Pyrene Metabolism Research Articles

Pyrene and benzo(a)pyrene metabolism by an Aspergillus terreus strain isolated from a polycylic aromatic hydrocarbons polluted soil.

A strain of Aspergillus terreus was isolated from a polycyclic aromatic hydrocarbons (PAHs) polluted soil. The metabolism of pyrene and benzo(a)pyrene by this fungus was investigated in liquid submerged culture added of 50 and 25 ppm respectively of each compound. Depletion of pyrene and Benzo(a)pyrene was evident during the first stages of growth and was 60% and 27.5% respectively of the added amount after nine days of culture. Solvent extracts of the fermentation broth and mycelium were analysed for presence of metabolites by HPLC-MS technique. Under the present cultural conditions pyrene was mainly metabolised to pyrenylsulfate similarly to benzo(a)pyrene that led to benzo(a)pyrenylsulfate. The structure of 1-pyrenilsulfate was determined after purification of extracts and H-NMR analysis. The result show that the isolated A. terreus strain metabolises PAHs by reaction similar to those previously reported for non lignolinolytic fungi with a mechanism that suggests the hydroxylation by a cytochrome P-450 monooxygenase followed by conjugation with sulfate ion.

Interaction of tributyltin with hepatic cytochrome P450 and uridine diphosphate-glucuronosyl transferase systems of fish: in vitro studies.

Hepatic microsomes of red mullet (Mullus barbatus) and flounder (Platichthys flesus) were preincubated in the presence of a concentration range of the antifouling agent tributyltin (TBT) chloride, and the interactions of TBT with cytochrome P450 and uridine diphosphate-glucuronyl transferase systems were investigated. The enzyme systems were examined in terms of cytochrome P4501A (CYP1A)-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity and benzo[a]pyrene (BaP) metabolism and in terms of glucuronidation of testosterone and 17beta-estradiol, respectively. Ethoxyresorufin O-deethylase and BaP hydroxylase (BPH) activities of both fish species were progressively inhibited by increasing concentrations of TBT, and the effects were more pronounced for EROD than for BPH (maximal inhibition at 100 microM TBT for EROD and 250-500 microM TBT for BPH). Hydroxylated metabolites of BaP (3-hydroxy-, 7,8-dihydrodiol, and 9,10-dihydrodiol), representing 95% of the total metabolites formed, were reduced up to 75% in the presence of 100 to 500 microM TBT, whereas the formation of other metabolites was less affected. This may alter BaP toxicity and carcinogenicity. Overall, the results were consistent with a specific inhibitory effect of TBT on CYP1A in the two fish species. Additionally, the conjugation of testosterone was significantly inhibited (20%) at low TBT doses (5 microM), with no effect on the glucuronidation of estradiol.

Differential roles of Arg97, Asp293, and Arg108 in enzyme stability and substrate specificity of CYP2C9.

CYP2C9 metabolizes a wide range of drugs, many of which are negatively charged at physiological pH. Therefore, it has been thought that complementarily charged amino acid(s) are critically involved in substrate binding. Previous studies have implicated arginine residues at positions 97, 105, and 108 and aspartate at position 293 in the normal catalytic function of the enzyme. To elucidate the role of these amino acids in the substrate specificity of CYP2C9, a series of mutants were constructed and analyzed for functional activity, thermal stability, and ligand binding. Charge-modifying mutations at positions 97, 105, and 293 decreased catalytic activity toward diclofenac, (S)-warfarin, and pyrene in a substrate-independent manner with Arg105 the least, and Arg97 the most, sensitive amino acids in this regard. Decreases in functional activity paralleled thermal instability of the mutants, suggesting that loss of function reflects more generalized structural changes rather than the absence of a specifically charged amino acid at these three positions. The R108H mutant was inactive toward all three substrates because of unexpected nitrogen ligation to the heme. Conversely, the R108F mutant exhibited substrate-dependent catalytic behavior, with almost complete loss of activity toward (S)-warfarin and diclofenac, but preservation of pyrene metabolism. In addition, the R108F mutation abrogated the Type I difference spectra induced by flurbiprofen and benzbromarone, obligate anions at physiological pH. These data identify critical roles for Arg97 and Asp293 in the structural stability of the enzyme and demonstrate a selective role for Arg108 in the binding and metabolism of negatively charged substrates of CYP2C9.

Energetic costs of pyrene metabolism in isolated hepatocytes of rainbow trout, Oncorhynchus mykiss

The respiratory costs of pyrene exposure and biotransformation were examined in isolated hepatocytes of adult rainbow trout, Oncorhynchus mykiss. Baseline oxygen consumption rates measured at an acclimation temperature of 7.5 °C and during an acute temperature increase to 15 °C were 10.1±0.1 and 22.6±0.4 ng O 2/min/mg cells, respectively. Hepatocytes exposed to pyrene at 1, 5 and 10 μg/ml exhibited concentration-dependent increases in oxygen consumption. Respiration rates of cells exposed to these concentrations at their acclimation temperature were 12.5±0.1, 14.7±0.1 and 17.1±0.2 ng O 2/min/mg cells, respectively. Exposure of cells to pyrene at 15 °C also elevated oxygen consumption to a maximum of 34.4±0.3 ng O 2/min/mg cells, however, the relationship with pyrene concentration was biphasic. The major metabolite identified through a series of solvent extractions, acid hydrolysis, and synchronous fluorometric spectroscopy was conjugated 1-hydroxypyrene. At 7.5 °C, increased pyrene metabolism correlated with increased hepatocyte respiration rates. At 15 °C, however, pyrene metabolism reached a maximum at 5 μg/ml, suggesting saturation of detoxification enzymes, which correlated with maximum respiration rates at this concentration. Measures of respiration by isolated mitochondria indicated that changes in hepatocyte oxygen consumption were not through direct effects of pyrene on mitochondria. This study indicates that significant respiratory costs may be accrued by teleost hepatocytes actively metabolizing and secreting xenobiotic compounds.

In Vitro and In Vivo Studies on Oxygen Free Radical and DNA Adduct Formation in Rat Lung and Liver during Benzo[a]pyrene Metabolism

Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.

Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1.

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.

Identification of cytochrome P450 isoforms involved in 1-hydroxylation of pyrene

Urinary 1-hydroxypyrene (1-OHP) has been used as a biomarker for environmental polyaromatic hydrocarbon (PAH) exposure. However, it is known that there is an interindividual variability in metabolism of pyrene to 1-OHP depending on the activities of the metabolizing enzymes, especially cytochrome P450s (CYPs). In this study, we investigated the 1-hydroxylation of pyrene by 10 forms of cDNA-expressed human P450s in order to identify the principal isoforms of P450s that are involved in the major metabolic pathway of pyrene. The pyrene 1-hydroxylation activity was found to be the highest in CYP1A1 at both 0.5 and 50 μM of pyrene, followed by CYP1B1 and 1A2, whereas other enzymes, including CYP2A6, 2C8, 2C9*1, 2C19, 2D6, 2E1, 3A4, and control microsomes, showed very low or undetectable rates of 1-hydroxylation. In conclusion, CYP1A1, 1B1, and 1A2 are major metabolizing enzymes in 1-hydroxylation of pyrene in vitro. This suggests that the individual difference of these enzymes must be included in epidemiological studies to evaluate PAH exposure using urinary 1-OHP.

Pyrene metabolism in the springtailOrchesella cinctaL. (Collembola, Entomobryidae)

A novel, solvent-free spiking method was used to contaminate foodstuff (Desmococcus spp. algae) with pyrene for bioassays with the springtail Orchesella cincta L. (Collembola, Entomobryidae). The primary metabolite 1-hydroxypyrene and two conjugates (pyrene-1-glucoside and one tentatively identified as pyrene-1-glucosidemalonate) were quantified in the insects by high-performance liquid chromatography (HPLC) with fluorescence detection. Individuals from an unpolluted site (SUS population) were capable of rapid metabolism of pyrene, reaching an apparent steady state after 24 h with a composite assimilation/transformation rate of approximately 25 pmol pyrene x g(-1) fresh weight x h(-1) at an exposure level of approximately 18 microg pyrene x g(-1) dry weight algae. A cadmium (Cd)-tolerant population (TOL), genetically distinct from SUS, was exposed in parallel and the populations' pyrene metabolism compared in order to gain insight into any potential cost of Cd-tolerance. The TOL animals assimilated and/or performed phase 1 transformation of pyrene approximately twice as fast as SUS (approximately 56 pmol x g(-1) x h(-1)), but there was no significant difference in the elimination rate constants between SUS and TOL animals. Attributing this difference to any specific cost of Cd-tolerance requires more work and may he coincidental to metal tolerance, but pyrene metabolism in these distinct genotypes, subjected to different selection pressures, was nevertheless significantly different.

Genetic effects on urinary 1-hydroxypyrene levels in a Korean population.

Urinary 1-hydroxypyrene (1-OHP) has been used as a biomarker for assessing the level of exposure to environmental carcinogenic polycyclic aromatic hydrocarbons (PAHs). In order to perform the appropriate biological monitoring for examining the level of exposure to PAHs, this study investigated whether or not genetic polymorphisms of the metabolic enzymes, which might be involved in the metabolism of pyrene, affected the urinary 1-OHP levels in a population of 661 Koreans (male, 63%; female, 37%; mean age, 36.5 +/- 11.1 years) who were not occupationally exposed to PAHs. Urinary 1-OHP was detected in 76% of the subjects (range 0.001-3.8 micro g/l). Among the physical and lifestyle factors, cigarette-smoking was found to be associated with the urinary 1-OHP levels (P < 0.05). After adjusting for these factors, we found that the GSTT1 genotypes affected the urinary 1-OHP levels, i.e. the GSTT1 present subjects had approximately 1.5 times the urinary 1-OHP level than the GSTT1 null subjects (P < 0.05). In the case of the subjects who were also GSTM1 null, this trend became stronger, i.e. the GSTT1 present subjects had approximately 2 times the urinary 1-OHP level (P < 0.01). However, the genetic polymorphism of the other metabolic enzymes, cytochrome P-450 (CYP)1A1, CYP1B1 and GSTM1 alone, did not affect the urinary 1-OHP level. Therefore, this study suggests that the GSTT1 genetic polymorphism has the potential to affect the biological monitoring of PAHs with urinary 1-OHP, and might act as a genetic factor in PAH-related toxicity.

Metabolism of pyrene by two clam species, Mya arenaria and Protothaca staminea.

Two species of marine clam, Mya arenaria and Protothaca staminea, were exposed to pyrene and 1-hydroxypyrene in small glass aquaria. After 10 days of exposure the clams were sacrificed, and both clam tissue and seawater were assayed for pyrene metabolites by using HPLC, fluorescence spectroscopy, HPLC-ESI-MS, GC-MS and 1H-NMR spectrometry. 1-Pyrenol-1-hydrogensulfate (pyrene-1-sulfate) was identified as the major water soluble metabolite formed from both pyrene and 1-hydroxypyrene by both species of clam. 1-Hydroxypyrene was identified as a minor metabolite of pyrene, and pyrenediol-hydrogen sulfate was identified as a minor metabolite of 1-hydroxypyrene.

Metabolism of 1-fluoropyrene and pyrene in marine flatfish and terrestrial isopods

Monofluorinated polycyclic aromatic hydrocarbons (F-PAHs) are useful reference compounds for a broad spectrum of PAH studies. The pyrene metabolite 1-hydroxypyrene is often used as a biomarker of PAH exposure. Two species, isopod ( Porcellio scaber) and flatfish ( Platichthys flesus), that produce 1-hydroxypyrene as the major intermediary metabolite and have distinct phase-II conjugates, were selected to investigate the cytochrome P450 catalyzed metabolism of 1-fluoropyrene. The fluorine atom blocks one of the four most favored carbon atoms, yielding different metabolite patterns and the results obtained in the selected species were compared with metabolite profiles obtained for unsubstituted pyrene. Charge densities in 1-fluoropyrene measured with 13C-NMR were used to predict possible positions of hydroxylation, which were confirmed by 19F-NMR. Additionally, the retention behaviour of conjugated 1-fluoropyrene metabolite isomers in reversed-phase liquid chromatography on a polymeric alkyl-bonded phase was interpreted based on the slot model. Whereas three phase-I metabolites were found in isopod hepatopancreas, only two were observed in flatfish bile. Phase-II metabolism appeared unaffected by the fluorine substituent. It was concluded that the phase-I enzyme cytochrome P450 is non-regioselective in the isopod: the activation is mostly influenced by the electron density distribution. In contrast, the enzymatic oxidation in the flounder is more selective. These differences will affect to what extent pyrene metabolite measurements can be used to assess the impact of PAHs to different species.

URINARY EXCRETION KINETICS OF 1-HYDROXYPYRENE IN RATS SUBCHRONICALLY EXPOSED TO PYRENE OR POLYCYCLIC AROMATIC HYDROCARBON MIXTURES

The urinary excretion kinetics of 1-hydroxypyrene (1-OHP) were studied in male Sprague-Dawley rats exposed orally, on Tuesdays and Fridays for 10 consecutive weeks, to 0.046 mg/kg/d of pyrene or 0.3, 1 or 3 mg/kg/d of polycyclic aromatic hydrocarbon (PAH) mixtures containing pyrene (0.046, 0.15, and 0.46 mg/kg/d, respectively). During the subchronic exposure, 24-h urine samples were collected on Mondays (prior to exposure) and Tuesdays (after exposure) for all exposure groups. During a 14-d period following last exposure in rats treated with 3 mg/kg/d of PAH mixture, 24-h urine samples were collected at frequent time intervals (0-24, 48-72, 96-120, 144-168, 193-217, 313-338 h). Whatever the administered dose, repeated exposures to pyrene and PAH mixtures resulted in a progressive time-dependent increase in the daily urinary excretion of 1-OHP. After 10 wk of treatment, daily excretion rates were on average 5 times higher than those observed after the first administration. Following the subchronic exposure to 3 mg/kg/d of PAH mixture, the time profile of 1-OHP excretion in rat urine showed a multiphasic elimination. An average first-order apparent elimination half-life of 26.5 h could be estimated for the 48-168 h period following the end of the exposure. The observed time-dependent increase in 1-OHP excretion values upon repeated exposures to PAHs does not appear to result from a PAH enzyme induction effect of pyrene metabolism to 1-OHP. Rather, the slow release of residual pyrene accumulated in a long-term compartment and/or the enterohepatic recirculation of 1-OHP and other pyrene metabolites may play significant roles in the observed urinary excretion kinetics of 1-OHP. Furthermore, the absence of mixture effect on the urinary excretion of 1-OHP suggests that 1-OHP is a good bioindicator of exposure to complex PAH mixtures, in the dose range used in this study.

Effects of the polychaetes Nereis diversicolor and Arenicola marina on the fate and distribution of pyrene in sediments

Effects of the polychaetes Nereis diversicolor and Arenicola marina on the distribution, metabolism and removal of [ 14 C-4,5, 9 ,10]-pyrene in sediment microcosms were studied over 42 and 53 d, respectively. Unmetabolized pyrene, water- and organic-soluble pyrene metabolites were quantified in sediment, interstitial water and overlying water, and 14 CO 2 production was also assessed. Both N. diversicolor and A. marina enhanced fluxes of sediment-associated pyrene and metabolites into overlying water but to different extents. The major pathway of pyrene removal from sediments with N. diversicolor was release of water-soluble metabolites, probably formed by metabolism in N. diversicolor. In contrast, flushing of unmetabolized pyrene due to bioturbation was the major removal route from sediments with A. marina. Additionally, increased 14 CO 2 production and concentrations of water-soluble metabolites were also observed in overlying water indicating that A. marina also stimulated the metabolism of pyrene. The high production of water-soluble metabolites was probably due to endogenous metabolism in the lugworm itself, whereas the increased 14 CO 2 production indicated a stimulation of microbial metabolism. After 42 d, only 75 % of the originally added amount of pyrene remained associated to sediment particles in microcosms inhabited by N. diversicolor, whereas no significant reduction was observed in microcosms with A. marina due to the necessity of using large amounts of sediment in the lugworm experiment. The actual flux of pyrene and metabolites to overlying water was higher in microcosms with A. marina than with N. diversicolor. Both A. marina and N. diversicolor significantly enhanced the removal pyrene from the sediment, but differences in feeding and life strategies resulted in different removal paths and extents.

Cytochrome P4502C9 genotype in Southeast Anatolia and possible relation with some serum tumour markers and cytokines.

Substrates for CYP2C9 include fluoxetine, phenytoin, warfarin, losartam and numerous nonsteroidal anti-inflammatory drugs. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino-acid residues 144 Arg/Cys and 359 Ile/Leu of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, the frequency of this allele is, however, rather low. Consistently with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. The polymorphic enzyme CYP2C9 takes part in the metabolism of alkylating agents and polycyclic aromatic hydrocarbons like benzo(a)pyrene, a carcinogen present in tobacco smoke. Although the impact of impaired enzyme activity in metabolism of carcinogens and procarcinogens has not been fully defined, an association of CYP2C9 variant alleles to DNA adduct levels in lung tissues as well as to lung cancer risk have been reported. In this study 64 healthy subjects (44M/22F) were analysed for CYP2C9 genotype with PCR-RFLP and for serum carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA 19-9, CA 15-3, ferritin, IL-6, IL-8 concentrations by chemiluminescence or electrochemiluminescence methods. CYP2C9*1 was found to be the most prevalent allele and CYP2C9*1/CYP2C9*1 was the most frequent genotype represented in 64% of the population in southeastern Anatolia (Gaziantep). Although slight differences in serum tumour marker and cytokine concentrations were observed for CYP2C9 genotypes the differences were statistically insignificant (P > 0.05). This could be due to the complexity of the role of CYP2C9 in benzo(a)pyrene metabolism as well as from other contributing factors like interindividual variability of diverse enzymes participating in the same metabolic pathway, unequal expression of the variant alleles and differences in exposure to carcinogens. However, determination of CYP2C9 phenotypes in a larger group of subjects might clarify these slight differences.

Effects of occupation, lifestyle and genetic polymorphisms of CYP1A1, CYP2E1, GSTM1 and GSTT1 on urinary 1-hydroxypyrene and 2-naphthol concentrations.

This study was undertaken to determine the effects of occupation, lifestyle and the genetic polymorphisms of cytochrome P450 1A1 (CYP1A1), cytochrome P450 2E1 (CYP2E1) and glutathione S-transferases micro1 (GSTM1) and 1 (GSTT1) on the concentrations of urinary 1-hydroxypyrene (1-OHP) and 2-naphthol among Korean coke oven workers and university students. The study subjects included 90 coke oven workers and 128 university students. A questionnaire was used to obtain detailed data about the work area, smoking habits and food intake of subjects. Associations between urinary polycyclic aromatic hydrocarbon (PAH) concentrations and occupation, smoking status, total airborne PAH level and genetic polymorphisms were tested. Urinary 1-OHP and 2-naphthol concentrations were higher in coke oven workers than in students and correlated significantly with work area. Urinary 2-naphthol concentrations increased with an increase in the level of cigarette smoking in students. Total airborne PAH level correlated with urinary 1-OHP concentration in coke oven workers. Urinary 1-OHP and 2-naphthol concentrations were higher in coke oven workers with the c1/c2 or c2/c2 genotype of CYP2E1 than in those with the c1/c1 genotype. Urinary 2-naphthol concentrations were higher in GSTM1-null workers than in GSTM1-positive workers. In multiple regression analysis CYP2E1 was a significant factor determining urinary 1-OHP concentrations in coke oven workers. CYP2E1 and GSTM1 were significant determinants for urinary 2-naphthol concentrations in coke oven workers and GSTM1 and smoking were prognosticators among university students. Urinary 1-OHP is a better indicator of occupational exposure to PAH in coke oven workers than 2-naphthol, whereas urinary 2-naphthol may be more sensitive for non-occupational inhalation exposure to PAH. In occupationally exposed populations CYP2E1 and GSTM1 appear to play an important role in the metabolism of pyrene and naphthalene. In individuals not occupationally exposed to PAHs GSTM1 and smoking seem to influence the urinary concentration of 2-naphthol.

Lung carcinogenesis: Resveratrol modulates the expression of genes involved in the metabolism of PAH in human bronchial epithelial cells

Studies suggest that resveratrol (trans-3,4',5-trihydroxystilbene), which is a diphenolic antioxidant found in plants and foods, has cancer chemopreventive and chemotherapeutic potential. A lower risk of lung cancer among consumers of wine compared with consumers of other beverages has been observed, which may be partly attributed to the high content of resveratrol particularly in red wine. We have studied the effect of resveratrol on the expression of genes involved in the metabolism of polycyclic aromatic hydrocarbons in the human bronchial epithelial cell line BEP2D. Expression of the cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase P1 (GSTP1) genes was measured by quantitative reverse transcriptase-polymerase chain reaction. The cells were treated either with benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin in the presence or absence of resveratrol. Resveratrol inhibited both the constitutive and the induced expression of CYP1A1 and CYP1B1 in a dose-dependent manner. In contrast, the expression of the mEH gene was increased in response to resveratrol and no change in the expression of GSTP1 was found. The altered gene expression in response to resveratrol was reflected in a reduced overall level of benzo[a]pyrene metabolism. These data indicate that resveratrol may exert lung cancer chemopreventive activity through altering the expression of genes involved in the metabolism of polycyclic aromatic hydrocarbons, resulting in altered formation of carcinogenic benzo[a]pyrene metabolites in human bronchial epithelial cells.

Monoclonal Antibody-Directed Analysis of Benzo[

Monoclonal antibody (MAb) 1-7-1 against 3-methylcholanthrene (MC)-induced forms of cytochrome P-450 (CYP) was used to characterize benzo[a]pyrene (B[a]P) metabolism in rat liver and extrahepatic tissues and its modulation by phenolic antioxidants, propyl and octyl gallates. Male Wistar rats were treated with these food additives (50 mg/kg body wt ip) twice weekly for 14 days alone or in combination with MC. Immunochemical inhibition of aryl hydrocarbon hydroxylase (AHH) and [<O*>14C]B[a]P metabolism (analyzed by high-performance liquid chromatography) were measured in liver, kidney, and lung microsomes. Organ-specific changes in levels of MAb-mediated inhibition of microsomal metabolism of B[a]P were observed. In liver microsomes from untreated rats, AHH was not affected by MAb, but in kidney and lung, there was 70% and 50% inhibition, respectively. In MC-treated rats, MAb reduced AHH activity by 43% in liver. Kidney and lung AHH was inhibited up to 80% by this MAb. Formation of B[a]P metabolites in MC-induced microsomes from liver and kidney was affected by MAb in a similar way. In lung, the total metabolism was inhibited by 50% by MAb treatment, but significant differences in inhibition of individual metabolites were observed. Treatment with propyl or octyl gallate alone had no effect on MAb inhibition of AHH activity in liver and lung but decreased the level of inhibition in kidney. Combined treatment with MC and propyl or octyl gallate slightly reduced the effect of MAb on AHH activity in liver and significantly reduced the level of inhibition in kidney but did not affect AHH activity in lung. The same treatment regimen dramatically reduced MAb inhibition of B[a]P metabolism in kidney but had no effect on B[a]P metabolite formation in liver. Inhibition by MAb of renal 3-hydroxy-B[a]P, 7,8-B[a]P-dihydrodiol, and 1,6-quinone-B[a]P was the most affected. In lung, treatment with gallates affected only formation of 7,8-B[a]P-dihydrodiol. These results suggest that treatment with gallates affects the CYP 1A and may change the CYP isozyme composition and, thus, alter the tissues' susceptibility to tumor induction by B[a]P.

Products from the Incomplete Metabolism of Pyrene by Polycyclic Aromatic Hydrocarbon-Degrading Bacteria

Products from the Incomplete Metabolism of Pyrene by Polycyclic Aromatic Hydrocarbon-Degrading Bacteria

Cloning, expression, and characterization of the katG gene, encoding catalase-peroxidase, from the polycyclic aromatic hydrocarbon-degrading bacterium Mycobacterium sp. strain PYR-1.

A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.

A survey of in vivo benzo[α]pyrene metabolism in small benthic marine invertebrates

A micro-extraction technique was used to examine in vivo polycyclic aromatic hydrocarbon (PAH) metabolism in 10 small invertebrate species exposed to sediments amended with 3H-benzo[α]pyrene (BaP). Phyla examined included Mollusca ( Hydrobia totteni, Ilyanassa obsoleta, Yoldia limatula, and Gemma gemma), Annelida ( Nereis succinea, Pectinaria gouldii, Haploscolopolous sp., and Capitella sp. 1) and Arthropoda ( Edotea triloba, and Gammarus mucronatus). Organisms were exposed to BaP-labeled sediments, harvested, and parent BaP separated from all polar metabolites by liquid extraction The percent of BaP-derived radioactivity present as polar metabolites ranged from 96% for N. succinea to 7% for P. gouldii. Wide ranges in metabolic capability were also observed between species in the other two phyla examined. Reverse-phase HPLC analysis of extracts of representative species from each phyla indicated that all these organisms form bay region metabolites, with two species forming the 7,8-dihydrodiol ( N. succinea and G. mucronatus). In light of the high variability in metabolic capability observed within each phylum, species-specific information on metabolic ability should be obtained before assessing bioaccumulation, critical body burdens, or trophic transfer of PAHs in invertebrates.