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Metabolite repression inhibits degradation of benzo[a]pyrene and dibenz[a,h]anthracene by Stenotrophomonas maltophilia VUN 10,003.

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10-15 mg (-1) of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25-100 mg l(-1), by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation.

Benz(a)Anthracenes↗

Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases.

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.

Amino Acid Sequence↗

Relationship between activities of cytochrome P-450 monooxygenases in human placental microsomes and binding of benzo(a)pyrene metabolites to calf thymus DNA.

Benzo(a)pyrene metabolism in human placental microsomes from smokers was studied. Benzo(a)pyrene metabolites were separated using high pressure liquid chromatographic technique. Reaction of benzo(a)pyrene with a microsomal fraction of placenta from individuals who smoke cigarettes during pregnancy yields 7,8 dihydroxy benzo(a)pyrene as a major metabolite, while 3'-hydroxy benzo(a)pyrene, 4,5 dihydroxy benzo(a)pyrene and quinones constitute minor metabolites. The activities of arylhydrocarbon hydroxylase and 7-ethoxycoumarin deethylase exhibited much higher activities in smokers than in nonsmokers. Examination of specific binding of monoclonal antibodies to cytochrome P-450 isozymes in placental microsomes revealed that cigarette smoking specifically enhanced the level of cytochrome P-450 c and d isozymes in human placental microsomes. Coincubation of 3H-benzo(a)pyrene and calf thymus DNA with placental microsomes yielded acid insoluble 3H-B(a)P from smokers, suggesting that cigarette smoking may induce placental enzymes which convert benzo(a)pyrene into ultimate metabolites to form carcinogen-DNA adducts.

Animals↗

[Co-metabolic degradation of pyrene in soil].

High molecular weight Polycyclic Aromatic Hydrocarbons (PAHs) are always degraded by means of co-metabolism. This study compared the degradation process of pyrene as sole source of carbon and energy and co-metabolic degradation process of pyrene. The degradation rate of pyrene after 25 days in the first process was 57%, while the degradation rate of pyrene in the co-metabolic processes were about 80%. The half-life of pyrene in the co-metabolic process was shorter than those in the processes without co-metabolism. Salicylic acid, phthalic acid, sodium succinate could serve as co-metabolic substrate to enhance the degradation rate of pyrene, and sodium succinate has the best effect. There was co-metabolic relationship between pyrene and phenanthrene which was a kind of low molecular weight PAHs, phenanthrene accelerated the degradation of pyrene, while naphthalene didn't. The principle of the co-metabolism and the optimal material used as co-metabolic substrate were demonstrated in this paper.

Environmental Pollution↗

Embryotoxicity of benzo(a)pyrene and some of its synthetic derivatives in Swiss mice.

We have studied the teratogenicity of benzo(a)pyrene (BP), benzo(a)pyrene-4,5-oxide, and a racemic mixture of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, a proximal metabolite and ultimate carcinogenic metabolite of BP, respectively, and of 6-methylbenzo(a)pyrene after direct injection into embryonal Swiss mice. The compounds were dissolved in acetone and trioctanoin (1:1) and injected at doses ranging from 0.4 to 16.0 nmol/embryo on days 10, 12, and 14 of development. The transplacental effects of BP given at the same gestational days and at comparable dose levels were also evaluated. The control groups received 0.5, 1.0, or 2.0 microliter/embryo of vehicle on days 10, 12, or 14 of pregnancy, respectively. The fetuses were examined when they were 18 days old. On the basis of gross external and internal malformations, 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene appeared to be the most potent embryotoxic and teratogenic compound tested, causing 85% of embryolethality and 100% of malformed fetuses in the group treated on day 10 of intrauterine development. There were 61 and 27% of malformed fetuses following 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene treatment on days 12 and 14 of gestation, respectively. The effects of this BP metabolite were very specific and malformations such as exencephaly, thoraco- and gastroschisis, phocomelia, and edema were found. The administration of BP (both transplacental and direct intraembryonal injection) and benzo(a)pyrene-4,5-oxide caused no significant increase of malformed fetuses in any of the developmental stages considered. 6-Methylbenzo(a)pyrene induced multiple malformations (among these a high percentage of protruding tongue) in 50, 46 and 31% of the fetuses treated on days 10, 12, and 14 of gestational age, respectively. These results combined with previous data concerning the induction of lung tumors by the tested compounds in 15-day-old Swiss mouse embryos, emphasize the requirement of a common metabolic derivative of BP to induce both teratogenesis and carcinogenesis in mice. Furthermore present data show that midgestation Swiss embryos are also highly sensitive to the 6-methyl derivative of BP.

Abnormalities, Drug-Induced↗

Effects of chronic ethanol consumption on benzo(a)pyrene metabolism and glutathione S-transferase activities in Syrian golden hamster cheek pouch and liver.

The metabolism of benzo(a)pyrene (BaP) by hepatic or cheek pouch epithelium microsomes obtained from Syrian golden hamsters which had been consuming an ethanol-containing liquid diet for 4 wk and from pair-fed controls was measured. Glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene or (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene as substrates was measured in cytosol obtained from the liver or cheek pouch epithelium of the same animals. Cytosolic hepatic glutathione levels were measured in both ethanol-consuming and control animals. The metabolism of BaP to 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene (BaP-4,5-diol), 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (BaP-7,8-diol), 9-hydroxybenzo(a)pyrene (9-OH-BaP), and 3-hydroxybenzo(a)pyrene (3-OH-BaP) by hepatic microsomes from ethanol-consuming hamsters was significantly reduced (40-52%) (P less than 0.05) compared to control microsomes. However, a 2-fold increase (P less than 0.05) in the metabolism of BaP to BaP-7,8-diol and 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene was measured with microsomes from the cheek pouch epithelium of ethanol-consuming animals. There was no significant change in the production of BaP-4,5-diol, 9-OH-BaP, or 3-OH-BaP by cheek pouch epithelium microsomes of ethanol-consuming hamsters compared to controls. No difference in glutathione S-transferase activity of hepatic or cheek pouch epithelium cytosol between control and ethanol-consuming hamsters towards 1-chloro-2,4-dinitrobenzene or (+/-)-r-7,t-8-dihydroxy-t-9,10- epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was observed. Hepatic glutathione content was significantly (P less than 0.05) decreased after 2 wk (23%) and 4 wk (33%) of ethanol consumption. The results suggest a mechanism by which ethanol might enhance BaP tumorigenesis in the hamster cheek pouch.

Alcoholism↗

Variations among untreated rabbits in benzo(a)pyrene metabolism and its modulation by 7,8-benzoflavone.

The metabolism of benzo(a)pyrene by rabbit liver microsomes can be stimulated or inhibited by 7,8-benzo(a)flavone (ANF) depending on the distribution of specific P-450 enzymes present within the microsomes. Treatment of rabbits with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or rifampicin leads to an increase of hepatic microsomal metabolism of benzo(a)pyrene. ANF stimulates the rate of benzo(a)pyrene metabolism catalyzed by microsomes isolated from rabbits treated with rifampicin by 3-fold. In contrast, ANF moderately inhibits the activity of microsomes from TCDD-treated rabbits. Variations in the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits apparently reflect differences in the expression of P-450 1, a constitutive form of P-450. Thus, the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits, which varies from 0.40 to 1.5 nmol/min/mg of protein, is directly correlated with the microsomal concentration of P-450 1. The metabolism of benzo(a)pyrene by microsomes containing high concentrations of P-450 1 is inhibited by a monoclonal antibody specific for this cytochrome to approximately the rate exhibited by microsomes with a low concentration of P-450 1. The benzo(a)pyrene activity stimulated by ANF in microsomes with a low concentration of P-450 1 is not inhibited by the monoclonal antibody. The activity of P-450 1 is inhibited by ANF at concentrations that stimulate other constitutive forms of P-450. Thus, ANF produces offsetting effects on benzo(a)pyrene metabolism in microsomes from untreated animals by stimulating the activity of at least one cytochrome and inhibiting P-450 1-mediated activity.

Animals↗

Effect of intratracheally instilled benzo(a)pyrene on the pulmonary and hepatic drug-metabolizing enzymes in normal and vitamin A deficient rats.

The effect of intratracheal instillation of different doses of benzo(a)pyrene (0.1, 1.0 and 2.0 mg) on the drug metabolizing enzymes of lung and liver was analysed in rats fed diet with or without vitamin A for 5-6 weeks. Benzo(a)pyrene exposure at 2.0 mg dose only elevated the level of cytochrome P-450 and b5, and activity of benzopyrene hydroxylase in liver, and extent of increase was similar in normal and vitamin A deficient groups. Contrary to this, pulmonary contents of cytochrome P-450 and b5, and benzopyrene hydroxylase activity increased over control values in both the groups even at lower doses of benzo(a)pyrene. Moreover, their values were higher in vitamin A deficient-treated groups compared to normal-treated controls. Increase in these parameters was greater in lung as compared to increase in liver. NADPH cytochrome C-reductase in lung and liver was not affected either by inducing vitamin A deficiency or exposing these rats further to benzo(a)pyrene. Uridine-diphospho-glucuronosyl-transferase (UDP-GT) activity in normal and vitamin A deficient groups was enhanced following exposure to benzo(a)pyrene both in lung and liver. However, activity of this enzyme remained impaired in vitamin A deficient groups, benzo(a)pyrene exposed or not exposed when compared to respective normal controls. Glutathione S-transferase activity remained unchanged following exposure to benzo(a)pyrene both in lung and liver. The apparent increase in hepatic glutathione S-transferase and decrease in pulmonary glutathione S-transferase activity in vitamin A deficiency was only due to vitamin A deficient status of rats with no further effect of benzo(a)pyrene.

Animals↗

Comparative benzo[a]pyrene metabolite patterns in fish and rodents.

Benzo[a]pyrene is converted to 3-hydroxybenzo[a]pyrene, 9-hydroxybenzo[a]pyrene, benzo[a]pyrene-4,5-dihydrodiol, benzo[a]pyrene-7,8-dihydrodiol, benzo[a]pyrene-9,10-dihydrodiol, and benzo[a]pyrene quinones by postmitochondrial supernatant or microsomes in such fish as the rainbow trout, flounder, salmon, mullet, little skate, Fundulus grandis, and sea catfish. It is also now well-established that many fish convert benzo[a]pyrene to potent mutagenic metabolites as has been demonstrated with the Ames test, especially when the fish are induced with Aroclor 1254 or 3-methylcholanthrene. The metabolite patterns obtained at different substrate concentrations levels indicate that the metabolism is more complex at low concentrations when metabolites are recycled in the in vitro system.

Animals↗

Effect of low-density lipoprotein on the incorporation of benzo(a)pyrene by cultured cells.

Benzo(a)pyrene added to human plasma in vitro associated with the plasma lipoproteins, especially the low-density fraction. The influence of plasma low-density lipoprotein on cellular uptake of benzo(a)pyrene was studied using WI-38, a human embryonic lung fibroblast line, and GM 1915, a skin fibroblast line derived from a patient with homozygous familial hypercholesterolemia. The WI-38 cells were low-density-lipoprotein receptor positive, and the familial hypercholesterolemia cells were receptor negative by standard binding studies with 125I-labeled low-density lipoprotein. Following 2 hr of incubation at 37 or 4 degrees, cell association of benzo(a)pyrene was determined with benzo(a)pyrene bound to lipoprotein or added at the same concentration to serum-free medium or medium containing delipidated serum. Uptake from delipidated or serum-free medium by both cell lines was linear with concentration, while incorporation of benzo(a)pyrene bound to low-density lipoprotein was much less and nonlinear at higher concentrations of lipoprotein. While low-density lipoprotein apparently influenced the availability of benzo(a)pyrene to the cell, no differences were noted in the incorporation of benzo(a)pyrene by WI-38 and familial hypercholesterolemia cells. Thus, benzo(a)pyrene entered the cells from low-density lipoproteins despite the absence of specific receptors, apparently by a rapid redistribution between the lipoprotein and cell membrane.

Benzo(a)pyrene↗

Relationship between benzo(a)pyrene-induced DNA base modification and frequency of reverse mutations in mutant strains of Salmonella typhimurium.

Salmonella typhimurium cells (TA98 and TA100) were incubated with [3H]benzo(a)pyrene ([3H]BP) and induced rat liver microsomes. The BP-induced cytotoxicity and His+ reverse-mutation frequencies were determined, and bacterial DNA hydrolysates were chromatographed on Sephadex LH-20. Analysis indicated three principal DNA adducts formed from two diastereoisomeric BP diol-epoxides and a 9-hydroxy-benzo(a)pyrene metabolite. An 8.6-fold increase in TA100 cell concentration in the microsome incubation was paralleled by a 7.2-fold decrease in total adducts per cell and a 7.4-fold decrease in mutation frequency. Separate TA98 incubations were titrated with increasing concentrations of [3H](+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene ([3H]anti BP diol-epoxide), [3H](+/-)-7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [3H]syn BP diol-epoxide), or [3H]9-hydroxybenzo(a)pyrene. Linear, nonsaturated increases in DNA adduct levels were seen up to the highest observed concentrations of 4.00 microM BP diol-epoxide or 6.00 microM 9-hydroxybenzo(a)pyrene in both TA98 and TA100 cells. The increasing adduct levels were accompanied by linearly increasing mutation frequencies. At equivalent concentrations of the two DP diol-epoxides, an average of 8.2-fold more base substitution mutations (TA100) were seen than frameshift mutations (TA98). The results also indicate significant differences in absolute mutagenic efficiency (mutation frequency/unit modified DNA) between these three covalent DNA ligands (TA98, syn BP diolepoxide greater than 9-hydroxy-4,5-epoxy-benzo(a)pyrene greater than anti BP diol-epoxide; TA100, 9-hydroxy-4,5-epoxy-benzo(a)pyrene greater than syn BP diol-epoxide greater than anti BP diol-epoxide).

Animals↗

Induction of cytotoxicity, mutation, cytogenetic changes, and neoplastic transformation by benzo(a)pyrene and derivatives in C3H/10T1/2 clone 8 mouse fibroblasts.

Benzo(a)pyrene (BaP), a series of its metabolic derivatives, and benzo(e)pyrene, a very weakly carcinogenic isomer, were tested for their biological effects on transformable C3H/10T1/2 cells. These cells were used as targets in a series of assays designed to measure oncogenic transformation, mutation to ouabain resistance, cytotoxicity, and induction of cytogenetic changes, as evidenced by chromosomal aberrations and sister chromatid exchange. Of all the compounds tested, only the parent hydrocarbon, BaP, an (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene were found to be significantly active in producing transformation and cytogenetic alterations. BaP, (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, and (+/-)-7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, however, were all effective inducers of mutation in C3H/10T1/2 cells. (+/-)-7 alpha, 8 beta-Dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was the most potent agent in tests for cytotoxicity, Benzo(e)pyrene was inactive in all assays examined. Among the compounds tested, there was a correlation between the ability to induce cytogenetic changes and the ability to produce mutation and transformation. These results support the demonstrated role of (+/-)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene as a proximal carcinogenic form of BaP and illustrate the utility of the C3H/10T1/2 cell system as an important tool for the detection of genotoxic damage by carcinogenic chemicals.

Animals↗

Benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae. Substrate binding, spectral and kinetic data.

Saccharomyces cerevisiae, brewer's yeast, produces a microsomal benzo(a)pyrene hydroxylase when grown at high glucose concentrations of which the haemoprotein, cytochrome P-450 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating) EC 1.14.14.1) is a component. We report here kinetic data derived from Lineweaver-Burk plots of benzo(a)pyrene hydroxylation. The Michaelis constant was decreased by growth of the yeast in the presence of benzo(a)pyrene showing the induction of a form of the enzyme more specific for this compound. NADPH or cumene hydroperoxide could be used as cofactors by this enzyme, although with different Km and V values for benzo(a)pyrene. A solubilised and a solubilised, immobilised enzyme preparation were capable of benzo(a)pyrene hydroxylation, using cumene hydroperoxide but not NADPH as the cofactor. Benzo(a)pyrene was found to produce a modified type I spectral change with yeast and rat liver microsomes. The interaction of benzo(a)pyrene with cytochrome P-450 was investigated further by means of an equilibrium gel filtration technique. There appeared to be 20 binding sites per mol ofcytochrome P-450 for benz(a)pyrene, in both yeast and rat liver microsomes.

Animals↗

Urinary benzo[a]pyrene and its metabolites as molecular biomarkers of asphalt fume exposure characterized by microflow LC coupled to hybrid quadrupole time-of-flight mass spectrometry.

As a step to study the health effects of asphalt fume exposure, an analytical method was developed to characterize benzo[a]pyrene and its hydroxy metabolites in the urine of asphalt fume-exposed rats. This method is based on microflow liquid chromatography (LC) coupled to hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry (Q-TOFMS). Twenty-four female Sprague-Dawley rats were used in the experiment, with 8 as controls and 16 exposed to asphalt fumes in a whole-body inhalation chamber for 10 days (4 h/day). Generated at 150 degrees C, the asphalt fume concentration in the animal exposure chamber ranged 76-117 mg/m(3). In the urine of the asphalt fume-exposed rats, benzo[a]pyrene and its metabolites of 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol(+/-), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(+/-) were identified, and their concentrations were determined at 2.19 +/- 0.49, 16.17 +/- 0.3, 6.28 +/- 0.36, and 29.35 +/- 0.26 ng/100 mL, respectively. The metabolite concentrations from the controlled group, however, were either under the detection limits or at a relatively very low level (0.19 +/- 0.41 ng/100 mL for benzo[a]pyrene-7,8,9,10-tetrahydrotetrol metabolite). The results clearly indicate that the benzo[a]pyrene and its hydroxy metabolites were significantly elevated (p < 0.001) in the urine of asphalt fume-exposed rats relative to controls. The study also demonstrated that the combination of microflow LC separation and collision-induced dissociation leading to a characteristic fragmentation pattern by hybrid Q-TOFMS offers a distinct advantage for the identifications and characterizations of the benzo[a]pyrene metabolites.

Journal Article↗

Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene.

Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene.

Animals↗

Identification of a rat oltipraz-inducible UDP-glucuronosyltransferase (UGT1A7) with activity towards benzo(a)pyrene-7,8-dihydrodiol.

Previous work has shown that polycyclic aromatic hydrocarbons and oltipraz both induce an unidentified rat liver UDP-glucuronosyltransferase with activity toward benzo(a)pyrene-7, 8-diol, the proximate carcinogenic form of benzo(a)pyrene. Here we report the isolation of a benzo(a)pyrene-7,8-diol transferase-encoding cDNA, LC14, from an adult rat hepatocyte-derived cell line (RALA255-10G LCS-3). The predicted amino acid sequence of LC14 is nearly identical (5 differences out of 531 residues) to that deduced from UGT1A7, recently cloned at the genomic DNA level (Emi, Y., Ikushiro, S., and Kyanagi, T. (1995) J. Biochem. (Tokyo) 117, 392-399). Northern analysis of RNA from female F344 rat liver and LCS-3 cells revealed over a 40-fold and 4.4-fold enhancement by oltipraz treatment, respectively. Benzo(a)pyrene-7, 8-diol glucuronidating activity was detected (0.4 nmol/10(6) cells/16 h) in AHH-1 cells transfected with the LC14 expression vector, pMF6-LC14-3. The LC14-encoded transferase exhibited even higher activity toward certain benzo(a)pyrene phenols, including the major 3- and 9-phenol metabolites (4.1 and 2.8 nmol/10(6) cells/16 h, respectively). The Km of the enzyme for (-)-trans benzo(a)pyrene-7, 8-diol and 3-OH-BP was 15.5 and 12.3 microM, respectively. Northern analyses of total RNA revealed expression of LC14 or LC14-like RNA in all extrahepatic tissues tested. Marked inducibility by oltipraz was observed only in liver and (to a lesser extent) intestine. The results suggest that induction of UGT1A7 may explain the increased glucuronidating activities toward benzo(a)pyrene-7,8-diol and other metabolites that occur following treatment with polycyclic aromatic hydrocarbon-type inducing agents and oltipraz. UGT1A7 appears to represent an important cellular chemoprotective enzyme which mediates conjugation and elimination of toxic benzo(a)pyrene metabolites.

Amino Acid Sequence↗

Fluorescence study of DNA-binding metabolites of benzo(a)pyrene formed in hepatocytes isolated from 3-methylcholanthrene-treated rats.

Hepatocytes and liver microsomes isolated from 3-methylcholanthrene-treated rats metabolize benzo(a)pyrene to products that bind to endogenous DNA and exogenously added calf thymus DNA, respectively. By using a sensitive fluorescence technique, it has been possible to characterize the major DNA-binding metabolite in hepatocytes as being produced by further metabolism of 9-hydroxybenzo(a)pyrene. In microsomes, two products binding to calf thymus DNA were recovered, a major species formed by activation of 9-hydroxybenzo(a)pyrene and a minor fraction formed by further metabolism of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene. The available evidence indicates that the ultimate products responsible for binding to DNA were identical to 9-hydroxybenzo(a)pyrene 4,5-oxide and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide, respectively. Our data further suggest that metabolic activation of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene results in quite different DNA:metabolite complexes. The former product(s) seems to be strongly associated with hydrophobic regions in DNA, whereas the latter metabolite(s) appears to be more exposed to the exterior.

Animals↗

Influence of inducers and inhibitors of mixed-function oxidasts on benzo(a)pyrene binding to the DNA of rat liver nuclei.

Benzo(a)pyrene-conjugated DNA was isolated after benzo(a)pyrene was incubated in vitro with liver nuclei from control, phenobarbital-treated, and methylcholanthrene-treated rats and the reduced nicotinamide adenine dinucleotide phosphate-generating system. Aryl hydrocarbon hydroxylase, the levels of activity of which varied with the different nuclear systems, was highly specific in the activation of the benzo(a)pyrene to forms that bind to DNA. Sephadex LH-20 chromatography of the degreded DNA from liver nuclei of pretreated rats revealed an in vivo DNA-bound product previously shown to be derived from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide (Product A). The nuclei from phenobarbital-pretreated rats also contained a DNA-bound product corresponding to that derived from the binding of benzo(a)pyrene 4,5-oxide (Product C), but they completely lacked the DNA-bound product derived from futher metabolism of 0-hydroxybenzo(a)pyrene (Product D). In contrast, nuclei from methylcholanthrene-treated rats yielded large amounts of Product D. No bound products were found with the control nuclei. 7,8-Benzoflavone caused an 80% inhibition of benzo(a)pyrene binding to DNA in the nuclei of methylcholanthrene-pretreated rats, but it had no effect on control nuclei or those from phenobarbital-pretreated rats. It inhibited formation of Product A, but not Products B or C, in nuclei from phenobarbital-pretreated rats. With nuclei from methylcholanthrene-pretreated rats. With nuclei from methylcholanthrene-pretreated animals, 7,8-benzoflavone led to an overall loss of products, but small amounts of Products A and B persisted. 1,1,1-Trichloropropene 2,3-oxide strongly inhibited nuclear aryl hydrocarbon hydroxylase activity and the phenol fraction measured by thin-layer chromatography in all nuclear systems, but it significantly increased the benzo(a)pyrene binding in all nuclei. With nuclei from phenobarbital-treated rats, it inhibited the formation of Product B but not of Products A and C; whereas with nuclei from methylcholanthrene-pretreated rats, it inhibited the fromation of all the hydrocarbon-deoxyribonucleoside products.

Animals↗