relative to the spontaneous rate. When V79 cells were exposed to 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE, the mutation rate increased by 73% and 210%, respectively, over that elicited by anti-BPDE alone. Sulfite itself was moderately cytotoxic, but caused no increase in mutation over the spontaneous rate. Characterization of the dose- and time-dependance of this enhancement of diol epoxide mutagenicity by sulfite closely resembled the effects seen previously in the bacterial system. In particular, enhancement by sulfite was evident when sulfite was added to the cells between 60 min and 1 min prior to the addition of the diol epoxide. Concurrent addition of sulfite and the diol epoxide attenuated the enhancement, and the effect was lost altogether when sulfite was added 10 min after the diol epoxide. The specificity of this effect of sulfite was shown by comparison with sulfate, which at concentrations of either 1 or 10 mM exhibited modest cytotoxicity, but neither was directly mutagenic nor able to enhance the mutagenic effect of anti-BPDE. Binding studies with labeled anti-BPDE showed that the addition of 10 mM sulfite increased binding of anti-BPDE to DNA by over 43%, corresponding to the observed increase in mutant frequency. Interestingly, this difference in level of DNA modification was not apparent after 30 min to 2 h exposures, but only emerged at the 4 h time point. The 4 h point was routinely used for all mutagenicity studies. Binding of anti-BPDE-derived materials to cellular RNA was not altered by 10 mM sulfite. The emergence of increased DNA modification at the latest time point suggests either a more prolonged period of active DNA binding than would occur with diol epoxide, or a difference in the ability to recognize and clear specific DNA adducts. Both possibilities are discussed in regard to the observed formation of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a] pyrene-10c-sulfonate (BPT-10-sulfonate) in those incubations. BPT-10-sulfonate is a relatively stable BP derivative which retains the ability to covalently modify DNA. The role of this derivative in the enhancement of diol epoxide mutagenicity by sulfite is strongly suggested by these data.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Evidence of anti-benzo[a]pyrene diolepoxide-DNA adduct formation in human colon mucosa.

As risk factors for colorectal cancer include consumption of foods potentially contaminated with polycyclic aromatic hydrocarbons (PAHs), 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 human colon mucosa samples was quantified by a sensitive and specific HPLC/fluorescence method (Alexandrov et al., Cancer Res. 51, 6248-6253, 1992). (+)-anti-BPDE-DNA adducts were detected in four out of seven colon mucosa samples but not in any of 11 human pancreas samples from smokers and non-smokers. Adduct levels in human colon varied between 0.2 and 1.0 (+)-anti-BPDE-DNA adducts/10(8) nucleotides. Our results provide evidence that: (i) the DNA in human colon cells can be damaged by benzo[a]pyrene, possibly derived from diet and/or tobacco smoke; (ii) DNA adduct formation in human colon epithelium proceeds via the diol epoxide pathway; (iii) benzo[a]pyrene and other PAHs could play a role in the etiology of human colorectal cancer.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Benzo[a]pyrene coated ferric oxide and aluminum oxide particles: uptake, metabolism and DNA binding in hamster pulmonary alveolar macrophages and tracheal epithelial cells in vitro.

Ferric oxide (Fe2O3) and aluminum oxide (Al2O3) particles are widely encountered in occupational settings. Benzo[a]pyrene (B[a]P), a well-characterized environmental carcinogen, is frequently adsorbed onto particles. It has been shown that B[a]P-coated Fe2O3 particles (B[a]P-Fe2O3) significantly increased lung tumors in the hamster in contrast to B[a]P-coated Al2O3 (B[a]P-Al2O3) or B[a]P alone. In order to determine the genotoxic effects of these particles on the metabolism of B[a]P, pulmonary alveolar macrophages (AM) from male Syrian golden hamsters were incubated with 5 microg (19.8 nmol) B[a]P-coated respirable size (99% < 5 microm) Fe2O3 and Al2O3 particles with loads from 0.5 to 2.0 mg. Intracellular uptake of B[a]P by AM at 24 h was higher with B[a]P-Fe2O3 than that of B[a]P alone (P < 0.05) or B[a]P-Al2O3 (P < 0.05). Total B[a]P metabolism was significantly greater in AM exposed to B[a]P-coated Fe2O3 at 1.0 and 1.5 mg than in the AM exposed to B[a]p-al2O3 (0.5, 1.0 and 1.5 mg) (P < 0.05) or B[a]P alone (P < 0.05). Similar significant differences for Fe2O3 relative to Al2O3 and B[a]P alone were also apparent for total dihydrodiols, quinones and phenolic metabolites. Co-administration of 5 microg alpha-naphthoflavone (alpha-NF, an inhibitor of cytochrome P-4501A1 and P-4501A2) and 10(-3) M cyclohexene oxide (CO, an inhibitor of epoxide hydrolase) significantly reduced B[a]P metabolism in B[a]P-Fe2O3 (P < 0.05) and B[a]P-Al2O3 (P < 0.05) treated groups relative to B[a]P alone. AM were co-cultured with hamster tracheal epithelial cells (HTE) and treated as described above for metabolism studies to assess the DNA binding of B[a]P metabolites in the target cells, using 32P-postlabeling techniques. Two adducts were observed that had chromatographic behavior similar to 7R,8S,9S-trihydroxy-10R-(N2-deoxyguanosyl-3'-phosphate)-7,8,9,10-t etrahydrobenzo[a]pyrene [(+)-anti-BPDE-dG, adduct 1, major adduct representing 70-80% of total adducts] and 7S,8R,9R-trihydroxy-10S-(N2-deoxyguanosyl-3'-phosphate)-7,8,9,10-t etrahydrobenzo[a]pyrene [(-)-anti-BPDE-dG, adduct 2, representing 20-30% of total adducts]. B[a]P-Fe2O3 treatment enhanced the levels of the two B[a]P-DNA adducts in the HTE compared with B[a]P-Al2O3 (P < 0.05) or B[a]P alone. The inhibitors alphaNF and CO significantly reduced total adduct levels in the HTE (P < 0.05) in the B[a]P and B[a]P-Fe2O3 treatments as well as adduct 1 and adduct 2 levels. Our data suggest that the cocarcinogenic effect of B[a]P-Fe2O3 relative to B[a]P-coated Al2O3 can be due to: (i) the enhancement of B[a]P metabolism in AM by Fe2O3 associated with the increased uptake of B[a]P; and (ii) augmentation of DNA adduct formation in epithelial cells.

Aluminum Oxide↗