Metabolism Of Benzo Research Articles

Preliminary evidence for the metabolism of benzo(a) pyrene by Plantago lanceolata.

Polycyclic aromatic hydrocarbons (PAHs) have long been recognised as potential carcinogens in animals in which biotransformation into reactive metabolites can lead to DNA damage. In animals PAHs metabolism mainly occurs in hepatic microsomes and is associated with the cytochrome p-450 mediated mixed functional oxidase (MFO) system. PAH metabolism in plants has been shown to occur via a similar enzyme system, but has received relatively little attention. This study is looking at how the plant species Plantago lanceolata metabolizes benzo(a)pyrene (B(a)P), which is one of the PAHs whose metabolism has been studied extensively in animals. The aim of the work is to establish firstly that the B(a)P is taken up and secondly that it is biotransformed by the plant to products possibly similar to those found in animals. This work is achieved by using C-14-B(a)P along with whole body autoradiography, scintillation analysis and chromatography techniques to locate the B(a)P and its metabolites.

Toxicological characterization of a novel in vivo benzo[a]pyrene metabolite, 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid anhydride.

Recently, we described a new in vivo pathway in the metabolism of benzo[a]pyrene (BP) that involves an opening of the aromatic ring system. One of the products of this pathway, isolated from rat urine, was the anhydride of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid (ABADA). We have now investigated the effect of ABADA on several cellular targets, known to be important in tumor formation. ABADA was as efficient as BP-7,8-diol-9,10-epoxide in inducing direct strand breaks but not alkali labile sites in DNA in HT-29 cells and exhibited weak mutagenic activity in Salmonella typhimurium strain TA 102. The cytotoxicity of ABADA to HCT 116 cells appeared to be due to apoptosis, as caspase-3 activity and poly-ADP-ribose polymerase (PARP) cleavage was observed. COX-2 promoter activity was induced by ABADA in HCT 116 cells. In conclusion, this novel metabolic pathway may also be contributing to the carcinogenicity of BP.

Species-Dependent Metabolism of Benzo[c]Chrysene Mediated by c-DNA-Expressed Human, Rodent and Fish Cytochrome P450 Enzymes

The metabolism of benzo[c]chrysene (B[c]Ch) with various cytochrome P450 (CYP) enzymes including rat 1A1, 1A2, 2B1 and 2E1, human 1A1, 1A2, 2A6, 1B1, 3A4 and 2E1, mouse 1B1, and scup fish 1A1 expressed in Chinese hamster V79 cells has been investigated to clarify the role of individual enzymes in the regioselective oxidation of B[c]Ch and the species dependency. In six cell lines expressing individual CYP enzymes from four different species B[c]Ch was metabolized to several isomeric phenols and trans−dihydrodiols. However, cell lines expressing human 3A4, 2A6 and 2E1 or rat 1A2, 2B1 and 2E1 were metabolically in-competent towards B[c]Ch. Among the trans−dihydrodiols the 9,10-isomer could be detected in cells expressing human, rat and fish CYP 1A1 and to a minor extent in cells with human 1A2, but not in cells expressing human and mouse CYP 1B1. The latter two cell lines produced high amounts of the bay region 3,4-dihydrodiol, whereas the K-region 7,8-dihydrodiol was a minor metabolite. Oxidation of B[c]Ch to the 1,2-dihydrodiol could not be catalyzed by any of the CYP enzymes investigated except fish 1A1. Our results suggest that metabolic activation of B[c]Ch is initiated predominantly by CYP 1A1 to result selectively in the formation of fjord region 9,10-dihydrodiol 11,12-epoxides regardless of the species involved. The activation of B[c]Ch appears to be limited by a low regioselectivity for the 9,10-oxidation.

Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites.

The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have studied the integrated metabolism of BP in vivo in germ-free rats given (14)C-labeled BP. Urinary metabolites were separated into groups according to acidity using lipophilic ion exchangers. The groups were analyzed by mass spectrometry and were further fractionated by high-performance liquid chromatography. The fraction of urinary metabolites previously shown to contain N-acetylcysteine and glucuronic acid conjugates was found to contain derivatives of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. These compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surprisingly, ring opening is a major pathway for metabolism of BP in the germ-free rat. The dicarboxylic acid may be excreted in urine as an ester glucuronide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3, 4-dicarboxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.

Metabolism of benzo(a)pyrene by duck liver microsomes.

The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.

Purification and Partial Characterization of Peroxidase from Human Term Placenta of Non-smokers: Metabolism of Benzo(a)pyrene-7,8-dihydrodiol

Peroxidase (Donor: H2O2oxidoreductase EC 1.11.1.7) from human term placentae of non-smokers was purified to homogeneity by a combination of NH4Cl extraction, affinity chromatography, (NH4)2SO4precipitation, ion-exchange and gel filtration chromatography. The homogeneity of purified human placental peroxidase (HTPP) was confirmed by gel filtration, reverse phase high performance liquid chromatography (HPLC) and SDS-PAGE. Peroxidase was found to be a membrane bound enzyme. A high concentration of NH4Cl (1.2m) was needed to extract and solublize the enzyme. Removal of the salt resulted in irreversible precipitation of the enzyme. The protein exhibited a molecular mass of 126000kDa according to gel filtration and approximately 60000kDa by SDS-PAGE, indicating that the peroxidase is a homodimer. The purified peroxidase showed an optimum pH range of 7 to 8.5 and the Kmfor H2O2and guaiacol were found to be 0.08m m and 10.0m m, respectively. The purified peroxidase oxidized several substrates, namely potassium iodide, tetramethyl benzidine, guaiacol, ortho dianisidne and tyrosine. The enzyme was resistant to thermal denaturation up to 70°C and also to chaotropic agents, guanidinium chloride and urea. Spectral properties indicated the presence of Soret band at 433 which shifted to 451nm on complexation with cyanide. The circular dichroism studies showed that HTPP has a predominantly helical secondary structure. The enzyme showed similarities to the myeloperoxidase with regard to spectral and catalytical properties but differed significantly in amino acid composition, the Rzvalue and molecular mass. Purified HTPP differed from eosinophil peroxidase in all physico-chemical properties indicating that it is not of eosinophil origin, but may represent a distinct, constitutive peroxidase in human placenta. Further, purified peroxidase catalyzed oxidation of benzo(a)pyrene-7,8-dihydrodiol in presence of tyrosine and hydrogen peroxide to BP-tetrols, the hydrolytic products of BP-diol-epoxides, demonstrating the ability of peroxidase in bioactivation of benzo(a)pyrene in human placenta.

Evidence for Dialdehyde/Imine Intermediate Formation During the Metabolism of Benzo[A]Pyrene-7,8-Dihydrodiol by Horseradish Peroxidase

Several enzymatic systems can activate the proximate carcinogen benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) to a reactive species which results in a chemiluminescence (CL) response and genotoxic lesions. This CL response is thought to be produced by the formation of a dioxetane from BP-7,8-diol which upon ring-opening forms a highly reactive dialdehyde intermediate. When BP-7,8-diol is incubated with horseradish peroxidase (HRP) and hydrogen peroxide, the CL observed was increased and prolonged by the addition of the endogenous amine, taurine. This CL response cannot be attributed to either BP-7,8-diol-9, 10-epoxide or its interaction with taurine, but is similar to the response of a known Schiff base formation. HPLC analysis of the metabolism products of the HRP reaction with taurine using radiolabeled BP-7,8-diol showed the existence of a new UV peak with an associated radioactive peak Because aldehydes have been shown to interact with cellular macromolecules, both the increased production of photons and the formation of a new electrophilic species from this activation mechanism may cause significant DNA and/or protein damage.

Species-dependent Metabolism of Benzo[c]phenanthrene and Dibenzo[a, l]pyrene by Various CYP450 Isoforms

The metabolism of benzo[c]phenanthrene (B[c]Ph) and dibenzo[a, l]-pyrene (DB[a, l]P) with various CYP isoforms including rat 1A1, 1A2, 2B1, 2E1, human 1A1, 1A2, 1B1, 2A6, 3A4, 2E1 and fish 1A expressed in Chinese hamster V79 cells has been compared. Major differences in the catalytic activities and in the regioselectivity of the eleven CYP isoforms with B[c]Ph and DB[a, l]P as substrates have been observed. There have been found substantially species-specific differences between homologous CYP isoforms at least when human, rat and fish are compared, which have to be taken into account when animal experiments are extrapolated to human. In particular, complementary catalytic activities of human CYP isoforms 1A1 and 1B1 could be demonstrated with B[c]Ph as substrate, i.e. CYP 1A1 is the major isoform involved in the formation of the proximate B[c]Ph 3,4-dihydrodiol metabolite, whereas its further oxidation to the ultimately carcinogenic fjord-region B[c]Ph-3,4-diol 1,2-epoxide metabolite is predominantly catalyzed by CYP 1B1.

Lung cancer risk in relation to genetic polymorphisms of microsomal epoxide hydrolase among African-Americans and Caucasians in Los Angeles County

Microsomal epoxide hydrolase participates in the metabolism of benzo[a]pyrene, an important carcinogen in tobacco smoke. Two relatively common polymorphisms of the microsomal epoxide hydrolase gene that influence enzyme activity have been described. An association between genetic variation in microsomal epoxide hydrolase and lung cancer risk has been reported in one of two studies of Caucasians. We examined the relation between these two polymorphisms and lung cancer risk among 337 incident cases and 700 population controls of African-American and Caucasian ethnicity enrolled in a case-control study in Los Angeles County. African-Americans, homozygous for the exon 3 variant allele conferring reduced activity, were at decreased risk of lung cancer (odds ratio (OR)=0.08, 95% CI 0.01–0.62). When data from both the exon 3 and exon 4 polymorphisms were combined into indices of predicted microsomal epoxide hydrolase activity, a decreased risk was seen among African-American subjects with very low predicted activity OR=0.10 (95% CI 0.01–0.83). No comparable association was seen among Caucasians. Although the three published results for Caucasians are somewhat variable, the association among African-Americans in these data provides some support for the hypothesis that genetically reduced microsomal epoxide hydrolase activity may be protective against lung cancer.

Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase.

Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High-performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of approximately 3 nmol min(-)(1) nmol of P450(-)(1) only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min(-)(1) nmol of P450(-)(1); the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]pyrene carcinogenesis.

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

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

Development of cytochrome P450 as a biomarker of organic pollution in Mytilus sp.: field studies in United Kingdom ('Sea Empress' oil spill) and the Mediterranean Sea

Literature on the properties, forms and regulation of cytochrome P450 (CYP) in digestive gland of Mytilus sp., including studies indicating the existence of an organic contaminant-inducible CYP1A-like protein, are briefly reviewed. Laboratory and field studies show increases in digestive gland microsomal CYP1A-immunopositive protein levels and/or benzo[a]pyrene hydroxylase (BPH) activity (i.e. metabolism of benzo[a]pyrene to phenols) with exposure of Mytilus sp. to certain polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls. In order to examine further the relationship between these two parameters, M. edulis were collected 25 and 130 days after the release of oil following the grounding of the tanker 'Sea Empress' in South Wales, UK (15 February 1996); and M. galloprovincialis were sampled from sites in south-western France and south-eastern Spain during a cruise aboard the IFREMER Research Vessel 'L'Europe' (2-18 August 1996). In both studies, sites with higher levels of CYP1A-immunopositive protein showed higher levels of BPH activities. Positive correlations were observed between the two measurements - R=0.65 (M. edulis) and 0.68 (M. galloprovincialis), and both fitted linear regression models (P < 0.05). The CYP1A-immunopositive protein levels and BPH activities tended to be highest at sites with greatest PAH body burden for the Mediterranean study. It is concluded that development of the CYP1A-like protein into a robust biomarker of exposure to organic contaminants will depend upon sequencing of the gene/protein and the subsequent production of Mytilus-specific cDNA and antibody probes. Such probes will then allow a full characterization of the enzyme's properties and gene regulation.

Regio- and stereoselectivity in the metabolism of benzo[ c]phenanthrene mediated by genetically engineered V79 Chinese hamster cells expressing rat and human cytochromes P450

Regio- and stereoselective metabolism mediated by cytochrome P450 (CYP) and metabolite-dependent cytotoxicity of benzo[ c]phenanthrene (B[ c]Ph) and its trans-3,4-dihydrodiol, the metabolic precursor of the carcinogenic fjord-region B[ c]Ph-3,4-dihydrodiol 1,2-epoxides (B[ c]PhDE), were investigated with V79 Chinese hamster cells genetically engineered for three rat and six human CYP isoforms. The order of the capabilities of the CYP isoforms to metabolize B[ c]Ph was as follows: h1A1>r1A1>r1A2>h1B1>h1A2>r2B1>>h2E1>h2A6>h3A4. Regardless of the species, all individual CYP isoforms preferentially catalyzed the oxidation of B[ c]Ph at the 5,6-position (K-region) except human CYP1A1 and human CYP1A2, which oxidized both the 5,6- and the 3,4-position with similar efficiency. While human CYP1A1, rat CYP1A1 and rat CYP1A2 formed almost exclusively the (−)-B[ c]Ph-3 R,4 R-dihydrodiol, human CYP1A2 produced both the (−)-3 R,4 R- and the (+)-3 S,4 S-dihydrodiol enantiomers in a ratio of 2:1. Stereoselective activation of B[ c]Ph, the (±)-B[ c]Ph-3,4-dihydrodiol and its (−)-3 R,4 R-enantiomer to the fjord-region (−)- anti-B[ c]PhDE occurred upon incubation with rat CYP1A1 and rat CYP1A2 as indicated by the formation of two stereoisomeric tetraols, the hydrolysis products of the labile anti-B[ c]PhDE. The formation of tetraols in the culture medium was accompanied by a concentration-dependent increase in cytotoxicity indicating that this effect was mediated by the fjord-region (−)- anti-B[ c]PhDE formed as reactive intermediate. All human and rat CYP-expressing V79 cell lines investigated did not show any significant capacity to metabolize the (+)-3 S,4 S-dihydrodiol. The present study indicates that the human CYP isoforms 1A1 and 1B1 have complementary catalytic properties to activate B[ c]Ph to its fjord-region B[ c]PhDE, whereas other human isoforms play a minor role. Activation of B[ c]Ph by human CYP1A1 and 1B1 is less efficient than by rat CYP1A1 or rat CYP1A2, but proceeds with similar stereoselectivity via the (−)-3 R,4 R-dihydrodiol to the strong carcinogen (−)- anti-B[ c]PhDE with ( R, S, S, R)-configuration.

Comparison of Fresh and Room-Aged Cigarette Sidestream Smoke in a Subchronic Inhalation Study on Rats

Two experimental types of cigarette sidestream smoke (SS) were compared in a subchronic inhalation study on rats. Fresh SS (FSS) was generated continuously from the reference cigarette 2R1. Room-aged SS (RASS) was generated by aging FSS for 1.5 h in a room with noninert surfaces with materials typically found in residences or offices. Male Sprague-Dawley rats were head-only exposed to three dose levels of each SS type and to filtered, conditioned fresh air (sham-exposure) for 6 h/day, 7 days/week, for 90 days. Room-aging resulted in decreased concentrations of various SS components, e.g., total participate matter (TPM) and nicotine, while other components, such as carbon monoxide (CO), were not affected. The CO concentrations were 6, 13, and 28 ppm for both SS types. TPM concentrations were between 0.6 and 8.7 (μg/liter and thus up to 100-fold above the maximum of average concentrations of respiratory suspended particles reported for environmental tobacco smoke. Slight reserve cell hyperplasia in the anterior part of the nose as well as hyperplastic and metaplastic epithelial changes in the larynx were the only observed dose-dependent findings. The metabolism of benzo(a)-pyrene-as a proxy for polycyclic aromatic hydrocarbon metabolism-was induced in the nasal respiratory epithelium and in the lungs while no effect was seen in the nasal olfactory epithelium. The lowest-observed effect level was 6 ppm CO or 0.6 μg TPM/liter. Most of the effects seen were less expressed in RASS- than in FSS-exposed rats when compared on the basis of the CO concentrations. When compared on the basis of TPM, these effects were equally pronounced for both SS types, suggesting a major role of particulate matter-associated compounds. All findings reverted to sham control levels following a 42-day postinhalation period.

The uptake, distribution and metabolism of benzo[a]pyrene in coho salmon (Oncorhynchus kisutch) during the parr‐smolt transformation

AbstractBenzo[a]pyrene (BaP) uptake, distribution, and metabolism patterns were investigated from February to June during the transformation of freshwater coho salmon (Oncorhynchus kisutch) parr to smolts. At a BaP concentration of 5 μg/L, uptake rates increased significantly from 0.01 ± 0.000 μg/g/h in February to 0.04 ± 0.003 μg/g/h in May and declined to 0.035 ± 0.004 μg/g/h in June. Following a 24‐h exposure to BaP, the highest percent of body burden of BaP was found in the liver, gills, skin, and bile. The proportion of BaP in the liver and gills increased in fish from February to May and declined in June, whereas the proportion of BaP in the bile continued to rise until June when it reached a maximum of 49% of the body burden. The percent body burden of BaP in tissues such as the stomach, intestine, visceral fat, muscle, and brain did not show significant changes through the duration of the study. An analysis of bile suggests that both coho salmon parr and smolts are capable of metabolizing BaP via phase I and II biotransformation reactions to glucuronide, sulfate, and other conjugated metabolites. No significant changes occurred in the proportions of metabolite classes during the parr–smolt transformation process.

Uptake and metabolism of benzo(a)pyrene absorbed to sediment by the freshwater invertebrate species Chironomus riparius and Sphaerium corneum.

The polyaromatic hydrocarbon (PAH) Benzo(a)pyrene (BP) is a widespread contaminant, which is known to be carcinogenic in mammals after ic activation. BP is released into the environment and the water as a by-product of combustion of fossil and recent material (fuel, wood) in industry, traffic and households and is also released by natural sources. Most of the PAHs are highly lipophilic and therefore bound to humic substances, dissolved macromolecules and particulate matter which are at least deposited in the aquatic sediments. The BP concentrations in sediments of pristine waters do normally not exceed 1 {mu}g/g dry weight (dw). In polluted waters of industrial areas, the BP concentration may increase up to 100 {mu}g/g dw. The risk for environmental health caused by such sediment bound PAHs can be assessed by using BP as a model substance. One aim of this study was to investigate if the sediment bound BP is bioavailable to sediment dwelling organisms. For this purpose we examined the uptake of sediment bound BP. The metabolism of PAHs in insects has been investigated, however, only little is known about the Phase I and Phase II metabolism in clams, especially in freshwater species. The organisms choosen were two sediment inhabitingmore » invertebrates, the larvae of the midge Chironomus riparius and the European fingernail clam Sphaerium corneum. Also investigated was the question of whether the BP taken up by the test organisms undergoes metabolic activation, since the toxicity of BP is modulated by metabolism. 11 refs., 3 figs., 4 tabs.« less

Metabolism Of Benzo[a]Pyrene in Fish Hepatocytes Cultured on Microplates

Abstract The metabolism of benzo[a]pyrene (BP) by trout and catfish hepatocytes seeded on 96-well microplates was examined. The 9,10-; 4,5-; and 7,8-dihydrodiols, 1,6-; 3,6- quinones and 3-OH BP were found to be the major metabolites in both the trout and catfish hepatocytes exposed to BP for 4 h. The results were compared with the corresponding long term culture of hepatocytes on porous membranes and in microsomes isolated from the whole liver. It was found that cells cultured on microplates retained their BP metabolizing activities for at least 6 d after seeding. This method has the major advantage that only a small number of cells are required and in parallel, it is possible to perform cytotoxicity and cytogenetic microassays on cell populations characterized for their metabolic capacity.

Contribution of human cytochrome P450 to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast.

The metabolism of benzo[a]pyrene (B[a]P) and its proximate mutagen B[a]P-7,8-dihydrodiol (7,8-diol) was investigated in the presence of human microsomal epoxide hydrolase and P450 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 expressed in the yeast Saccharomyces cerevisiae. P450 1A1 had the highest turnover numbers for the formation of all B[a]P metabolites, including phenols and dihydrodiols. P450 1A2, 2C8, 2C9, 2C18, 2C19 and 3A4, which are well represented in the liver, gave rise to the formation of appreciable amounts of 3-hydroxy-B[a]P and of some dihydrodiols from B[a]P. When 7,8-diol was used as substrate, P450 1A1 also exhibited the highest turnover numbers for the formation of tetrols, the hydrolysis products of the diolepoxides, whereas P450 1A2, 2C8, 2C19 and 3A4 showed moderate activities. In order to test the validity of the yeast system, the contribution of each P450 isoform to B[a]P and 7,8-diol metabolism was evaluated as the product of the turnover numbers of recombinant P450s by specific contents of each P450 in human liver microsomes. Calculated formation rates for each B[a]P and 7,8-diol metabolite globally matched experimental values. There is evidence that P450 3A4 and 2C9 play a major role in the formation of 3-hydroxy-B[a]P from B[a]P. Accumulation of the proximate mutagen 7,8-diol was predicted to be mainly driven by P450 1A2, 2C9 and 2C19, while formation of the genotoxic diolepoxides from 7,8-diol appeared to be dependent on P450 1A2 and 3A4 in the liver.

Regio-Selective Metabolism of Benzo(a)pyrene by Microsomes from Rat and Sea Bass: Evidences for the Occurrence of the 1-Hydroxybenzo(a)pyrene as a Major Metabolite

Abstract The aim of the study was (i) to improve benzo(a)pyrene (BaP) metabolite analysis, (ii) and to establish that 1-hydroxybenzo(a)pyrene (1-phenol) is a major BaP metabolite produced by vertebrate xenobiotic metabolism. The species studied were sea bass (Discentrarcus labrax) and male wistar rat treated with 3-methylcholanthrene (MC). Comparison of the different conditions concerning the oxidized positions in the BaP molecule, was performed and supported by statistical calculations. BaP 9,10-, 4,5-, 7,8-diols, 1,6-, 3,6-, 6,12-quinones and 9-, 1-, 3-phenols in the elution order are the major BaP metabolites detected with both species. This was confirmed by the retention time of cold standards and by the measurement of the UV spectrum of the 9-, 1- and 3-phenols of BaP extracted from incubations of MC treated rat microsomes in the presence of 1,1,1-trichloro-2-propeneoxide (TCPO), an epoxide hydrolase inhibitor. These results support that cytochrome P450 is able to oxidize BaP in six positions, the 3≫...

Bioaccumulation and metabolism of benzo[A]pyrene in three species of polychaete worms

AbstractPolychaete worms which inhabit and ingest contaminated sediments may be exposed to a suite of sediment‐sorbed xenobiotics, including polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP). This study compared the accumulation and metabolism of sediment‐sorbed BaP among various populations of polychaetes, including Leitoscoloplos fragilis, Nereis diversicolor, and Scolecolepides (= Marenzellaria) viridis. After exposure to trace amounts of BaP in sediment, N. diversicolor and S. viridis were found to metabolize BaP extensively, but metabolism of BaP appeared to be much slower in L. fragilis. Within each species, no consistent, significant differences in extent of BaP metabolism were observed between worms collected from contaminated versus reference sites. This result suggests that metabolism of BaP was not induced in worms from contaminated sites. To examine the extent to which metabolism of BaP is inducible in these species, worms were exposed in the lab to 3‐methylcholanthrene (3MC), a potent inducer of PAH metabolism in other species. Only one species, S. viridis, showed a small increase in the percent of total tissue BaP that was biotransformed after treatment with 3MC. Whether or not worms were treated with 3MC, biota‐sediment accumulation factors (lipid‐ and organic carbon‐normalized tissue to sediment ratios) were highest for L. fragilis.