Pyrene Metabolism Research Articles

Concentration dependent effects of tobacco particulates from different types of cigarettes on expression of drug metabolizing proteins, and benzo(a)pyrene metabolism in primary normal human oral epithelial cells

The ability of tobacco smoke (TS) to modulate phase I and II enzymes and affect metabolism of tobacco carcinogens is likely an important factor in its carcinogenicity. For the first time several types of TS particulates (TSP) were compared in different primary cultured human oral epithelial cells (NOE) for their abilities to affect metabolism of the tobacco carcinogen, (BaP) to genotoxic products, and expression of drug metabolizing enzymes. TSP from, reference filtered (2RF4), mentholated (MS), reference unfiltered, (IR3), ultra low tar (UL), and cigarettes that primarily heat tobacco (ECL) were tested. Cells pretreated with TSP concentrations of 0.2–10μg/ml generally showed increased rates of BaP metabolism; those treated with TSP concentrations above 10μg/ml showed decreased rates. Effects of TSPs were similar when expressed on a weight basis. Weights of TSP/cigarette varied in the order: MS≈IR3>2RF4>ECL>UL. All TSPs induced the phase I proteins, cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), phase II proteins, NAD(P)H dehydrogenase quinone 1 (NQO1), and microsomal glutathione S-transferase 1 (MGST1), and additionally, hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2), as assessed by qRT-PCR. The pattern of gene induction at probable physiological levels favored activation over detoxification.

Influence of cultivation conditions on pyrene degradation by the fungus Pleurotus Ostreatus D1

For the first time the dependence of completeness of pyrene degradation by the white-rot fungus Pleurotus ostreatus D1 on cultivation conditions was found. In Kirk’s medium about 65.6 ± 0.9% of the initial pyrene was metabolized after 3 weeks, with pyrene-4,5-dihydrodiol accumulating. This process was accompanied by laccase production only. In basidiomycetes rich medium, P. ostreatus D1 metabolized up to 89.8 ± 2.3% of pyrene within 3 weeks without pyrene-4,5-dihydrodiol accumulation throughout the time of cultivation. Phenanthrene and phthalic acid were identified as the metabolites produced from pyrene degradation under these conditions. Accumulation of phenanthrene with its subsequent disappearance was observed. One more metabolite probably was the product of phenanthrene degradation. Pyrene metabolism in basidiomycetes rich medium was accompanied first by laccase and tyrosinase production and later by versatile peroxidase production. The cell-associated activities of laccase, tyrosinase, and versatile peroxidase were found. The data obtained indicate that both enzymes (laccase and versatile peroxidase) are necessary for complete degradation of pyrene. Furthermore, both cell-associated and extracellular laccases can catalyse the first stages of pyrene degradation, and versatile peroxidase can be necessary for oxidation of the resulting metabolites.

Sulfotransferase Forms Expressed in Human Intestinal Caco-2 and TC7 Cells at Varying Stages of Differentiation and Role in Benzo[a]pyrene Metabolism

The Caco-2 cell line and its subclone TC7 are frequently used for studying human intestinal transport and metabolism of xenobiotics. We have investigated the expression of soluble sulfotransferases (SULT) in parental Caco-2 and TC7 cells by immunoblotting. SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C1, SULT1C2, and SULT2A1 were expressed in both cell lines. SULT2B1a, SULT2B1b, and SULT4A1 were absent. SULT1E1 protein was found in TC7 but not in Caco-2 cells. Other differences in SULT between the cell lines were minor. More important was the influence of differentiation. Expression of the various SULT forms was low or not detectable in cultures just reaching confluence but then increased strongly. Likewise, the rate of sulfation of the model substrate 3-hydroxybenzo[a]pyrene was increased with increasing culture duration. Benzo[a]pyrene-1-sulfate and -3-sulfate were formed in both cell lines when benzo[a]pyrene was used as a substrate. A further metabolite, 3-hydroxybenzo[a]pyrene-glucuronide, was detected in TC7 but not in parental Caco-2 cells. Cytochrome P450 inducers enhanced the conversion of benzo[a]pyrene to these metabolites without altering mRNA levels of major phenol-conjugating SULT forms (SULT1A1, SULT1A3, and SULT1B1). Overall, differentiated Caco-2 and TC7 cells are rich sources of SULT, as is human intestinal mucosa. The SULT pattern is most similar to that found in small intestine, although levels of SULT1A1 and SULT1B1 are lower, and those of SULT1C1 are higher in Caco-2 and TC7 cells than previously found in intestinal samples. The differentiation-dependent expression of SULT in the cultured cells reflects the in vivo situation, where SULT expression is focused to differentiated enterocytes.

Impact of multiple genetic polymorphisms on effects of a 4-week blueberry juice intervention on ex vivo induced lymphocytic DNA damage in human volunteers

Consumption of fruits and vegetables has been associated with a decrease in cancer incidence and cardiovascular disease, presumably caused by antioxidants. We designed a human intervention study to assess antioxidative and possible anti-genotoxic properties of fruit-borne antioxidants. We hypothesized that individuals bearing genetic polymorphisms for genes related to quercetin metabolism, benzo[a]pyrene metabolism, oxidative stress and DNA repair differ in their response to DNA protective effects of increased antioxidant intake. In the present study, 168 healthy volunteers consumed a blueberry/apple juice that provided 97 mg quercetin and 16 mg ascorbic acid a day. After a 4-week intervention period, plasma concentrations of quercetin and ascorbic acid and trolox equivalent antioxidant capacity (TEAC) were significantly increased. Further, we found 20% protection (P < 0.01) against ex vivo H(2)O(2)-provoked oxidative DNA damage, measured by comet assay. However, the level of ex vivo induced benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts was 28% increased upon intervention (P < 0.01). Statistical analysis of 34 biologically relevant genetic polymorphisms revealed that six significantly influenced the outcome of the intervention. Lymphocytes from individuals bearing variant genotype for Cyp1B1 5 seemed to benefit more than wild-types from DNA damage-protecting effects upon intervention. Variants for COMT tended to benefit less or even experienced detrimental effects from intervention. With respect to GSTT1, the effect is ambiguous; variants respond better in terms of intervention-related increase in TEAC, but wild-types benefit more from its protecting effects against oxidative DNA damage. We conclude that genotyping for relevant polymorphisms enables selecting subgroups among the general population that benefit more of DNA damage-modulating effects of micronutrients.

Characterizing and Biological Monitoring of Polycyclic Aromatic Hydrocarbons in Exposures to Diesel Exhaust

Polycyclic aromatic hydrocarbons (PAHs) are one of the most important and carcinogenic components in diesel exhaust (DE). Therefore, ambient PAHs concentrations were measured and characterized for work areas in a locomotive engine inspection plant. Pre- and post-shift urine samples and concurrent air samples were collected on 17 workers to measure the concentration of urinary 1-hydroxypyrene (1-OHP), a metabolite of pyrene. Increased urinary 1-OHP concentrations were observed over at least three consecutive sampling days. The biological kinetics of pyrene metabolism was studied with a one-compartment pharmokinetic model. The conversion rate and elimination rate of 1-OHP were estimated using nonlinear mixed-effects model, and validated with multiple nonlinear regression models by assessing the pattern of elimination rates of each worker separately. Urinary 1-OHP was confirmed to be a sensitive marker of PAHs exposure with mean half-life of 29 h in this population of Chinese workers. The study results would be beneficial to future occupational and environmental studies of PAH exposure.

Effects of sediment organic matter quality on bioaccumulation, degradation, and distribution of pyrene in two macrofaunal species and their surrounding sediment

Sediment dwelling macrofauna (infauna) are important vectors for the transfer of sediment-associated contaminants to higher trophic levels. Sedimenting organic matter constitutes an important food source for all benthic organisms and changes seasonally in terms of quantity and quality. Sediment organic matter (SOM) quality affects organism activity and feeding behaviour, and is therefore also likely to affect contaminant fate in benthic systems. We investigated the impact of SOM quality (enrichment with either labile Tetraselmis sp. or refractory lignin) on the accumulation and metabolism of sediment-associated pyrene in Nereis diversicolor (Annelida) and Amphiura filiformis (Echinodermata), as well as the combined effect of SOM quality and infaunal bioturbation on pyrene distribution and metabolism in the sediment. After 45 d of exposure, SOM quality almost doubled pyrene bioaccumulation in both species, while pyrene metabolism remained unaffected. Metabolites comprised approximately 80% of the total tissue pyrene in N. diversicolor and approximately 40% in A. filiformis. A. filiformis arms contained one fifth of the disk pyrene concentration. Approximately 20% of the pyrene found in A. filiformis arms was found to be covalently bound to, e.g. DNA, RNA or proteins, thus reducing pyrene bioavailability to arm-cropping predators. The sedimentary pyrene distribution and metabolism was species-dependent, but correlated poorly with prevailing knowledge on species-specific bioturbation patterns. This was attributed to the comparably high biodegradability of the contaminant thus altering its sorptive characteristics and function as inert tracer. Subduction of pyrene and metabolites occurred, and the fraction of pyrene covalently bound to SOM increased with depth, thereby removing pyrene from the bioavailable pool. Our results imply that bioaccumulation and trophic transfer of sediment-associated PAH should increase following fresh organic matter input, e.g. after sedimentation of phytoplankton blooms. We stress the importance of considering behavioural characteristics of infauna and the trophic situation of the system when assessing fate and effects of sediment-associated contaminants.

Metabolism of benzo[a]pyrene in peroxynitrite/Fe(III) porphyrin system

The peroxynitrite/porphyrin biomimetic system was established to investigate the effects of peroxynitrite on benzo[a]pyrene (B[a]P) metabolism. Three model systems consisting of different iron porphyrins were compared, and the results showed that the peroxynitrite/T( p-Cl)PPFeCl system was the highest catalytic efficiency in the metabolism of B[a]P. We analyzed the B[a]P metabolites produced from this system by RP-HPLC method and firstly identified the formation of nitrobenzo[a]pyrenes which are the special metabolites of B[a]P induced by peroxynitrite.

Complete and Integrated Pyrene Degradation Pathway in Mycobacterium vanbaalenii PYR-1 Based on Systems Biology

Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the beta-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.

Intermoult duration affects the susceptibility of shore crabs Carcinus maenas (L.) to pyrene and their ability to metabolise it

We have studied pyrene (PYR) toxicity and the ability to metabolise and eliminate PYR in two colour forms of shore crabs Carcinus maenas (Linnaeus, 1758). In addition, we analysed differences in the expression of expressed sequence tags (ESTs) encoding specific cytochrome P450s (CYPs) by quantitative realtime PCR. Green and red intermoult crabs are considered to represent different adaptational life stages, allocating energy into growth (green) and reproduction (red), respectively. PYR injection resulted in significantly higher mortality in red crabs than in green crabs during a 51 days period. PYR is an ideal model PAH compound as only 1 phase I metabolite, 1-hydroxypyrene (1-HP) is formed, which is further conjugated to form various phase II metabolites. In this study, 1-HP was detected only after deconjugation of total PYR derived metabolites indicating that PYR hydroxylation (putatively CYP catalysed) conceivably is the rate-limiting step in PYR metabolism. Investigation of the accumulation of PYR and 1-HP (after deconjugation), in different tissues revealed a significantly higher accumulation of PYR in muscle and epidermis of red crabs compared to green crabs. Consistent with this observation, green crabs had significantly higher levels of 1-HP in the hepatopancreas than red crabs. This indicates that a larger portion of the injected PYR was metabolised into 1-HP in green crabs compared to red crabs. 1-HP was mainly detected in the hepatopancreas confirming its major role in the biotransformation of lipophilic compounds. CYP enzymes typically mediate the phase I hydroxylations of lipohilic contaminants such as PYR. In agreement with the higher rate of conversion of PYR into 1-HP in green compared to red crabs, increased abundance of several CYP transcripts was observed in green crabs. Furthermore, in vitro pyrene hydroxylase assays revealed significantly higher NADPH-depedent pyrene hydroxylase activity in hepatopancreas microsomes of green crabs (18.4 ρmol min −1 mg −1 protein) compared to red crabs (8 ρmol min −1 mg −1 protein). The present study demonstrates that the susceptibility of shore crabs to PYR and their ability to metabolise it is life stage dependent, conceivably due to life stage related differences in the expression of certain CYP genes, suggesting a mechanistic explanation of the observed life stage differences in PYR toxicity.

Metabolism of pyrene by aquatic crustacean, Daphnia magna

The aquatic crustacean Daphnia magna is an important species for ecotoxicological study, and is often used as a test organism for environmental risk assessment. However, the mechanism of xenobiotic metabolism by this species has not been studied in detail. In the present study, pyrene was used as model substance to investigate the mechanism of xenobiotic metabolism in D. magna. The results of 24-h exposure experiments showed that D. magna could metabolize pyrene and biotransform it into water-soluble metabolites. On the other hand, the metabolism of pyrene was significantly inhibited by SKF-525A as the cytochrome P450 (CYP) inhibitor. These observations indicated that oxidation by CYP participated in the biotransformation of pyrene by D. magna. We also identified the pyrene metabolites formed by D. magna by HPLC with an electrospray ionization triple quadrupole mass spectrometry detector (LC/ESIMS/MS) and de-conjugation by sulfatase, β-glucuronidase, and β-glucosidase. One of the metabolites was ionized in ESI negative mode and formed a dominant mass of m/ z 297 (MS) with the product ion of m/ z 217 (MS 2). Furthermore, this metabolite formed 1-hydroxypyrene on treatment with sulfatase. This metabolite was considered to be a sulfate conjugate of oxidized pyrene (1-hydroxypyrenesulfate). Furthermore, we quantified the deconjugated 1-hydroxypyrene formed by the above enzyme treatment. It showed that 52% of the total metabolized pyrene was biotransformed into 1-hydroxypyrene-sulfate, and more than 73% was biotransformed into oxidized pyrene conjugate. These results indicated that CYP and several conjugation enzymes participate in its biotransformation, and sulfation is important in D. magna for metabolism and elimination of xenobiotics.

Differential utilization of pyrene as the sole source of carbon by Bacillus subtilis and Pseudomonas aeruginosa strains: role of biosurfactants in enhancing bioavailability

Our goal is to compare the efficiency of utilization of pyrene as the sole source of carbon for growth and energy by two nonactinomycetous groups of bacteria viz., Bacillus subtilis DM-04 and Pseudomonas aeruginosa mucoid (M) and nonmucoid (NM) strains, isolated from a petroleum-contaminated soil sample of north-east India. Bacillus subtilis DM-04 and P. aeruginosa M and NM bacterial strains were capable of secreting biosurfactant in the culture medium while growing on pyrene and their pyrene utilizing efficiency was demonstrated by correlating the bacterial growth in the presence of pyrene as the sole source of carbon along with a concomitant decrease in pyrene content from the culture medium with respect to time. The biosurfactant secreted by the respective bacterial strains enhanced the apparent solubility of pyrene by factors of 5-7 and influenced the bacterial cell surface hydrophobicity resulting in higher uptake and utilization of pyrene by bacteria. The growth of B. subtilis DM-04 and P. aeruginosa M and NM strains at the expense of pyrene after 96 h showed an assimilation of about 48.0 +/- 1.1% (mean +/- SD) and 32.0 +/- 0.6% (mean +/- SD) of pyrene carbon, respectively, showing differences in metabolism of pyrene by these bacterial strains. Bacillus subtilis DM-04 strain exhibited higher utilization and cellular assimilation of pyrene compared with P. aeruginosa M and NM strains. Further, the biosurfactants produced by the bacteria under study are capable of enhancing the solubility of pyrene in aqueous media and can influence the cell surface hydrophobicity of the biosurfactant-producing strains that results in a higher uptake of pyrene. It may be suggested that the bacteria used in this study are suitable candidates for practical field application for effective in situ bioremediation of pyrene-contaminated sites.

Benzo(a)pyrene metabolism, DNA-binding and UV-induced repair of DNA damage in cultured skin fibroblasts from a patient with unilateral multiple basal cell carcinoma.

The metabolism of benzo(a)pyrene (BP), a ubiquitous environmental carcinogen, and its subsequent binding to DNA, and the repair of UV-induced DNA damage were studied in fibroblasts cultured from the skin of a 61-year-old male who had multiple BCC (greater than 100) on his left upper trunk. Biopsies were obtained and fibroblasts cultured from clinically normal tumour-free skin adjacent to tumour-bearing sites (TBS) and from visibly uninvolved normal skin (UNS) at distant sites. The cultured cells were incubated with [3H]-BP for 24 h and BP metabolism was assessed by HPLC and the formation of BP-diols, quinones and phenols verified. Total BP metabolism was 45% lower in TBS fibroblasts than in UNS fibroblasts. The formation of BP-7,8-diol, the precursor of the carcinogenic end product of BP metabolism, was 53% lower in TBS cells than in UNS cells. Pretreatment of UNS cells with benz(a)anthracene (BA) (x 10(-4) M) did not significantly affect BP metabolite formation whereas BA-treatment of TBS cells resulted in 55% and 76% increases in total BP metabolism and BP-7,8-diol formation, respectively. Treatment of TBS cells with BA also caused a substantial increase (95%) in BP-DNA adduct formation. Whereas DNA-binding in UNS cells was unaffected by this treatment. In response to irradiation with 2J/m2 UVC, total DNA repair was similar in both cell types; on alkaline elution it appeared that the TBS cells were more efficient in repairing UV-induced DNA strand breaks. These results suggest that BP metabolism and repair of DNA are altered in TBS cells and that patients with this type of metabolic profile may be at higher risk of the development of cutaneous neoplasms. It is also possible that fibroblasts from tumour bearing skin undergo some as yet unexplained alteration in carcinogen metabolism as a consequence of the induction of neoplasia.

Antioxidant responses of microalgal species to pyrene

The antioxidant response of four freshwater microalgal species, Chlorella vulgaris Beij., Scenedesmus platydiscus (G. M. Smith) Chod., Scenedesmus quadricauda(Turp.) Breb., and Selenastrum capricornutum Printz without pyrene addition (control) and at two pyrene concentrations (0.1 and 1.0 mgL−1) were investigated. Under the control condition, the values of the antioxidant parameters differed significantly among species and the difference was seemed not to be related to their susceptibility to pyrene. The antioxidant response to pyrene treatments also varied from species to species. Pyrene led to a significant increase in total glutathione (GSH) content in all species except C. vulgaris, a species did not exhibit any ability to metabolize pyrene. The glutathione-S-transferase (GST) activities also remained unchanged in pyrene treated C. vulgaris, increased greatly in S. platydiscus and Se. capricornutum (the two species with higher pyrene metabolism ability), but inhibited remarkably in S. quadricauda (the only species sensitive to pyrene toxicity). On the other hand, the glutathione reductase (GR) activities increased in C. vulgaris but remained at a similar level as the control in the other three species. The malondialdehyde (MDA) content, an indicator of lipid peroxidation, declined in S. quadricauda but showed no significant change in the other three species. The activities of glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) in pyrene treated cells remained almost the same as the controls for all microalgal species. Results suggested that the alterations of antioxidant systems in microalgae might not be useful indicators of pyrene exposure but pyrene-enhanced GSH metabolism might be important in pyrene biotransformation.

Induction of hepatic cytochrome P450 isozymes, benzo(a)pyrene metabolism and DNA binding following exposure to polycyclic aromatic hydrocarbon residues generated during repeated fish fried oil in rats

In the present study the effect of repeated fish fried oil (RFFO) and its extract (RFFE) on hepatic cytochrome P450 (CYP) isozymes, benzo(a)pyrene (BP) metabolism and DNA adduct formation was undertaken. HPLC analysis of RFFO showed the presence of several polycyclic aromatic hydrocarbons. CYP in microsomes from control and RFFO-treated animals showed a peak at 450 nm; however, a shift of 2 nm in the SORET region along with significant induction was observed in microsomes prepared from 3-methylcholanthrene (MC)- and RFFE-treated animals. Activities of hepatic ethoxyresorufin-O-deethylase, methoxyresorufin-O-deethylase, aryl hydrocarbon hydroxylase and erythromycin-N-demethylase were found to be significantly (P < 0.05) induced following exposure of RFFE, whereas none of these enzymes were altered in RFFO-treated group. Immunoblot analysis revealed that RFFE and MC were potent inducers of CYP1A1, 1A1/2 and 3A1 isozymes, where as RFFO showed no change in these protein levels. RT-PCR analysis showed induction of cDNA of CYP1A1 and CYP3A1 by RFFE treatment. Hepatic microsomes prepared from RFFE exposed animals enhanced BP metabolism with a concomitant increase in the relative proportion of BP 7,8-diol. Hepatic microsomes prepared from animals pretreated with RFFE and MC significantly enhanced the binding of [(3)H]-BP to calf thymus DNA. The overall results suggest that exposure to RFFE may induce hepatic CYP isozymes thereby producing enhanced reactive metabolites with a potential to bind with DNA that may result in cancer.

Characterization of Common CYP1B1 Variants with Different Capacity for Benzo[a]pyrene-7,8-Dihydrodiol Epoxide Formation from Benzo[a]pyrene

Cytochrome P450 1B1 (CYP1B1), an extrahepatic enzyme inducible by smoking, is overexpressed in many tumors and catalyzes the metabolic activation of procarcinogens such as polycyclic aromatic hydrocarbons. In human, CYP1B1 is genetically polymorphic and five common missense mutations causing amino acid substitution have been identified. In this study, we have investigated CYP1B1 haplotypes present in a Spanish population and carried out functional analyses of the corresponding enzymes in yeast using benzo[a]pyrene as a substrate. CYP1B1*1, CYP1B1*2, CYP1B1*3, CYP1B1*4, CYP1B1*6, and CYP1B1*7, encoding combinations of the Arg48Gly, Ala119Ser, Leu432Val, Asn453Ser, and Ala443Gly amino acid substitutions, were present at frequencies of 14.3%, 25.5%, 38.8%, 18.1%, 0.4%, and 2.6%, respectively. The variant CYP1B1 forms were heterologously expressed with human reductase in Saccharomyces cerevisiae and kinetic analyses of benzo[a]pyrene metabolism were carried out. CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, Leu432Val, and Ala443Gly, exhibited a significantly decreased capacity (P < 0.001) for the formation of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol from benzo[a]pyrene as indicated by lower intrinsic clearance (Vmax/Km). A somewhat decreased clearance was observed for CYP1B1.4, whereas no significant differences in kinetic properties among the remaining variant enzymes were observed as compared with CYP1B1.1. Thus, genetic polymorphism in the CYP1B1 gene, as defined by the haplotypes investigated, might cause interindividual differences in susceptibility (e.g., to lung cancer induced by smoking). The results indicate the necessity to make molecular epidemiologic investigations regarding the association of the specific CYP1B1 haplotypes and cancer risk.

Catalytic turnover of pyrene by CYP3A4: Evidence that cytochrome b5 directly induces positive cooperativity

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b 5 ( b 5) on turnover kinetics. In the absence of b 5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate–velocity curve where K m1 and V max1 were 1.3 μM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a π–π stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b 5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V max was 15.7 pmol/min/pmol P450, the K m was 7.5 μM, and the Hill coefficient was greater than two. This demonstrated that b 5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.

Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1

The effects of pH on the growth of Mycobacterium vanbaalenii PYR-1 and its degradation of phenanthrene and pyrene were compared at pH 6.5 and pH 7.5. Various degradation pathways were proposed in this study, based on the identification of metabolites from mass and NMR spectral analyses. In tryptic soy broth, M. vanbaalenii PYR-1 grew more rapidly at pH 7.5 (mu'=0.058 h(-1)) than at pH 6.5 (mu'=0.028 h(-1)). However, resting cells suspended in phosphate buffers with the same pH values displayed a shorter lag time for the degradation of phenanthrene and pyrene at pH 6.5 (6 h) than at pH 7.5 (48 h). The one-unit pH drop increased the degradation rates four-fold. Higher levels of both compounds were detected in the cytosol fractions obtained at pH 6.5. An acidic pH seemed to render the mycobacterial cells more permeable to hydrophobic substrates. The major pathways for the metabolism of phenanthrene and pyrene were initiated by oxidation at the K-regions. Phenanthrene-9,10- and pyrene-4,5-dihydrodiols were metabolized via transient catechols to the ring fission products, 2,2'-diphenic acid and 4,5-dicarboxyphenanthrene, respectively. The metabolic pathways converged to form phthalic acid. At pH 6.5, M. vanbaalenii PYR-1 produced higher levels of the O-methylated derivatives of non-K-region phenanthrene- and pyrene-diols. Other non-K-region products, such as cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic acid, 1,2-dicarboxynaphthalene and benzocoumarin-like compounds, were also detected in the culture fluids. The non-K-region polycyclic aromatic hydrocarbon oxidation might be a significant burden to the cell due to the accumulation of toxic metabolites.

Modulation of adult rat benzo(a)pyrene (BaP) metabolism and DNA adduct formation by neonatal diethylstilbestrol (DES) exposure

This study seeks to elucidate the role of diethylstilbestrol (DES), a synthetic estrogen on benzo(a)pyrene (BaP) metabolism in the male rat reproductive tissues. Offspring of timed-pregnant Sprague-Dawley rats were neonatally treated on days 2, 4, and 6 post-partum with 1.45 μmol/kg of DES. Ten weeks after birth, the adult rats were challenged with radiolabeled benzo(a)pyrene ( 3H BaP) (10 μmol/kg) and the rats were sacrificed 2 h after BaP exposure. Prostrate, testis, lung, liver, urine and feces samples were collected and extracted using a mixture of H 2O, MeOH and CHCl 3. The extracts were analyzed by reverse phase HPLC. The concentrations of BaP organic metabolites in DES rats were lower compared to controls (vehicle-treated rats). On the other hand, concentrations of aqueous metabolites were significantly increased in DES treated animals. The toxication to detoxication ratios were significantly decreased in DES rats compared to controls. This trend is also reflected in the decreased concentrations of BaP-DNA adducts in DES rats. Collectively these results suggest that DES is capable of modulating the metabolic pathway of BaP towards detoxification thereby preventing the manifestation of toxicity.

Metabolism of aromatic hydrocarbons by the filamentous fungus Cyclothyrium sp.

The metabolism of biphenyl, naphthalene, anthracene, phenanthrene, pyrene and benzo[ a]pyrene by Cyclothyrium sp. CBS 109850, a coelomycete isolated for the first time in Brazil from industrially polluted estuarine sediment, was studied. The metabolites were extracted and separated by high performance liquid chromatography (HPLC) and characterized by UV spectral analyses and mass, and proton nuclear magnetic resonance ( 1H NMR) spectrometry. Cyclothyrium sp. transformed biphenyl to 4-hydroxybiphenyl and anthracene to anthracene trans-1,2-dihydrodiol. This isolate metabolized 90% of [9- 14C]phenanthrene, producing phenanthrene trans-9,10-dihydrodiol as a major metabolite, phenanthrene trans-3,4-dihydrodiol, 1-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, and a novel metabolite, 2-hydroxy-7-methoxyphenanthrene. Circular dichroism spectra analyses indicated that the major enantiomers of phenanthrene trans-9, 10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol and pyrene trans-4,5-dihydrodiol, a pyrene metabolite produced previously by Cyclothyrium sp. CBS 109850, were predominantly in the ( R, R) configuration, revealing a high stereoselectivity for initial monooxygenation and enzymatic hydration of phenanthrene and pyrene by Cyclothyrium sp. CBS109850. The results also show a high regioselectivity since the K-regions of phenanthrene and pyrene were the major sites of metabolism.

Influence of diet and ration level on benzo[a]pyrene metabolism and excretion in rainbow trout (Oncorhynchus mykiss).

Juvenile rainbow trout (Oncorhynchus mykiss) were fasted or fed one of three isoenergetic diets varying in protein and lipid content at full satiation levels or half rations for up to 9 weeks. At 3, 6, and 9 weeks, fish in each treatment group were dosed intraperitoneally with 10 mg tritiated benzo[a]pyrene [3H]-B[a]P/kg (B[a]P) to examine the effects of diet composition and energy intake on xenobiotic biotransformation and excretion. The percent dose eliminated during the experiment did not differ among fish receiving the different diet compositions or rations (range 73% to 84%). However, it was significantly decreased (to 53%) in the group that was fasted for 9 weeks. Examination of fish fasted for 6 and 9 weeks showed a significant increase in the proportion of phase I metabolites and a concomitant decrease in the proportion of phase II metabolites found in bile compared with all other groups. Also, fish that were fasted for 9 weeks produced proportionately less 9,10-dihydroxy-benzo[a]pyrene-trans-9,10-diol, more 3-hydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene, and more glucuronic acid conjugates compared with all other groups. Thus, dietary protein and lipid concentration did not appear to affect either the rate of B[a]P metabolism or its excretion; however, prolonged fasting resulted in a shift in metabolite profiles and decreased excretion.