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PubMed1983

Benzo[a]pyrene induction of extrachromosomal viral DNA synthesis in rat cells transformed by polyoma virus.

The effect of the carcinogen benzo[a]pyrene (BP) on the production of specific extrachromosomal DNAs has been studied in transformed rat cells containing integrated polyoma virus DNA. Exposure of cells previously transformed by the polyoma virus mutant ts-a, which codes for a temperature sensitive T antigen, to a non-toxic dose of BP (0.25 micrograms/ml) enhanced the production of free polyoma DNA at the population level. This occurred at 33 degrees C (the permissive temperature) but not at 39 degrees C, indicating that the BP effect was dependent on the function of the T antigen of polyoma virus. In the same cell system, BP did not induce the production of extrachromosomal copies of two endogenous rat retrovirus genomes, the 30S RNA virus and rat leukemia virus. Thus, the effects on integrated polyoma DNA are quite specific and do not reflect a generalized increase in asynchronous DNA replication and excision. Further studies utilizing a carcinogenic metabolite of BP, benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide (anti) (BPDE), on a rat cell line transformed by wild type (WT) polyoma virus demonstrate that the enhancement of polyoma DNA synthesis persists for at least 5 days after a single exposure to BPDE, despite the rapid decay of this compound. In addition, enhanced synthesis of polyoma DNA can be induced by fusion of normal rat fibroblasts previously exposed to BPDE with polyoma transformed rat fibroblasts not exposed to BPDE. It appears, therefore, that the effects we have observed are not due solely to direct carcinogen damage at the level of the integrated polyoma virus DNA, but may instead involve the induction of cellular factor(s) that act indirectly to enhance asynchronous replication of polyoma DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1984

Quantitative significance of glutathione and glutathione-S-transferase in regulating benzo[a]pyrene anti diol-epoxide level in reconstituted C3H/10T1/2 cell lysates, and comparison to rat liver.

Induced 10T1/2 cell microsomes were independently reconstituted with [3H]benzo[a]pyrene (BP) and glutathione (GSH) or purified GSH-transferases. Levels of the primary BP anti 7,8-dihydrodiol 9,10-epoxide (r-7,t-8 dihydroxy-t-9,10-oxy-7,8,9,10 tetrahydrobenzo[a]pyrene) hydrolysis product, 7,10/8,9-tetrol, were measured in incubation extracts, enabling us to monitor the level of free anti diol-epoxide in incubations and to determine the independent effects of GSH or GSH-transferases upon it. GSH alone had no effect on anti diol-epoxide levels over the concentration range tested (0-4.0 mM), however, the addition of purified GSH-transferase from rat liver resulted in a dose-dependent conjugation of anti diol-epoxide as well as 9,10-epoxide and 7,8-epoxide with 50% conjugation occurring at 0.036, 0.039 and 0.17 units GSH-transferase/ml, respectively. Free anti diol-epoxide was reduced by greater than 95% when we reconstituted with the GSH-transferase concentration which we measured in 10T1/2 cells (0.15-0.27 units/ml cell cytosol); this GSH-transferase concentration represents only 6% of that found in rat liver. The results suggest that in both 10T1/2 cells and rat hepatocytes GSH-transferase catalyzed GSH conjugation is quantitatively significant in determining the intracellular level of anti diol-epoxide.

Animals↗
PubMed1984

Modifying effect of dietary fat on benzo[a]pyrene-induced respiratory tract tumours in hamsters.

Groups of 40 Syrian golden hamsters of both sexes were fed diets containing either 5% fat (control), 20% saturated fat (beef tallow) or 20% unsaturated fat (sunflower seed oil) from weaning and during the whole experimental period (up to 656 days). Respiratory tract tumours were induced by intratracheal instillation of benzo[a]pyrene attached to ferric oxide and suspended in saline. Mortality was slightly, but not statistically significantly higher in the high fat groups than in the low fat control group. Microscopic examination of the respiratory tract revealed an increased number of tumour bearing animals, an increased multiplicity of respiratory tract tumours and an increased total number of respiratory tract tumours in animals fed high fat diets. The tumour enhancing effect of fat was most pronounced in the high unsaturated fat group. Especially epidermoid papillomas, epidermoid carcinomas and combined epidermoid and adenocarcinomas contributed to the observed differences in tumour response amongst groups. It was concluded that dietary fat enhances benzo[a]pyrene-induced respiratory tract carcinogenesis in hamsters.

Adenocarcinoma↗
PubMed1984

Prostaglandin H synthase-dependent co-oxygenation of (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in hamster trachea and human bronchus explants.

The role of prostaglandin H synthase (PHS) in the metabolism of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) has been examined in short-term explant cultures of hamster and human tracheobronchial tissues. Labeled BP-7,8-diol was incubated with the explants in the presence and absence of the PHS substrate arachidonic acid (20:4) and the PHS inhibitor indomethacin. The addition of 10 microM to 200 microM 20:4 to incubations of hamster trachea with 5 microM BP-7,8-diol caused significant increases in the formation of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (anti-BPDE). These increases were not seen when 1 microM or 20 microM BP-7,8-diol was employed. The stimulation of anti-BPDE formation was observed after incubations of from 1 to 48 h. This stimulation was inhibited to the basal level by 20 microM indomethacin, supporting the role of PHS in the response. No effect of 20:4 was seen on the uptake of BP-7,8-diol by the tracheas or on the formation of water-soluble metabolites. Significant increases in covalent binding of BP-7,8-diol metabolites to DNA of the tracheal epithelium were also elicited by the addition of 20:4, however these increases were not well correlated quantitatively with the increases in anti-BPDE formation. H.p.l.c. profiles of deoxynucleoside adducts from basal and 20:4-stimulated incubations were qualitatively identical. Far greater variability of metabolism was seen in human bronchus explants, but 20:4-dependent increases in anti-BPDE formation could be demonstrated in those tissues as well. Inhibition of this stimulation by indomethacin was either absent or incomplete. This variation in the effect of indomethacin was explained by the examination of the products of 20:4 metabolism by the two tissues. Hamster trachea produced almost exclusively PHS metabolites whereas human bronchus yielded predominantly products of lipoxygenases, enzymes insensitive to indomethacin. In conclusion, this study indicates that co-oxygenation of chemical carcinogens can occur in hamster and human tracheobronchial tissues. The concentration-dependence observed with BP-7,8-diol, however, suggests that this pathway is of minor importance in the activation of BP in these tissues.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1984

Modifying effect of vitamin A on benzo[a]pyrene-induced respiratory tract tumours in hamsters.

Groups of 40 hamsters of each sex were fed natural ingredient diets containing 600, 4000 or 100 000 IU vitamin A/kg. After an adaptation period of 170 days on the diets, respiratory tract tumours were induced by intratracheal instillation of benzo[a]pyrene + ferric oxide suspended in saline. Mortality was statistically significantly increased in the high dose group as compared with the mid (control) and low dose group. Microscopy of the respiratory tract did not reveal any significant differences in tumour response between the various dose groups. However, the incidence of preneoplastic respiratory tract lesions was distinctly inversely correlated with the oral vitamin A dose. The serum retinol values were similar in the various dose groups, without any indication of a dose related increase. It was concluded that under the experimental conditions used vitamin A did not influence benzo[a]pyrene-induced respiratory tract cancer in hamsters.

Animals↗
PubMed1984

Spectroscopic studies of DNA complexes formed after reaction with anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide enantiomers of different carcinogenic potency.

Light absorption, fluorescence and linear dichroism (l.d.) spectroscopy and fluorescence lifetime measurements reveal characteristic differences that arise from structural differences between the DNA complexes with the optical enantiomers (+)- and (-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxides (BPDE), a strong and a weak carcinogen, respectively. Both types of complexes appear heterogeneous but can be described as composed of two major complex types I and II, in different proportions. Like previously observed for DNA modified by racemic anti-BPDE, the only distinguishable spectral component of (+)-anti-BPDE-DNA is the type II complex, whereas the (-)-anti-BPDE-DNA is a mixture of both types I and II complexes. The type I complex is characterized by negative I.d., a light absorption and excitation spectrum maximum (above 300 nm) at 354 nm and strong fluorescence quenching in native DNA, properties expected for an intercalation complex in the classical sense. The type II complex on the other hand is characterized by positive I.d., a light absorption and excitation spectrum maximum (above 300 nm) at 345 nm, and moderate fluorescence quenching in native DNA, properties not consistent with intercalation geometry. Rather, the BPDE chromophore forms less than 55 degree angle with the mean direction of the helix axis. Its interaction with the DNA bases seems to be less than in complex I, and is highly sensitive to Ag+ ions. The type II complex may be associated with local obstruction of base-pairing properties of native DNA. Since DNA-binding of chemical carcinogens is considered crucial for tumour initiation it follows that the unique properties of the type II BPDE-DNA complex may be of fundamental importance in benzo[a]pyrene carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Glutathione conjugation of the carcinogenic and mutagenic electrophile (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetra hydrobenzo[a]pyrene catalyzed by purified rat liver glutathione transferases.

The kinetics of the enzyme-catalyzed conjugation of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]-pyrene [(+/-)-anti-BPDE] with glutathione (GSH) by the following purified soluble rat liver GSH transferases: 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4 have been studied. When BPDE concentration was varied while GSH concentration remained constant (1 mM), linear Lineweaver-Burk plots were obtained: maximum rates of conjugation mediated by GSH transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4 were 105, 72, 83, 35, 179 and 357 nmol/min/mg protein, respectively. When GSH concentration was varied while BPDE concentration remained constant (40 microM), biphasic Lineweaver-Burk plots were obtained in each case with a break point of 0.2 mM GSH below which the affinity of these enzymes for GSH was apparently greater. These results are discussed with respect to the detoxication of benzo[a]pyrene (BP) in vivo.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Metabolism of benzo[a]pyrene by cultured rat and human buccal mucosa cells.

Primary cultures of epithelial and fibroblast cells derived from human oral mucosa were studied for the ability to activate a tobacco smoke carcinogen, benzo[a]pyrene (BP). The cells were exposed to benzo[a]pyrene for 18 h. The cell-free medium was extracted with ethylacetate/acetone, and high-pressure liquid chromatography analysis of this fraction revealed that BP tetrols and diols were the major metabolites formed by both epithelial and fibroblast cells. However, the epithelial cells had a much higher rate of biotransformation of BP as measured by binding to cellular DNA. The mean binding level to human buccal mucosal DNA was among the highest observed in stratified human epithelia. The major BP-DNA adduct was formed by the reaction of the 'bay-region' BP diolepoxide with the exocyclic 2-amino group in guanine. In contrast to human cells, BP phenols and BP 9,10-diol were the major metabolites produced by primary epithelial and fibroblast cells derived from rat buccal mucosa. The DNA binding levels of BP in the two rat cell types were identical, and the binding level was several-fold lower than in the human epithelial cells. When an established rat tongue epithelial cell line (RTE 2) was treated with polycyclic aromatic hydrocarbons--BP and 7,12-dimethylbenz[a]-anthracene--a slight toxic effect was observed. Our results indicate that primary cultures of oral mucosa are able to metabolize BP into its ultimate carcinogenic form at a rate similar to or higher than other potential target tissues for BP-induced carcinogenesis.

Animals↗
PubMed1985

Benzo[a]pyrene-DNA adduct formation in target and activator cells in a Wistar rat embryo cell-mediated V79 cell mutation assay.

To determine the relationship of the benzo[a]pyrene (BaP)-DNA adducts formed in the activator cells of a cell-mediated mutation assay to the adducts formed in the target cells and to mutation induction, irradiated second passage Wistar rat embryo (WRE) cells and V79 Chinese hamster lung cells were exposed to [3H]BaP for 5, 24 and 48 h under the conditions of a cell-mediated mutation assay. The V79 target cells were separated from the WRE activator cells by an immunoseparation procedure; the resulting V79 cell pellet contained less than 7% WRE cells. The percentage of the BaP-DNA adducts containing cis vicinal hydroxyl groups and the h.p.l.c profile of individual adducts in the V79 target cells were similar to those of the WRE activator cells for each time point. The transfer of reactive BaP metabolites from the activator cells to the target cell DNA was detectable after only 5 h of exposure to BaP, however, exposure for this length of time did not result in significant mutation induction. The (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE)-deoxyguanosine (dGuo) adduct was essentially absent after 5 h of exposure, but the amount of this adduct increased at longer times of exposure as did the mutation frequency. The complex mixture of BaP-DNA adducts formed in the WRE cells was also present in the V79 cells after all three times of exposure to BaP, a result which demonstrates the value of this cell-mediated mutation assay for investigating the role of species-specific differences in the activation of BaP. The correlation between the increase in mutation induction and the relative amount of the (+)-anti-BaPDE-dGuo adduct present in V79 cell DNA suggests the importance of this adduct in mutation induction by BaP.

Animals↗
PubMed1986

Mutagenicity of the mononitrobenzo[a]pyrenes in Chinese hamster ovary cells mediated by rat hepatocytes or liver S9.

Previous studies have shown that 1- and 3-nitrobenzo[a]pyrene (NBaP) were mutagenic in the Salmonella reversion assay without exogenous activation and that 1-, 3- and 6-NBaP were mutagenic in the presence of hepatocytes or liver homogenate (S9). In the present study, 1-, 3- and 6-NBaP were tested for mutagenicity in Chinese hamster ovary (CHO) cells under activation conditions similar to those used in the bacterial studies. None of the NBaPs was mutagenic without exogenous activation and none was mutagenically activated by hepatocytes from unpretreated rats or rats pretreated with Aroclor 1254 or 3-methylcholanthrene. Benzo-[a]pyrene (BaP), the parent compound, induced a strong mutagenic response under all hepatocyte mediation conditions. The NBaPs did produce positive mutagenic responses with S9 activation (3- = 1- greater than 6-NBaP), but these moderate responses were less than those of BaP. The difference between the bacterial and CHO results under the variety of activation conditions suggests the importance of the endogenous metabolism of the target cell as well as the source and the type of exogenous metabolic activation.

Animals↗
PubMed1988

Quantitation of protein adducts as a marker of genotoxic exposure: immunologic detection of benzo[a]pyrene--globin adducts in mice.

Immunologic methods have been developed for the determination of benzo[a]pyrene (BP)-protein adducts and validated in animals treated with [3H]BP. A previously developed antibody, 8E11, which recognizes 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-I)-modified DNA or protein as well as BPDE-I-tetraols, was used. The sensitivity of the assay was increased by enzymatic digestion of the modified protein with insoluble protease into peptides and amino acids before analysis. In a competitive enzyme-linked immunosorbent assay (ELISA) with digested BPDE-I-modified bovine serum albumin, 50% inhibition occurred at 400 fmol of adduct compared to 1450 fmol for the non-digested albumin. Analysis of globin (Gb) isolated from animals treated in vivo with 0.3-3 mg [3H]BP indicated that the ELISA could detect 90-100% of the adducts determined by radioactivity. Levels of adducts in lung and liver DNA and serum albumin were correlated with the levels of Gb adducts. Of the total radioactivity associated with hemoglobin, only less than or equal to 10% was from Gb while approximately 80% was from the heme fraction and the remainder from free BP metabolites. Significant cross-reactivity of antibody 8E11 was found with several BP-diols and phenols, suggesting that the immunoassay will not only be specific for BPDE-I adducts but will also detect adducts of other BP metabolites as well as other aromatic hydrocarbon diol epoxides. An immunoaffinity column of antibody 8E11 coupled to Sepharose 4B was used to isolate modified peptides from the digested Gb. About 65% of the applied radioactivity was retained on the column. Between 1 and 2 mg of non-modified digested Gb could be added to the sample without interfering with binding of adducts. Protein digestion and immunoaffinity chromatography should be useful for the measurement of protein adducts in biomonitoring studies.

Animals↗
PubMed1988

Suppression of keratinocyte differentiation in SSC-9 human squamous carcinoma cells by benzo[a]pyrene, 12-O-tetradecanoylphorbol-13-acetate and hydroxyurea.

In the human squamous carcinoma cell line SCC-9, the expression of two markers of keratinocyte differentiation, involucrin and transglutaminase, was greatly stimulated when growing cultures reached confluence. However, the two markers differed temporally in their induction, with transglutaminase reaching maximal levels shortly after confluence and involucrin a week later. If replication was arrested with hydroxyurea prior to confluence, transglutaminase induction occurred within several days but involucrin levels were completely suppressed. Such a striking degree of uncoupling also resulted when the cells were treated with polycyclic aromatic hydrocarbons such as benzo[a]pyrene but not with 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, or with pyrene. Chronic treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed expression of both transglutaminase and involucrin. However, suppression of the latter (evident in greatly reduced mRNA levels) was considerably more potent and powerful. These findings demonstrate uncoupling of keratinocyte differentiation, potentially useful in analysis of its multiple regulatory influences. They also emphasize the utility of sensitive keratinocyte targets for studying the mechanisms by which model carcinogens disturb the orderly progression of events in their differentiation program.

Benzo(a)pyrene↗
PubMed1988

Peroxidase-catalyzed oxidation of (bi)sulfite: reaction of free radical metabolites of (bi)sulfite with (+/-)-7,8-dihydroxy-7, 8-dihydroxy[a]pyrene.

The peroxidase-catalyzed metabolism of (bi)sulfite (hydrated sulfur dioxide) in the presence of (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was examined. Both horseradish peroxidase and prostaglandin peroxidase catalyze the one-electron oxidation of (bi)sulfite. This results in the formation of a sulfur trioxide radical anion which then reacts with molecular oxygen to form a peroxyl radical. This (bi)sulfite-derived peroxyl radical then reacts with BP-7,8-diol to form BP-7,8-diol-9,10-epoxides, the ultimate carcinogenic form of benzo[a]pyrene (BP). Addition of (bi)sulfite to incubations containing BP-7,8-diol and an active peroxidase resulted in significantly increased levels of BP diol-epoxide formation. This result may, in part, explain the reported co-carcinogenic effect of sulfur dioxide on BP-induced tumors in the respiratory tracts of rats and hamsters. The sulfur trioxide radical anion also reacts directly with BP-7,8-diol to form a sulfonate adduct. This reaction was particularly significant under conditions where molecular oxygen was depleted from the incubations. While the significance of this particular adduct is not known, its formation suggests that the sulfur trioxide radical anion generated during the peroxidase-catalyzed oxidation of (bi)sulfite could react with a wide assortment of compounds to form sulfonate adducts.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

Interlaboratory comparison of antisera and immunoassays for benzo[a]pyrene-diol-epoxide-I-modified DNA.

An interlaboratory comparison of immunoassays using antisera elicited against benzo[a]pyrene-diol-epoxide-modified DNA (BPDE-I-DNA) was carried out resulting in standardization of antisera, competitors and assay conditions. The assays used included competitive enzyme-linked immunosorbent assays (ELISA) with color and fluorescence endpoint detection and an ultrasensitive enzyme radioimmunoassay (USERIA) with a radioactive endpoint. Three different antisera were compared, two of which were obtained from different rabbits immunized with the same BPDE-I-DNA and a third from an animal immunized with another BPDE-I-DNA sample. Samples of standardized BPDE-I-DNA with high (36 pmol adduct/microgram DNA; 1.2 adducts/10(2) nucleotides) and low (4.5 fmol/microgram DNA; 1.5 adducts/10(6) nucleotides) modification levels were prepared and used in each laboratory. The antisera were all elicited against DNAs modified to a high extent, and it was therefore not surprising that they detected adducts in a slightly modified DNA sample with lower efficiency than those in highly modified DNA samples. The discrepancy of antibody recognition between the highly and slightly modified samples varied between 1.4- and 11.2-fold depending on the antiserum and assay. To ascertain the quantitative capability of the immunoassays, the modification level of DNA isolated from mouse keratinocytes treated with [3H]benzo[a]pyrene was determined by radioactivity and immunoassay. These results indicated that when a biological sample is assayed against a BPDE-I-DNA standard modified in the same range as the biological samples (4.5 fmol/microgram), quantitative recovery of adducts is achieved by immunoassay. These studies resulted in the realization that interlaboratory differences in immunoassay procedure can have significant consequences for data comparison and that where possible it is preferable for laboratories to use the same antisera and modified DNA standards.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

Suppressive activities of OGG1 and MYH proteins against G:C to T:A mutations caused by 8-hydroxyguanine but not by benzo[a]pyrene diol epoxide in human cells in vivo.

8-Hydroxyguanine (8OHG), an oxidatively damaged base, and benzo[a]pyrene-diol-epoxide (BPDE), a metabolite of benzo[a]pyrene found in cigarette smoke, are thought to be major causes for G:C to T:A transversions in DNA of human cells. In this study, we assessed the abilities of OGG1, MYH and APE1 proteins, which are components of a base excision repair pathway, to suppress G:C to T:A transversions caused by 8OHG or BPDE by a bacterial suppressor tRNA (supF) forward mutation assay using a shuttle plasmid, pMY189. The introduction of a single 8OHG residue at position 159 of the supF gene and treatment with BPDE led to a 65- and 34-fold increase in mutation frequencies of the pMY189 plasmid, respectively, after replication in the NCI-H1299 human lung cancer cell line. G:C to T:A transversions were predominantly induced in these plasmids. Both the mutation frequency of the 8OHG-containing plasmid in NCI-H1299 cells and the occurrence of G:C to T:A transversions at position 159 in the supF gene were significantly reduced by overexpression of OGG1 and MYH proteins, but not by that of APE1 protein. In contrast, neither mutation frequency nor the occurrence of G:C to T:A transversion of the BPDE-treated plasmid was reduced by overexpression of OGG1, MYH and APE1 proteins. These results indicate that OGG1 and MYH function as suppressors for G:C to T:A transversions by 8OHG but not by BPDE in human cells.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

Nickel (II) enhances benzo[a]pyrene diol epoxide-induced mutagenesis through inhibition of nucleotide excision repair in human cells: a possible mechanism for nickel (II)-induced carcinogenesis.

Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

The determination of benzo[a]pyrene in the total particulate matter of cigarette smoke.

A procedure for the isolation and determination of benzo[a]pyrene in the total particulate matter of cigarette smoke is described. Two high-pressure liquid chromatographic (HPLC) techniques are employed: a normal-phase, mu Bondapak-NH2, amino column is used for isolation of the benzo[a]pyrene fraction and a reversed-phase, Vydac 201TP54, polymeric octadecyl silane column is used for quantitation. Fluorescence detection is used in both modes of chromatography. The wavelengths of excitation and emission are evaluated for analytical detection. Extraction media and various isolation techniques are compared for their extraction efficiency and isolation from interferences, respectively. The procedure is efficient, reproducible, sensitive (3 pg), and gives results that compare favorably with other techniques reported in the literature for the B[a]P content of reference cigarettes, 1R1 and 1R4F.

Benzo(a)pyrene↗
PubMed1986

Human fibroblast chromatin states as effectors of the DNA-binding characteristics of benzo[a]pyrene anti-7,8-dihydrodiol 9,10-epoxide and two nonalkylating DNA-binding molecules.

Pure populations of mitotic or nonmitotic diploid human fibroblasts (greater than 98% pure) were exposed to [3H]benzo [a]pyrene (CAS: 50-32-8) anti-7,8-dihydrodiol 9,10-epoxide: r-7,t-8 dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo [a]pyrene (or anti-diol-epoxide). In addition, metaphase chromosomes, interphase chromatin, or naked DNA was isolated from the pure cell populations and then titrated to saturation with anti-diol-epoxide, chromomycin A3, or 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide (ethidium bromide). At saturation, anti-diol-epoxide had covalently modified 1.5% of the total deoxyguanosine residues in naked DNA, and this was reduced to 29 and 15% of this level in saturating the available anti-diol-epoxide-binding sites in chromosomes or chromatin, respectively. A similar hierarchy of accessible binding sites (naked DNA greater than chromosomes greater than chromatin) was also observed for the noncovalent interaction of chromomycin A3 or ethidium bromide with the human cell DNA. Deproteinization of the chromosome or chromatin preparations returned the level of drug binding to that seen with naked DNA. The results clarify the association between proteins and DNA in human chromatin and suggest how cell-cycle-dependent changes in DNA-associated proteins or higher-order changes in protein-DNA conformation can act to alter the access of molecules to DNA-binding sites.

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