Metabolism and binding of benzo(a)pyrene and 2-acetylaminofluorene by short-term organ cultures of human and rat bladder

The ability of organ cultures of normal human and rat bladder to metabolize the polycyclic hydrocarbon, benzo(a)pyrene (BP), and the arylamine, 2-acetylaminofluorene, has been studied. Cultures were maintained for 0 to 6 days in a chemically defined medium before incubation with [3H]BP (0.3 to 0.5 microM) or 2-[14C]acetylaminofluorene (18 to 25 microM) for 24 hr. Ethyl acetate-soluble and water-soluble metabolites were produced from both compounds by both species. The ethyl acetate extracts from [3H]BP-treated human cultures contained 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, and 3-hydroxybenzo(a)pyrene. Rat bladder cultures produced similar metabolites but in slightly different proportions. Ethyl acetate-soluble products of 2-[14C]acetylaminofluorene from human cultures contained 7-hydroxy-2-acetylaminofluorene, 9-hydroxy-2-acetylaminofluorene, 2-aminofluorene, and N-hydroxy-2-aminofluorene. Rat bladder cultures produced similar metabolites, but 2-aminofluorene was found in relatively higher proportion. Hydrolysis by beta-glucuronidase of the water-soluble products produced from both carcinogens gave ethyl acetate-extractable derivatives. These hydrolyzable glucuronide conjugates were relatively more abundant following metabolism of the carcinogens by the rat than by the human cultures. Covalent binding to DNA occurred with [3H]BP in both human (19.7 +/- 13 pmol/mg DNA) and rat cultures (22.8 +/- 8.6 pmol/mg DNA). As with other human tissues, considerable variation (50-fold) was observed between individuals. The results demonstrate that both human and rat bladder epithelium can metabolize known potent carcinogens and, in the case of BP, can effect covalent binding between the products of metabolism and the urothelial cell DNA. In theory, carcinogenesis in the urinary bladder could thus be initiated by carcinogens produced or excreted in the urine without the necessity for their prior metabolism elsewhere in the body.     

Human and rat kidney cell metabolism of 2-acetylaminofluorene and benzo(a)pyrene

The metabolism and mutagenic activation of the model carcinogens benzo(a)pyrene [B(a)P] and 2-acetylaminofluorene (AAF) by human and rat kidney cells were measured. A slicing technique followed by enzyme digestion was utilized to obtain the kidney cells. Although levels of total metabolism of B(a)P by rat and human kidney cells were similar, analysis of specific metabolites of B(a)P indicated that species differences existed. Human kidney cells produced the organic-soluble metabolites B(a)P-9,10-diol, B(a)P-4,5-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, and B(a)P-9-phenol. Rat kidney cells produced organic-soluble B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-4,5-diol, and B(a)P-6,12-quinone. Both species produced sulfate and glucuronide conjugates of all products. For AAF, kidney cells from some human tissues produced up to four times the level of total metabolites compared to rat kidney cells. Organic-soluble metabolites were qualitatively similar between the species and consisted of 2-aminofluorene (AF), N-hydroxy-AAF and ring-hydroxylated products at the 1, 3, 5/9, 7, and 8 positions. Sulfate and glucuronide conjugates of these metabolites were also detected. Human interindividual variation with kidney cells was about 2.5-fold for total AAF metabolism and up to 6-fold for individual AAF metabolites. For B(a)P metabolism, human interindividual variation in total metabolism was low while for specific metabolites there was up to a 4-fold variation. Levels of AAF and AF cell-mediated Salmonella typhimurium mutagenesis were significantly higher with human cells as compared to rat kidney cells. It appears that the differences between human and rodent kidney cell metabolism of chemical carcinogens vary with the chemical class and understanding these differences will be necessary in the extrapolation of rodent carcinogenesis data to humans.     

Metabolism of benzo(a)pyrene by human epithelial cells in vitro.

Primary cell cultures derived from human skin epithelium metabolized benzo(a)pyrene to three classes of compounds: phenols, quinones, and dihydrodiols. The relative proportions of metabolites varied according to the skin donor but differed from the pattern of metabolites in rat liver microsome preparations. While appreciable amounts of 7,8- and 9,10-dihydrodiol; 1,6-, 3,6-, and 6,12-quinone; and 3- and 9-hydroxy derivatives were found in the medium, no 4,5 (K-region)-dihydrodiol or epoxide was detected. Reduced amounts of quinones were produced when the cultures were pretreated with hydrocortisone before exposure to the hydrocarbon. The cultures did not require a period of enzyme induction for efficient metabolism of the hydrocarbon. Cultures of fibroblasts derived from the same skin samples as the epithelial cells metabolized the hydrocarbon but to a much different extent. Preexposure of the epithelial cell cultures to mixtures of polycyclic hydrocarbons resulted in a decrease in the amounts of carcinogen metabolized to phenols and dihydrodiols. These findings suggest that the prevalence of carcinomatous disease in humans is due to the differential capacity of the epithelial cells to metabolize potential carcinogens to active forms, a capacity reduced in fibroblasts or other nonepithelial cells. This suggestion is supported by the observations that supposedly normal prostate cells also efficiently metabolize polycyclic hydrocarbons in a manner similar to that of epidermal cells. No evidence of neoplastic transformation was seen in cytological preparations of cells exfoliated into the medium.     

Metabolism of 2-acetylaminofluorene by two 3-methylcholanthrene-inducible forms of rat liver cytochrome P-450

The present study examines the contribution of two major 3-methylcholanthrene (3-MC)-inducible forms of rat liver cytochrome P-450 (P-448MC and P-448HCB) to the metabolism of 2-acetylaminofluorene (AAF). In a reconstituted enzyme system, purified rat liver P-448MC metabolized AAF at a 10-fold greater rate than P-448HCB. The major metabolites produced by cytochrome P-448MC were 3-hydroxy (OH) (30%), 5-OH (24%), 7-OH (22%), and 9-OH (10%). N-OH-AAF (3%) was a minor metabolite. In contrast, P-448HCB catalyzed the N-hydroxylation of AAF preferentially (15% of total metabolites). The other primary metabolites formed by this isozyme were 7-OH-AAF (30%), 5-OH-AAF (29%), and 9-OH-AAF (25%). Cytochrome P-448HCB catalyzed the formation of less 3-OH-AAF (7%) than did P-448MC (30%). Since cytochrome P-448HCB is immunochemically related to P-448MC, specific antisera to both isozymes were made by immunoabsorption with the appropriate antigen bound covalently to Sepharose. Anti-P-448MC inhibited AAF metabolism approximately 43% in microsomes from 3-MC-induced male rats and 30% in microsomes from rats treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), another 3-MC-type inducer. Anti-P-448HCB inhibited total metabolism of AAF by only 22 and 38% in microsomes from 3-MC- and HCB-induced rats. However, anti-P-448HCB inhibited N-hydroxylation by 60% in both 3-MC- and HCB-induced microsomes. Anti-P-448MC did not inhibit N-hydroxylation. Neither antibody affected AAF metabolism in control microsomes. These data suggest that, in rat liver, two 3-MC-inducible izozymes of cytochrome P-450 metabolize AAF; however, N-hydroxylation is catalyzed primarily by one of these isozymes, cytochrome P-448HCB.     

Metabolism of benzo(a)pyrene by variant mouse hepatoma cells

Four mouse hepatoma cell lines, a parent (Hepa-1c1c7) and three variants (MUL12, BPrc1, and TAOc1BPrc1) which had been derived from Hepa-1c1c7 by the fluorescence-activated cell sorter, were incubated with benzo(a)pyrene, and the metabolites were analyzed by high-pressure liquid chromatography. Among these four cell lines, Hepa-1c1c7 and MUL12 metabolized benzo(a)pyrene the most quickly and to the greatest extent, and BPrc1 had the weakest metabolic activity for this substrate. TAOc1BPrc1 had intermediate benzo(a)pyrene-metabolizing activity, depending on cell density and incubation time. At low cell density, the active variant TAOc1Bprc1 resembled the weakly active Bprc1 in accumulating a low amount of ethyl acetate-soluble metabolites in the medium while, at high cell density, TAOc1Bprc1 resembled the parent clone Hepa-1c1c7 and the highly active variant MUL12. At short incubation times, TAOc1Bprc1 also had low conjugating activity while, at longer incubation times, the conjugating activity approached that of Hepa-1c1c7 and MUL12. At low cell density, Bprc1 was able to produce phenols, but this variant did not seem to have this ability at high cell density. When the substrate concentration was 4 microM and the incubation time was 24 h, beta-glucuronidase treatment of water-soluble metabolites released about 5.3 times more pmol of quinones compared with phenols. But when the substrate concentration was 25 nM, beta-glucuronidase released about 2.0 times as many phenols compared with quinones. The parent and the two more actively metabolizing variants showed differences in the peak times of accumulation of 9,10-diol and 7,8-diol of benzo(a)pyrene, which may have implications for binding to DNA and nuclear proteins. It was concluded that BPrc1 has basal but not easily inducible aryl hydrocarbon hydroxylase activity, whereas Hepa-1c1c7, MUL12, and TAOc1Bprc1 have basal and inducible aryl hydrocarbon hydroxylase activity. These results show that variants of a single parent cell line can exhibit significant differences in the rate and extent of metabolism of benzo(a)pyrene.     

Metabolism of 2-acetylaminofluorene by eight different forms of cytochrome P-450 isolated from rat liver

Eight different forms of cytochrome P-450 have been isolatedfrom the liver microsomes of male Sprague-Dawley rats and comparedwith previously characterized forms on the basis of chromatographicbehavior, absorption maximum of the carbon monooxide complex,apparent molecular weight upon SDS-disc gel electrophoresis,and substrate specificity. With respect to 2-acetylaminofluorenemetabolism in a reconstituted system involving these cytochromesP-450, form c was {small tilde}16 times as active as form d,which was in turn at least 30 times as active as any of theother forms. The patterns of metabolites obtained with all ofthe cytochromes P-450 were determined by h.p.l.c. and were allunique. Form d was as active as form c in producing N-hydroxy-2-acetylaminofluorene.This metabolite was observed with only one other form of thecytochrome, i.e., form PB/PCN-E, which gave rise to it at avery low rate.     

Induction of monooxygenases in rhesus monkeys by 3-methylcholanthrene: metabolism and mutagenic activation of N-2-acetylaminofluorene and benzo(a)pyrene.

Induction of content and activity of hepatic and lung cytochrome P450-dependent monooxygenases by 3-methylcholanthrene (MCA) was studied in male rhesus monkeys. Hepatic microsomes were concomitantly studied for mutagenic activation of N-2-acetylaminofluorene (AAF) as well as with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatic N-hydroxylation of AAF was increased sixfold to sevenfold after MCA treatment; aryl hydrocarbon hydroxylase was similarly increased in both liver and lung. The hepatic cytochrome P450 content was increased by 50%, and the Soret maximum in the CO-hemoprotein complex was shifted to the blue by 2 nm after MCA induction. Electrophoresis of hepatic microsomes from MCA-treated monkeys showed an increase in a polypeptide band of 54,000 molecular weight. Mutagenic activation of AAF by liver microsomes from untreated rhesus monkeys was low but was increased 40-fold after MCA treatment. No effect was observed on the mutagenic activation of N-hydroxy-2-acetylaminofluorene by liver microsomes after MCA treatment. Rhesus monkeys may provide a model for evaluation of the role of polycyclic hydrocarbon induction in chemically caused cancer and toxicity in primates.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.      

Metabolism of benzo[a]pyrene by human melanocytes in culture

Benzo[a]pyrene (B[a]P) is a ubiquitous environmental pollutant and known skin carcinogen. In the present study, in vitro addition of [3H]B[a]P to normal human melanocytes in culture, isolated from adult human skin, resulted in the metabolism of [3H]B[a]P both intracellularly and extracellularly. HPLC analysis showed that [3H]B[a]P-9,10- and 7,8-diol were the major intracellular and extracellular metabolites followed by 3,6-quinone, 9-hydroxy and 3-hydroxy metabolites. Significant amounts of the [3H]B[a]P metabolites were found to be present in the sonicated cell suspension and culture medium as the glucuronide and sulfate conjugates. In total 37.3% of the [3H]B[a]P added in the culture medium was metabolized by melanocytes, of which 21.1% was quantified as the intracellular and 16.2% as the extracellular metabolites. Our data show that human melanocytes are capable of metabolizing B[a]P.     

Metabolism of the carcinogenic hydrocarbon benzo(a)pyrene in human fibroblast and epithelial cells

Metabolism of the carcinogenic hydrocarbon benzo(a)pyrene (BP) to water and alkali-soluble products was measured in cultured fibroblast and epithelial cells from human embryos. The alkali-soluble products represented only a small fraction of BP metabolism. Fibroblasts from different organs from the same embryo metabolized similar amounts of BP, while those derived from different embryos can metabolize different amounts. The fibroblasts were divided into three groups which metabolized an average of 350, 850 and 3,400 μμmoles of water soluble products/10⁶cells/3 days. No significant differences were found in relationship to the age of the embryos from 2.5 to 5 months. Fibroblasts from the endometrium of adult women metabolized less than embryonic fibroblasts. Cultures which contained more than 20% epithelial cells metabolized 3–25 times more BP than fibroblast cultures from the same embryo. Epithelial cells from different embryos also varied in the degree of BP metabolism and low epithelial activity was not necessarily associated with low fibroblast activity in the same embryo. It is suggested that there is a genetic heterogeneity in BP metabolism in fibroblast and epithelial cells, and that the higher activity of epithelial cells might be related to the higher incidence of carcinomas rather than sarcomas in humans.     

The metabolic activation of benzo(a)pyrene and 9-hydroxybenzo(a)pyrene by liver microsomal fractions.

A rat liver microsome-mediated bacterial mutagenicity test showed 9-hyroxybenzo(a)pyrene to be significantly more effective as a pre-mutagen than benzo(a)pyrene. Experiments measuring the ability of these compounds to be metabolically activated to moieties that alkylate exogenous DNA demonstrated that 9-hydroxybenzo(a)pyrene was almost six times more effective than benzo(a)pyrene itself. Addition of trichloropropene-2,3-oxide to the reaction mixture enhanced the mutagenicity and DNA alkylation by benzo(a)pyrene but had little or no effect on the 9-hydroxybenzo(a)pyrene-mediated mutagenicity and alkylation. On the other hand, 7,8-benzoflavone inhibited the microsome-mediated mutagenicity and DNA alkylating activity of both hydrocarbons.     

Metabolism of benzo(e)pyrene by rat liver microsomal enzymes

Metabolites of benzo(e)pyrene (B[e]P) formed upon incubationof [³H]-B[e]P with hepatic microsomes from control and inducedrats have been separated by high-pressure liquid chromatographyand identified by comparison of retention times, absorbanceand fluorescence spectra with those of synthetic standards.The major metabolite produced was B[e]P-4, 5-dihydrodiol, accountingfor 20–30% of the total metabolism depending on the sourceof the microsomes. This was followed by a phenolic metabolite(shown not to be 4-OH-; 9-OH-; or 10-OH-B[e]P). A possible proximatecarcinogenic derivative of B[e]P, B[e]P-9, 10-dihydrodiol, wasidentified, but was found to constitute less than 1% of thetotal metabolites. Similar results were obtained with a purifiedand reconstituted mixed-function oxidase system. In these laterincubations, production of the dihydrodiols was dependent onthe addition of purified epoxide hydrase to the incubation mixtures. These results suggest that formation of the reactive diol-epoxide,9, 10-dihydroxy-11, 12-epoxy-9, 10, 11,-12-tetrahydro-B[e]P,a potential ultimate carcinogenic metabolite of B[e]P, is notfavored by rat liver enzymes. This provides a partial explanationfor the lack of carcinogenicity of B[e]P within the frameworkof the bay region theory of chemical carcinogenesis.     

The glucuronidation of hydroxylated metabolites of benzo[{alpha}]pyrene and 2-acetylaminofluorene by cDNA-expressed human UDP-glucuronosyltransferases

The capacity of four cDNA-expressed human liver UDP-glucuronosyltransferases(UGT), UGT1*6, UGT2B7, UGT2B10 and UGT2B11, to glucuronidatehydroxylated metabolites of benzo[     

Metabolism of benzo(e)pyrene by rat liver microsomal enzymes

Carcinogensis, 1, 165–173, February 1980. Page 165, Introduction, lines 18–19: should read "...is the fact that the epoxide group forms part of the bayregion of the hydrocarbon..." Page 166, Paragraph on Microsomal preparations, lines 5–6:should read "Cytochrome P450 contents of the microsomal preparations usedwere: ..." The incorrectly used "cytochrome P450 (P448)" refersto a specific form of cytochrome P450 and not total cytochromeP450 content. Page 171, Discussion, line 3 from bottom of page: should read "...Can efficiently form B(e)P bay region diol expoxides..."     

Metabolism of benzo(a)pyrene and 1-naphthol in cultured human tumorous and nontumorous colon

The oxidative metabolism of benzo(a)pyrene and the conjugative metabolism of 1-naphthol by explant cultures of normal human colon and colonic tumor tissue, obtained at surgery, have been studied. After 24 hr in culture, the explants were exposed to either [1-14C]-1-naphthol (20 to 100 microM) or [3H]-benzo(a) pyrene (1.5 microM) for a further 1.5 to 24 hr. Both normal-appearing tissue and tumor tissue metabolized benzo(a)pyrene to a wide variety of organic solvent-soluble metabolites, including monohydroxybenzo(a)pyrenes, dihydrodiols, and tetrols. 1-Naphthol was metabolized by cultured human colonic mucosa and tumor tissue to both its glucuronic acid and sulfate ester conjugates. In the normal tissues, with naphthol (20 microM), sulfate ester conjugation predominated. However, with the tumor tissue, sulfate ester conjugation decreased; thus, the percentage of glucuronic acid conjugates, expressed as a percentage of total metabolites formed, was increased significantly compared to normal tissue. The relationship, if any, of these changes to neoplastic transformation is unclear. The technique of explant culture described in this study may be of use for the study of other facets of the pathobiology of solid tumors.     

Metabolism and formation of DNA adducts of benzo(a)pyrene in human diploid fibroblasts.

Cultured human diploid skin fibroblasts incubated with [G-3H]benzo(a)pyrene yielded about 10 times more H2O-=soluble benzo(a)pyrene metabolites and DNA adducts of stationary growth phase than did proliferating cultures. This increased formation could be blocked by alpha-naphthoflavone. Trichloropropenoxide and cyclohexenoxide, inhibitors of the epoxide hydratase, inhibited predominantly the formation of DNA adducts. Cultures from older individuals formed significantly more benzo(a)pyrene metabolites and DNA adducts, but control cultures from patients with either lung cancer or melanoma did not. The age influence was not apparent when the ratio of DNA adducts to H2O-soluble metabolites was determined for each individual cell line. However, the proportion of DNA-bound material in the cells from patients with lung cancer was significantly increased compared to cells from melanoma patients or healthy individuals.