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Metabolite production in degradation of pyrene alone or in a mixture with another polycyclic aromatic hydrocarbon by Mycobacterium sp.

Degradation of pyrene (PYR) alone and in the presence of phenanthrene or fluoranthene by Mycobacterium sp. strain A1-PYR isolated from mangrove sediments was investigated. When PYR was the only polycyclic aromatic hydrocarbon compound and the sole carbon source, only 33% of the added PYR was slowly degraded during 7 d of incubation. Seven metabolites were obtained, including four-ring metabolites (monohydroxypyrene and three different dihydroxypyrenes) and three-ring metabolites (dihydroxyphenanthrene, 4-phenanthrene-carboxylic acid, and 4-phenanthrol), of which more four-ring metabolites accumulated compared with three-ring metabolites. To our knowledge, this is the first report in which PYR was initially attacked by Mycobacterium sp. to form three different dihydroxypyrenes. Pyrene degradation was significantly stimulated when mixed with phenanthrene or fluoranthene. In the presence of fluoranthene, PYR was rapidly degraded (up to 57%), and significant amounts of dihydroxypyrene were formed within 3 d of incubation, followed by a period of minimal PYR degradation from 3 to 7 d with disappearance of four-ring metabolites and accumulation of three-ring metabolites. In contrast, PYR was removed completely, and little evidence of metabolites was detected in the presence of phenanthrene. These results showed that PYR was degraded to a larger extent when mixed with another polycyclic aromatic hydrocarbon concomitant with a higher turnover of PYR metabolites. The induction of complex enzyme systems and increase in biomass possibly affected the transformation of PYR metabolites in the mixture with phenanthrene or fluoranthene.

Biodegradation, Environmental↗

Identification of 1-hydroxypyrene as a major metabolite of pyrene in pig urine.

1-Hydroxypyrene is a major metabolite of pyrene given orally to pigs. A method for isolating the metabolite from urine is described, utilizing C18-adsorbent cartridges, h.p.l.c. separation, and fluorescence detection. Unconjugated 1-hydroxypyrene can be detected in the urine of pigs following oral administration of as little as 1 mg pyrene. Identity of the 1-hydroxypyrene was confirmed by u.v.-absorption and fluorescence spectrometry, mass spectrometry, and by comparison with the retention time characteristic of synthetic 1-hydroxypyrene.

Animals↗

Selenium suppresses the metabolism of benzo[a]pyrene by rat-liver extracts, and exerts a dual effect on its mutagenicity.

Liver homogenates from rats injected with 3-methylcholanthrene were employed for metabolism of benzo[a]pyrene (BP) and in assays of aryl hydrocarbon hydroxylase (AHH) activity in vitro. Sodium selenite inhibited AHH activity to a maximum of approximately 70%. It suppressed the overall metabolism of benzo[a]pyrene; a distinct reduction in the products was evident on h.p.l.c. analysis. Sodium thiosulphate also inhibited AHH activity by approximately 47%. Inclusion of S2O3(2-) and SeO3(2-), in combination, led to a cumulative inhibition of 87%. The mutagenicity of BP in the Salmonella auxotroph reversion system (Ames test) was enhanced by SeO3(2-) at concentrations below 0.2 mM. Above this level a significant antimutagenic effect was observed.

Animals↗

Studies on the induction of aryl hydrocarbon(benzo[a]pyrene) hydroxylase in Neurospora crassa, and its suppression by sodium selenite.

Six fungal species were grown in the presence of benzo[a]pyrene (BP); four showed benzo[a]pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) activity. Penicillium sp. and Neurospora crassa metabolized BP to a limited extent. N. crassa AHH activity was induced by BP, the major product of metabolism being 3-hydroxy-BP. Both induction of AHH activity and metabolism of BP were suppressed by sodium selenite in the growth medium. Two polypeptides, unique to BP-grown cells, were revealed by two-dimensional electrophoretic separation of [35S]methionine-labelled proteins in N. crassa cell extracts. In selenium-grown cells the synthesis of BP-specific polypeptides was severely inhibited.

Aryl Hydrocarbon Hydroxylases↗

Benzo[a]pyrene metabolism in the Mongolian gerbil: influence of chlordecone and mirex induction.

1. The metabolism of benzo[a]pyrene (BP) by gerbil hepatic microsomes is increased following induction by phenobarbital (PB), chlordecone, mirex and 3-methylcholanthrene (3-MC). 2. By several criteria including the influence of alpha-naphthoflavone (alpha-NF) on BP-hydroxylase activity and BP-metabolite profiles, the cytochromes P-450 responsible for benzo[a]pyrene metabolism appear to be similar in microsomes isolated from PB-, chlordecone-, or mirex-treated gerbils. The cytochromes P-450 present in microsomes isolated from control animals and those treated with 3-MC are different from each other and from those present in PB, chlordecone, or mirex microsomes by the same criteria. 3. Of the inducers used, only PB induced microsomal epoxide hydrolase activity.

Animals↗

Suppression of humoral and cell-mediated immune responses in vitro by benzo(a)pyrene.

The effect of benzo(a)pyrene (BaP) at different molar (M) concentrations on the in vitro anti-sheep red blood cell (SRBC) plaque (antibody) forming cell (PFC) response and the one-way mixed lymphocyte response (MLR) was tested. Inhibition of the PFC response and the MLR occurred when spleen cells were exposed to a wide range of BaP concentrations from 10(-4) M to 10(-8) M. Maximum depression of the responses occurred at 10(-5) M for PFC production (47% of controls) and for the MLR (19% of controls) as measured by a stimulation index. No significant loss in cell viability was observed at this or lower molar concentrations of BaP. The non-carcinogenic analog of BaP, benzo(e)pyrene, did not suppress PFC responses at comparable concentrations. This in vitro system will facilitate manipulations of T and B lymphocytes and macrophages (adherent cells) in a controlled culture environment for precisely characterizing the sensitivity of these cells and their subpopulations on exposure to BaP.

Animals↗

Redox properties of phenols, their relationships to singlet oxygen quenching and to their inhibitory effects on benzo(a)pyrene-induced neoplasia.

The inhibitory effects of synthetic phenolic compounds on benzo(a)pyrene-induced neoplasia of the mouse forestomach have been measured by Wattenberg et al. The efficiency of this inhibition has been estimated for each phenol, using R, the ratio of the number of tumors per mouse in the protected group over the number of tumours per mouse in the control group. We have observed a linear correlation between the chemoprotection efficiency R and the logarithm of the rate of quenching of singlet oxygen, k, by this family of phenols, log k being itself correlated with the one-electron oxidation potential of the phenols. These correlations suggest a charge transfer mechanism for the inhibition of neoplasia induced by benzo(a)pyrene. The correlations described provide a theoretical basis for scaling the inhibitors of mutagenicity induced by polycyclic aromatic compounds in terms of their oxidation potentials.

Animals↗

High benzo[a]pyrene diol-epoxide DNA adduct levels in lung and blood cells from individuals with combined CYP1A1 MspI/Msp-GSTM1*0/*0 genotypes.

Levels of anti-benzo[a]pyrene diol-epoxide DNA adducts were analysed by high-pressure liquid chromatography/fluorimetric detection in non-tumorous lung tissues from 20 lung cancer patients and in white blood cells from 20 polycyclic aromatic hydrocarbon exposed coke oven workers. All were current tobacco smokers. CYP1A1 mutations (MspI at 6235 nt, Ile-Val462) and GSTM1 deletion polymorphisms in each individual were analysed in genomic DNA by PCR/restriction fragment length polymorphism. Independently of the CYP1A1 genotype (1) all 23 samples in the two groups with non-detectable adducts (< 0.2 per 10(8) nt) were of GSTM1 active genotype; (2) the 17 samples with detectable adducts (> or = 0.2 per 10(8) nt) in the two groups were GSTM1*0/*0. The difference in adduct levels between GSTM1*0/*0 and GSTM1 active genotype was highly significant (p < 0.00005). Among GSTM1-deficient individuals (n = 17), a subgroup of 14 individuals with CYP1A1*1/*1 (wild-type, n = 7) or heterozygous genotype (*1/*2A or *1/*2B, n = 7) showed low levels of BPDE DNA-adducts (range: 0.2-1.3 per 10(8) nt). (3) Three individuals with the rare combination CYP1A1*2A/*2A or *2A/*B and GSTM1*0/*0 showed significantly higher adduct levels (median: 17.4 adducts/10(8) nt, range 1.9-44; p = 0.017). Therefore, combination of homozygous mutated CYP1A1 and GSTM1*0/*0 genotypes lead, at a similar or even lower smoking dose, to a stronger increase of anti-benzo[a]pyrene diol-epoxide DNA adduct levels than found in individuals with CYP1A1 and GSTM1 wild-type. These data provide a mechanistic understanding of epidemiological studies that correlated these 'at risk' genotypes with increased smoking-related lung cancers.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

Treatment of lacZ plasmid-based transgenic mice with benzo[a]pyrene: measurement of DNA adduct levels, mutant frequencies, and mutant spectra.

The evaluation of the relationship between the dose to DNA of a genotoxic carcinogen and in vivo somatic cell mutagenesis may provide important information on the mechanisms leading to induced mutational events. The induction of DNA adducts and DNA mutations in different tissues of lacZ plasmid-based transgenic mice has been investigated following a single intraperitoneal administration of the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). DNA adducts were measured by (32)P-postlabeling at times between 1 and 28 days following injection and DNA mutations in the lacZ reporter gene were quantified using a highly efficient immunomagnetic purification of the lacZ-containing pUR288 plasmid, followed by electrotransformation of circularized plasmids into a host-restriction negative E. coli C (DeltalacZ, galE (-)) host. The major DNA adduct formed by B[a]P, N (2)-(10beta-[7beta,8alpha, 9alpha-trihydroxy-7,8,9, 10-tetrahydrobenzo(a)pyrene]yl-deoxyguanosine, reached maximum levels between 5 and 7 days followed by a gradual decrease. The induced mutant frequency reached a maximum between 7 and 14 days, after the DNA adduct level in a particular tissue had reached its apparent maximum between 5 and 7 days. The mutant spectrum, defined as the percentage of no-change and size-change mutations in a particular tissue, changed from predominantly size-change mutations in untreated tissues to predominantly no-change mutations in tissues displaying the highest B[a]P-induced mutant frequencies. Contrary to the assumption that there are no factors that can eliminate mutations once they exist, it was observed that mutant frequencies in the organs studied declined to background levels, which was accompanied by a change in the mutant spectrum from predominantly no-change mutations to predominantly size-change mutations.

Animals↗

Detection of benzo[a]pyrene-DNA adducts in leukocytes of coke oven workers.

Coke oven workers are often heavily exposed to polynuclear aromatic hydrocarbons (PAHs) and particularly to benzo[a]pyrene (B[a]P), which has been associated with a high incidence of cancer. B[a]P is metabolically activated to its diol-epoxide derivative, benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE-I), a potent carcinogen which binds covalently to DNA, thereby producing BPDE-I-DNA adducts. In this study, an investigation was made of the exposure of coke oven workers to PAH via the measurement of urinary 1-hydroxypyrene (1-OHP) levels, and of exposure to B[a]P by the analysis of BPDE-I-DNA adducts in leukocytes using an ELISA competitive immunoassay. 1-OHP levels measured in post-shift samples correlated with those of DNA adducts detected in leukocytes, with a mean value (140.11 fM/50 micrograms of DNA) which differed significantly from the control value (P < 0.001). It is concluded that measurement of BPDE-I-DNA adduct levels in coke plant workers is essential in determining cancer risk due to high exposure to PAHs, and in particular of B[a]P.

Air Pollutants, Occupational↗

NAD(P)H:quinone oxidoreductase 1 deficiency increases susceptibility to benzo(a)pyrene-induced mouse skin carcinogenesis.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes the metabolic detoxification of quinones and their derivatives. This protects cells against quinone-induced oxidative stress, cytotoxicity, and mutagenicity. C57BL6 NQO1-/- mice, deficient in NQO1 RNA and protein, were generated in our laboratory. To investigate the role of NQO1 in chemical carcinogenesis, the dorsal skin of NQO1-deficient (NQO1-/-) and wild-type (NQO1+/+) mice were treated with a single dose of benzo(a)pyrene, followed by twice weekly applications of phorbol-12-myristate-13-acetate. The NQO1-/- mice showed a much higher frequency of skin tumor development when compared with their wild-type littermates. Interestingly, the male NQO1-/- mice were slower to develop skin tumors than their NQO1-/- female littermates. Histological analysis of the NQO1-/- tumors showed proliferative activity. These results demonstrate that NQO1 acts as an endogenous factor in protection against benzo(a)pyrene carcinogenicity.

Animals↗

[Specific response to benzo(a)pyrene treatment depending on mode of administration in rats].

Mathematical and statistical procedures were used to identify individual response in rats to benzo(a)pyrene (BP) treatment versus dose and regimen (single or repeated administration). Primary experimental data were suitably processed to exclude extreme values. A significant correlation was established between life-span and urine level of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (7,8-BP) after BP administration. It was found that, with single administration regimen, individual response to the carcinogenic agent correlated primarily with efficiency of excretion of the active forms of BP while in chronic experiment--with deactivation of the agent by enzymes.

Animals↗

Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung.

p53 mutations are common in lung cancer. In smoking-associated lung cancer,the occurrence of G:C to T:A transversions at hotspot codons, e.g., 157, 248, 249,and 273, has been linked to the presence of carcinogenic chemicalsin tobacco smoke including polycyclic aromatic hydrocarbons suchas benzo(a)pyrene (BP). In the present study, we have used a highly sensitive mutation assay to determine the p53 mutation load in nontumorous human lung and to study the mutability of p53 codons 157, 248, 249, and 250 to benzo(a)pyrene-diol-epoxide (BPDE), an active metabolite of BP in human bronchial epithelial BEAS-2B cells. We determined the p53 mutational load at codons 157, 248, 249, and 250 in nontumorous peripheral lung tissue either from lung cancer cases among smokers or noncancer controls among smokers and nonsmokers. A 5-25-fold higher frequency of GTC(val) to TTC(phe) transversions at codon 157 was found in nontumorous samples (57%) from cancer cases (n = 14) when compared with noncancer controls (n = 8; P < 0.01). Fifty percent (7/14) of the nontumorous samples from lung cancer cases showed a high frequency of codon 249 AGG(arg) to AGT(ser) mutations (P < 0.02). Four of these seven samples with AGT(ser) mutations also showed a high frequency of codon 249 AGG(arg) to ATG(met) mutations, whereas only one sample showed a codon 250 CCC to ACC transversion. Tumor tissue from these lung cancer cases (38%) contained p53 mutations but were different from the above mutations found in the nontumorous pair. Noncancer control samples from smokers or nonsmokers did not contain any detectable mutations at codons 248, 249, or 250. BEAS-2B bronchial epithelial cells exposed to doses of 0.125, 0.5, and 1.0 microM BPDE, showed G:C to T:A transversions at codon 157 at a frequency of 3.5 x 10(-7), 4.4 x 10(-7), and 8.9 x 10(-7), respectively. No mutations at codon 157 were found in the DMSO-treated controls. These doses of BPDE induced higher frequencies, ranging from 4-12-fold, of G:C to T:A transversions at codon 248, G:C to T:A transversions and G:C to A:T transitions at codon 249, and C:G to T:A transitions at codon 250 when compared with the DMSO-treated controls. These data are consistent with the hypothesis that chemical carcinogens such as BP in cigarette smoke cause G:C to T:A transversions at p53 codons 157, 248, and 249 and that nontumorous lung tissues from smokers with lung cancer carry a high p53 mutational load at these codons.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

[Degradation of phenanthrene and pyrene in contaminated soil by immobilized Zoogloea sp. and Fusarium sp].

Immobilized with PVA, sodium alginate and activated carbon, both Zoogloea sp. and Fusarium sp. strains could degrade phenanthrene and pyrene efficiently. The optimal carrier was made of 100 rho.g-1 L PVA, 5 sodium alginate rho.g-1 L and 50 activated carbon rho.g-1 L. The degradation rates of phenanthrene and pyrene in 10 days were 87.48% and 75.34% by the immobilized bacterium, 37.04% and 20.85% higher than those by the free bacterium, and the rates in 15 days were 84.36% and 74.87% by the immobilized fungus, 5.35% and 11.23% higher than those by the free fungus.

Biodegradation, Environmental↗

Role of glutathione conjugate efflux in cellular protection against benzo[a]pyrene-7,8-diol-9,10-epoxide-induced DNA damage.

Glutathione (GSH) conjugation of (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE], the activated metabolite of benzo[a]pyrene, is believed to be an important mechanism in detoxification of this environmental and dietary carcinogen. Here, we demonstrate that the intracellular accumulation of GSH conjugate of (+)-anti-BPDE (BPD-SG) caused a statistically significant increase in (+)-anti-BPDE-induced DNA adduction. The relationship between intracellular accumulation of BPD-SG and (+)-anti-BPDE-induced DNA adduction was studied using a canine kidney epithelial cell line (MDCKII) and its variants overexpressing multidrug resistance transporter (MDR1) or canalicular multispecific organic anion transporter (cMOAT; also known as multidrug resistance protein 2). MDR1 and cMOAT are implicated in ATP-dependent efflux of anticancer drugs or GSH-xenobiotic conjugates, or both. The GST activity toward (+)-anti-BPDE in parental MDCKII cells did not differ from that in subline overexpressing MDR1 (MDCKII-MDR1) or cMOAT (MDCKII-cMOAT). Intracellular accumulation of BPD-SG, after a 5- or 10-min incubation with 1 microM (+)-anti-BPDE, was significantly higher in parental (41- to 67-fold) and MDCK II-MDR1 cells (31- to 43-fold) than in the MDCKII-cMOAT cells. Interestingly, the levels of DNA adducts of (+)-anti-BPDE, after a 30-min incubation with 0.1 or 0.5 microM [(3)H](+)-anti-BPDE, were significantly higher (about 2.1- and 1.7-fold, respectively) in parental cells than in the MDCKII-cMOAT cells. The results of the present study indicate that in addition to GSH conjugation, the efflux of BPD-SG may be essential for cellular protection against (+)-anti-BPDE-induced DNA damage.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

[The degradation of phenanthrene and pyrene contaminated soil with immobilized technique].

The phenanthrene and pyrene contaminated soil was degradaded by immobilized-imbedding the selected Zoogloea sp. The degradation rate was determined under various amounts of inoculation and concentration, and the result showed that there was the best degradation rate at 5%. After 168 h, the degradation rate of immobilized Zoogloea sp. to phenanthrene and pyrene was 84.89% and 76.94% respectively. While, the degradation rate of the native bacterium was only 27.85% and 19.65% at the same condition. So the Zoogloea sp. had the better degradation ability. And, Zoogloea sp. distributing shape in immobilized carrier was studied with electric scan-photo, it inferred that immobilized bacterium had the better degradation rate.

Environmental Pollution↗

Toxicokinetics of sediment-sorbed benzo[a]pyrene and hexachlorobiphenyl using the freshwater invertebrates Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus.

This study investigated the effect of long-term sediment aging on the toxicokinetics of benzo[a]pyrene (BaP) and hexachlorobiphenyl (HCBP) using three freshwater benthic invertebrates. Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus were exposed to BaP- and HCBP-spiked sediments that were aged for 7 d or 1.5 years. The toxicokinetics of the two compounds were determined for each test organism using a two-compartment model. The modeling of BaP was more complex because biotransformation was included within the model. The results of this study showed that the HCBP uptake clearance rates (k(s)) for each species were generally an order of magnitude greater than those determined for BaP and this difference was most likely due to preferential and rapid binding of BaP to sediment particles. Overall, the bioavailability of HCBP in spiked sediments tended to decrease with duration of aging, based on k(s) values and bioaccumulation factors (BAFs). However, the decreases in bioavailability appear to be species specific. Benzo[a]pyrene did not decline in bioavailability for the species tested because it may resist movement into the micropores of the sediment due to its large size. In addition to the bioassays, this article outlines a method for toxicokinetic modeling of biotransformed compounds and methods for statistical comparisons of kinetic parameters (i.e., k(s), k(d)...) and BAF values.

Adsorption↗

[Effects of aromatics on the degradation of benzo(a) pyrene in slurry reactors].

Benzo(a) pyrene (BaP) can't be biodegraded in the soil easily, the degradation of BaP is know as a cometabolic process. This paper dealed with the effects of some polycyclic aromatic hydrocarbons (PAHs) and two kinds of monoaromatics on the degradation of BaP. The test soil was pre-incubated with the aromatics for 3d and spiked with BaP stock solution, which eventually formed into slurry system in Erlenmeyer flasks and was incubated on rotary shaker. 42 d experiment demonstrated that lag period in degradation process of BaP disappeared due to the preexposure to phenanthrene, and degradation rate of BaP was enhanced. On the contrary, degradation pattern of BaP was not obviously improved by the preexposure to anthracene or pyrene, and was even inhibited by benz(a) anthracene. The lag period also disappeared after the preexposure to salicyclic acid or phthalic acid, and the degradation of BaP was accelerated by both monoaromatics, even though the impact of mass difference on the degradation of BaP was not detected.

Benzo(a)pyrene↗