Metabolism Of Benzo Research Articles

Metabolism of benzo[a]pyrene after low-dose subchronic exposure to an industrial mixture of carcinogenic polycyclic aromatic hydrocarbons in rats: a cocktail effect study.

Polycyclic aromatic hydrocarbons (PAHs) are interesting environmental pollutants for understanding cocktail effects. High-molecular-weight-PAHs (HMW-PAHs) are classified as probable or possible carcinogens; only benzo[a]pyrene (B[a]P) is a certain carcinogen in humans. Their toxicity depends on their metabolic activation. While 3-hydroxybenzo[a]pyrene (3-OHB[a]P) represents its detoxification pathway, trans-anti-7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (tetrol-B[a]P) represents the carcinogenicity pathway. The objective was to study the metabolism of B[a]P and HMW-PAHs during chronic low-dose exposure to B[a]P or a PAH mixture. Rats were exposed orally 5 times/week for 10weeks to low-levels of B[a]P (0.02 and 0.2mg.kg-1.d-1) or to an industrial mixture extracted from coal tar pitch (CTP) adjusted to 0.2mg.kg-1.d-1 B[a]P. Urinary levels of monohydroxy-, diol-, and tetrol-PAH were measured during weeks 1 and 10 by HPLC-fluorescence and GC‒MS/MS. After 1 week, the percentages of B[a]P eliminated as 3-OHB[a]P and tetrol-B[a]P were not different depending on the dose of B[a]P, whereas they were reduced by half in the CTP group. Repeated exposure led to an increase in the percentages of the 2 metabolites for the 0.02-B[a]P group. Moreover, the percentage of B[a]P eliminated as 3-OHB[a]P was equal in the 0.2-B[a]P and CTP groups, whereas it remained halved for tetrol-B[a]P in the CTP group. The percent elimination of HMW-PAH metabolites did not vary between weeks 1 and 10. Thus, dose, duration of exposure and chemical composition of the mixture have a major influence on PAH metabolism that goes beyond a simple additive effect. This work contributes to the reflection on determination of limit values and risk assessments in a context of poly-exposures.

Biodegradation kinetics and metabolism of Benzo(a)fluorene by Pseudomonas strains isolated from refinery effluent

The final sinkers of polyaromatic hydrocarbons are water sources, where they undergo bioaccumulation and biomagnification, leading to adverse mutagenic, carcinogenic, and teratogenic effects on exposure in flora, fauna, and humans. Two indigenous strains, Pseudomonas sp. WDE11 and Pseudomonas sp. WD23, isolated from refinery effluent, degraded over 97.5% of benzo(a)fluorene (10 mg/L) in 7 days. On growth at concentration dependent amounts (50 mg/L and 100 mg/L), the degradation reduced to approximately 90% and 80% respectively in 56 days. Degradation kinetics was concentration dependent, as degradation followed first-order and second-order kinetics for 50 mg/L and 100 mg/L respectively. The half-life for degradation of benzo(a)fluorene ranged between 11.64 - 12.26 days and 13.11–14.5 days for strains WDE11 and WD23 respectively. The values of Andrew-Haldane kinetic parameters i.e. μmax, Ks, and Ki were 0.306 day−1, 11.11 mg/L, and 120.41 mg/L for strain WDE11 respectively, while for strain WD23, the respective values were 0.312 day−1, 9.97 mg/L, and 152 mg/L. Degradation metabolites were identified by their MS patterns as 3,4-dihydroxy fluorene, 2-(1-oxo-2,3-dihydro-1H-inden-2-yl) acetic acid, 3,4-dihydrocoumarin, salicylic acid, catechol, and oxalic acid. Metabolic pathway of degradation constructed, revealed that benzo(a)fluorene was metabolized via the formation of fluorene, further metabolized by salicylate pathway forming catechol. The catechol formed was degraded into simpler metabolites by meta-cleavage pathway, which was validated by catechol 2,3 dioxygenase enzyme activity.

081 Modulation of the aryl hydrocarbon receptor by adipose tissue: Implications for skin carcinogenesis

The aryl hydrocarbon receptor(AhR) is a ligand-activated transcription factor that responds to chemical carcinogens. An endogenous AhR agonist, 6-Formylindolo[3,2-b]carbazole, is produced following ultraviolet light(UV) absorption by tryptophan. Epidemiological studies show a correlation between exposure to AhR agonists and non melanoma skin cancer(NMSC). As an example, AhR activation promotes metabolism of pro-carcinogens to carcinogens such as the metabolism of benzo(a)pyrene to benzo[a]pyrene diol epoxide(BPDE).

Benzopyrene induces oxidative stress and increases expression and activities of antioxidant enzymes, and CYP450 and GST metabolizing enzymes in Ulva lactuca (Chlorophyta).

Benzopyrene is rapidly incorporated and metabolized, and induces oxidative stress and activation of antioxidant enzymes, and CYP450 and GST metabolizing enzymes in Ulva lactuca. To analyze absorption and metabolism of benzo[a]pyrene (BaP) in Ulva lactuca, the alga was cultivated with 5µM of BaP for 72h. In the culture medium, BaP level rapidly decreased reaching a minimal level at 12h and, in the alga, BaP level increased until 6h, remained stable until 24h, and decreased until 72h indicating that BaP is being metabolized in U. lactuca. In addition, BaP induced an initial increase in hydrogen peroxide decreasing until 24h, superoxide anions level that remained high until 72h, and lipoperoxides that initially increased and decreased until 72h, showing that BaP induced oxidative stress. Activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (AP), glutathione reductase (GR) and glutathione peroxidase (GP) were increased, whereas dehydroascorbate reductase (DHAR) activity was unchanged. The level of transcripts encoding these antioxidant enzymes was increased, but transcripts encoding DHAR remained unchanged. Interestingly, the activity of glutathione-S-transferase (GST) was also increased, and inhibitors of cytochrome P450 (CYP450) and GST activities enhanced the level of BaP in algal tissue, suggesting that these enzymes participate in BaP metabolism.

Cytochrome b5 impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation: studies in hepatic cytochrome b5/P450 reductase null (HBRN) mice

Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b5, which can also act as an electron donor from cytochrome b5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP–DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP–DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.

Research Article Genetic expression of pobA and fabHB in Bacillus licheniformis M2-7 in the presence of benzo[a]pyrene

Bacillus licheniformis M2-7 is a heat-resistant bacterium able to biotransform polycyclic aromatic hydrocarbons. It can transform a wide range of these compounds as naphthalene, phenanthrene, pyrene and benzo[a]pyrene. Benzo[a]pyrene is a polycyclic aromatic hydrocarbon of high molecular weight considered as potentially toxic and carcinogenic for humans. Aiming to discover the genes involved in the biotransformation of benzo[a]pyrene, we made a B. licheniformis M2-7 genomic library in E. coli. We isolated two E. coli strains that were able to grow in minimal salt medium supplemented with benzo[a]pyrene. From the analysis of the DNA fragments in the clones H23 and H38, we identified open reading frames coding for 5 possible genes, among them pobA and fabHB, which products are the enzymes 4-hydroxybenzoate 3-monooxygenase and the ketoacyl-ACP synthase III, respectively. To evaluate the role of these genes in the metabolism of benzo[a]pyrene in B. licheniformis M2-7, we estimated their relative expression through reverse transcription quantitative PCR. Finally, we observed that the genes pobA and fabHB were overexpressed after 3 h under induction with benzo[a]pyrene, suggesting that this strain could use these genes during the metabolism of this PAH, plus it does it in a faster time than that reported for other bacterial genera

Incorporation of a metabolizing system in biodetection assays for endocrine active substances

The use of in vitro assays for the biodetection of endocrine active substances (EAS) aims to reduce and replace in vivo studies required for regulatory assessment. However, this approach often fails to take into account the role of biotransformation on the activity of the test substances. A method introducing an S9 metabolic system into the CALUX-reporter gene assays for estrogen receptor α- and anti-androgen receptor-mediated activities was developed. Methoxychlor, which exhibits increased estrogenic and anti-androgenic activities after biotransformation, was used to establish the method in ERα and anti-AR CALUX. For the anti-androgenic assay, stanozolol was used as a competing agonist not metabolized by S9. The method was first applied in both agonist and antagonist modes to methoxychlor and bisphenol A, as positive and negative controls, respectively. Then, benzo(a)pyrene and flutamide were also tested for their bioactivation potential. Co-treatment with S9 successfully increased the ERα agonist and AR antagonist potency of methoxychlor; no change was observed for bisphenol A. Incubation with S9 also enhanced the anti-androgenic activity of flutamide. Interestingly, the metabolism of benzo(a)pyrene by S9 resulted in a greatly increased estrogen receptor-mediated transcriptional activation, while the shift in EC50 was only minor. It is likely that both enzyme kinetics and metabolite stability affect the composition of the final metabolite mixture and thus influence the observed effects. Together these results demonstrate the relevance of including biotransformation in in vitro bioassays for the detection of EAS.

Incorporation of a metabolizing system in biodetection assays for endocrine active substances.

The use of in vitro assays is important for the biodetection of endocrine active substances (EAS), reducing and replacing the in vivo studies required for regulatory assessment. However, this approach often fails to take into account the role of biotransformation on the activity of the test substances. A method incorporating an S9 metabolic system into the CALUX-reporter gene assays for estrogen receptor α- and anti-androgen receptor -mediated activities has been developed. Methoxychlor, which is known to exhibit increased estrogenic and anti-androgenic activities after biotransformation, was used to set up the method in ERa and anti-AR CALUX. For the anti-androgenic assay, stanozolol was used as a competing agonist not metabolized by S9. The method was first applied in both agonist and antagonist modes to methoxychlor and bisphenol A, as positive and negative controls, respectively. Then, benzo(a)pyrene and flutamide were also tested for their potential of bioactivation. Co-treatment with S9 successfully increased the ERα agonist and AR antagonist potency of methoxychlor; no change was observed for bisphenol A. Incubation with S9 also enhanced the anti-androgenic activity of flutamide. Interestingly, the metabolism of benzo(a)pyrene by the S9 resulted in an increased estrogen receptor-mediated transcriptional activation; any increase in the potency was only minor. It is likely that both enzyme kinetics and metabolite stability have influenced these effects, which would affect the composition of the final metabolite mixture. Together these results demonstrate the relevance of including biotransformation in in vitro bioassays for the detection of EAS.

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

Heterologous expression of human cytochrome P450 2S1 in Escherichia coli and investigation of its role in metabolism of benzo[a]pyrene and ellipticine.

Cytochrome P450 (CYP) 2S1 is “orphan” CYP that is overexpressed in several epithelial tissues and many human tumors. The pure enzyme is required for better understanding of its biological functions. Therefore, human CYP2S1 was considered to be prepared by the gene manipulations and heterologous expression in Escherichia coli. Here, the conditions suitable for efficient expression of human CYP2S1 protein from plasmid pCW containing the human CYP2S1 gene were optimized and the enzyme purified to homogeneity. The identity of CYP2S1 as the product of heterologous expression was confirmed by dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. To confirm the presence of the enzymatically active CYP2S1, the CO spectrum of purified CYP2S1 was recorded. Since CYP2S1 was shown to catalyze oxidation of compounds having polycyclic aromatic structures, the prepared enzyme has been tested to metabolize the compounds having this structural character; namely, the human carcinogen benzo[a]pyrene (BaP), its 7,8-dihydrodiol derivative, and an anticancer drug ellipticine. Reaction mixtures contained besides the test compounds the CYP2S1 enzyme reconstituted with NADPH:CYP reductase (POR) in liposomes, and/or this CYP in the presence of cumene hydroperoxide or hydrogen peroxide. High performance liquid chromatography was employed for separation of BaP, BaP-7,8-dihydrodiol, and ellipticine metabolites. The results found in this study demonstrate that CYP2S1 in the presence of cumene hydroperoxide or hydrogen peroxide catalyzes oxidation of two of the test xenobiotics, a metabolite of BaP, BaP-7,8-dihydrodiol, and ellipticine. Whereas BaP-7,8,9,10-tetrahydrotetrol was formed as a product of BaP-7,8-dihydrodiol oxidation, ellipticine was oxidized to 12-hydroxyellipticine, 13-hydroxyellipticine, and the ellipticine N2-oxide.Graphical abstractElectronic supplementary materialThe online version of this article (doi:10.1007/s00706-016-1738-2) contains supplementary material, which is available to authorized users.

Metabolism of benzo(a)pyrene by aortic subcellular fractions in the setting of abdominal aortic aneurysms

As exposure to polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) have been implicated in cardiovascular diseases, the ability of the aortic tissue to process these toxicants is important from the standpoint of abdominal aortic aneurysms and atherosclerosis. Benzo(a)pyrene (B(a)P), a representative PAH compound is released into the environment from automobile exhausts, industrial emissions, and considerable intake of B(a)P is also expected in people who are smokers and barbecued red meat eaters. Therefore, knowledge of B(a)P metabolism in the cardiovascular system will be of importance in the management of vascular disorders. Toward this end, subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) were isolated from the aortic tissues of Apo E mice that received a 5mg/kg/week of B(a)P for 42 days and 0.71mg/kg/day for 60 days. The fractions were incubated with 1 and 3μM B(a)P. Post incubation, samples were extracted with ethyl acetate and analyzed by reverse-phase HPLC. Microsomal B(a)P metabolism was greater than the rest of the fractions. The B(a)P metabolite levels generated by all the subcellular fractions showed a B(a)P exposure concentration-dependent increase for both the weekly and daily B(a)P treatment categories. The preponderance of B(a)P metabolites such as 7,8-dihydrodiol, 3,6-, and 6,12-dione metabolites are interesting due to their reported involvement in B(a)P-induced toxicity through oxidative stress.

Metabolism of benzo(a)pyrene by subcellular fractions of gastrointestinal (GI) tract and liver in Apc Min mouse model of colon cancer

Given the fact that increased dietary intake of polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) leads to the formation and development of colon tumors, the ability of the gastrointestinal tract to process these compounds is important from the viewpoint of toxicity/carcinogenesis. Benzo(a)pyrene (BaP), a prototypical PAH compound is released into the environment from automobile exhausts, cigarette smoke, and industrial emissions. Additionally, considerable intake of BaP is expected in people who consume barbecued foods and a diet rich in saturated fat. In exposed animals, BaP becomes activated to potent metabolites that interfere with target organ function and as a consequence cause toxicity and cancer. Therefore, knowledge of BaP metabolism in the digestive system will be of importance in the management of cancers of the digestive tract. The objective of our study was to study the metabolism of BaP by subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) of the gastrointestinal tract and liver. Subcellular fractions were isolated by differential centrifugation from the stomach, jejunum, colon, and liver tissues of Apc(Min) mice that received a subchronic dose of 25 μg/kg BaP. The fractions were incubated with 1 and 3 μM BaP. Subsequent to incubation, samples were extracted with ethyl acetate and analyzed for BaP metabolites by reverse-phase HPLC equipped with fluorescence detection. Among the different fractions tested, microsomal BaP metabolism was higher than the rest of the fractions in all the samples analyzed. Additionally, a BaP exposure concentration-dependent effect on metabolite levels generated by the subcellular fractions was recorded. The BaP metabolites identified were the following: BaP-9,10-diol; BaP-4,5-diol; BaP-7,8-diol; 9(OH) BaP; 3(OH) BaP; BaP-3,6-dione; and BaP-6,12-dione. While the diol group of metabolites was frequently detected, among diones, the 3,6 and 6,12-dione metabolites were infrequently detected. Among the diol metabolites, the preponderance of BaP-7,8-dihydrodiol is interesting, since this metabolite is a precursor to the DNA-reactive BaP-7,8-dihydrodiol epoxide (BPDE) that has been linked to BaP-induced cancer.

Screening reactive metabolites bioactivated by multiple enzyme pathways using a multiplexed microfluidic system.

A multiplexed, microfluidic platform to detect reactive metabolites is described, and its performance is illustrated for compounds metabolized by oxidative and bioconjugation enzymes in multi-enzyme pathways to mimic natural human drug metabolism. The device features four 8-electrode screen printed carbon arrays coated with thin films of DNA, a ruthenium-polyvinylpyridine (RuPVP) catalyst, and multiple enzyme sources including human liver microsomes (HLM), cytochrome P450 (cyt P450) 1B1 supersomes, microsomal epoxide hydrolase (EH), human S9 liver fractions (Hs9) and N-acetyltransferase (NAT). Arrays are arranged in parallel to facilitate multiple compound screening, enabling up to 32 enzyme reactions and measurements in 20-30 min. In the first step of the assay, metabolic reactions are achieved under constant flow of oxygenated reactant solutions by electrode driven natural catalytic cycles of cyt P450s and cofactor-supported bioconjugation enzymes. Reactive metabolites formed in the enzyme reactions can react with DNA. Relative DNA damage is measured in the second assay step using square wave voltammetry (SWV) with RuPVP as catalyst. Studies were done on chemicals known to require metabolic activation to induce genotoxicity, and results reproduced known features of metabolite DNA-reactivity for the test compounds. Metabolism of benzo[a]pyrene (B[a]P) by cyt P450s and epoxide hydrolase showed an enhanced relative DNA damage rate for DNA compared to cyt P450s alone. DNA damage rates for arylamines by pathways featuring both oxidative and conjugative enzymes at pH 7.4 gave better correlation with rodent genotoxicity metric TD(50). Results illustrate the broad utility of the reactive metabolite screening device.

Time-resolved fluorescence studies of the effects of metabolism on the In Vitro reversible binding and accumulation of benzo[a]pyrene in DNA

Time-resolved fluorescence techniques have been employed to compare the DNA association constant of the parent hydrocarbon benzo[a]pyrene versus that of trans-7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene. The latter molecule is a highly carcinogenic metabolite derived from benzo[a]pyrene. Solubilization experiments were also carried out to compare the maximum total uptake of benzo[a]pyrene and of the metabolite by DNA. In the solvent system used in the association constant measurements (20% methanol) the association constant for benzo[a]pyrene is 3.0+0.3 × 104 M-1 while that of the more polar proximate carcinogen is 7.5 times lower. The results of the solubilization studies carried out in buffered aqueous medium demonstrate that at a DNA concentration of 5 × 10-4 M in PO-4 the maximum uptake of benzo[a]pyrene into DNA is one molecule per 2.2+0.5 × 103 nucleotide bases. The maximum uptake of the more soluble proximate carcinogen is 4 to 25 times greater than that of the parent hydrocarbon. The present results indicate that metabolism of benzo[a]pyrene leads to the formation of a nonreactive proximate carcinogen which has a lower binding constant than the parent hydrocarbon, but which is better able to diffuse through a polar medium and to accumulate at ultimate biological target sites.

The role of aryl hydrocarbon receptor in regulation of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons in a model of rat liver progenitor cells

In contrast to hepatocytes, there is only limited information about the expression and activities of enzymes participating in metabolic activation of environmental mutagens, including polycyclic aromatic hydrocarbons (PAHs), in liver progenitor cells. In rat liver “stem-like” WB-F344 cell line, sharing many characteristics with rat liver progenitor cells, PAHs are efficiently activated to their ultimate genotoxic metabolites forming DNA adducts. The present study aimed to characterize expression/activities of enzymes of two major pathways involved in the metabolism of benzo[ a]pyrene (BaP): cytochrome P450 (CYP) family 1 enzymes and cytosolic aldo–keto reductases (AKRs). We report here that, apart from induction of CYP1A1 and CYP1B1 expression and the corresponding enzymatic activity, both BaP and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induced rat 3α-hydroxysteroid dehydrogenase (AKR1C9) expression and activity. In contrast, the aldehyde reductase AKR1A1 was not induced by either treatment. Thus, both CYP1 and AKR metabolic pathways were inducible in the model of liver progenitor cells. BaP and TCDD were efficient inducers of NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in WB-F344 cells, a principal enzyme of cellular antioxidant defense. Both compounds also induced expression of transcription factor NRF2, involved in control of enzymes protecting cells from oxidative stress. However, although BaP induced a significant formation of reactive oxygen species, it did not induce expression of heme oxygenase-1, suggesting that induction of oxidative stress by BaP was limited. Using shRNA against the aryl hydrocarbon receptor (AhR), we found that similar to CYP1A1 and CYP1B1, the AKR1C9 induction was AhR-dependent. Moreover, constitutive AKR1C9 levels in AhR-deficient rat BP8 hepatoma cells were significantly lower than in their AhR-positive 5L variant, thus supporting possible role of AhR in regulation of AKR1C9 expression. Taken together, both CYP1 and AKR1C9 appear to be AhR-regulated metabolic pathways, which may contribute to formation of pro-carcinogenic PAH metabolites in liver progenitor cells.

Pregnane X Receptor Protects HepG2 Cells from BaP-Induced DNA Damage

Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP-DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50-65% (p < 0.001), demonstrating that PXR protects liver cells from genotoxicity induced by exposure to BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p < 0.001), independent of BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.

The uptake and metabolism of benzo[ a]pyrene from a sample food substrate in an in vitro model of digestion

Food ingestion is the major route of exposure to many hydrophobic organic contaminants (HOCs) such as benzo[ a]pyrene (BaP). It has been proposed that food-bound HOCs may become bioavailable after their mobilization by gastrointestinal fluids. The purpose of this research was to measure the uptake efficiency of [ 14C]-BaP bound to skim milk powder using an in vitro model of gastrointestinal digestion followed by sorption to human enterocytes (Caco-2 cells). Neutralization of intestinal fluids released [ 14C]-BaP into the soluble fraction. Ageing of benzo[ a]pyrene onto skim milk for 6 months significantly decreased the mobilized fraction but did not affect the amount of benzo[ a]pyrene taken up into Caco-2 cells. Hence, significant differences in aqueous phase concentrations may not always be reflected in significant differences in uptake. We obtained evidence that the digestion/uptake of skim milk lipids is accompanied by the diffusive uptake of BaP (the fat flush hypothesis) as trans-cellular transfer of BaP was favoured in the apical to basolateral direction. These data support the theory that non-polar substances including HOCs are preferentially transferred from the lumen into the bloodstream and provide indirect evidence that the uptake is related to the fugacity gradient created by the unidirectional uptake of dietary lipids.

Tissue Distribution and Metabolism of Benzo[a]pyrene in Embryonic and Larval Medaka (Oryzias latipes)

The need to understand chemical uptake, distribution, and metabolism in embryonic and larval fish derives from the fact that these early life stages often exhibit greater sensitivity to xenobiotic compounds than do adult animals. In this study, a 6-h acute waterborne exposure immediately after fertilization was used to quickly load the egg with benzo[a]pyrene (BaP). This exposure was used to mimic the initial egg concentration of a persistent bioaccumulative toxicant that could result from maternal transfer. We used multiphoton laser scanning microscopy (MPLSM) in combination with conventional analytical chemistry methods to characterize the tissue distribution of BaP and its principal metabolites in medaka embryos and post-hatch larvae. Embryonic metabolism of BaP was evident by MPLSM prior to liver formation or heart development. A major product of this metabolism was identified by liquid chromatography/mass spectrometry as BaP-3-glucuronide. MPLSM showed that metabolites were sequestered within the yolk, biliary system, and gastrointestinal tract. When the gastrointestinal tract became patent a few days after hatch, the metabolites were rapidly eliminated. These findings indicate that some of the earliest embryonic tissues are metabolically competent and that redistribution of BaP and its metabolic products occurs throughout development. Rapid metabolism of BaP substantially reduces the body burden of parent chemical in the developing embryo, potentially reducing toxicity. It remains unclear whether metabolism of BaP in medaka embryos leads to the formation of DNA adducts associated with genotoxic effects or yields metabolites that later lead to other toxicity in juveniles or adults.

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.

Role of cell signaling in B[ a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[ a]P metabolites

Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[ a]pyrene (B[ a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor α-naphthoflavone (α-NF), the p53 inhibitor pifithrin-α (PFT-α), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[ a]P as measured by the generation of tetrols and by covalent binding of B[ a]P to macromolecules. In contrast, the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, had neither an effect on the cyp1a1 expression nor the B[ a]P-metabolism. In order to avoid these unspecific effects, we characterized the mechanisms involved in the apoptotic effects of B[ a]P-metabolites. B[ a]P and the B[ a]P-metabolites B[ a]P-7,8-DHD and BPDE-I induced apoptosis, whereas B[ a]P-4,5-DHD had no effect. B[ a]P, B[ a]P-7,8-DHD and BPDE-I induced an accumulation and phosphorylation of p53, while the Bcl-2 proteins Bcl-xl, Bad and Bid were down-regulated. Interestingly, the levels of anti-apoptotic phospho-Bad were up-regulated in response to B[ a]P as well as to B[ a]P-7,8-DHD and BPDE-I. Both p38 MAPK and JNK were activated, but the p38 MAPK inhibitors were not able to inhibit BPDE-I-induced apoptosis. PFT-α reduced the BPDE-I-induced apoptosis, while both the PI-3 kinase inhibitor and the ERK inhibitors increased the apoptosis in combination with BPDE-I. BPDE-I also triggered apoptosis in primary cultures of rat lung cells. In conclusion, often used cell signaling inhibitors both enhanced the expression and the level of cyp1a1 and more directly acted as inhibitors of cyp1a1 metabolism of B[ a]P. However, studies with the B[ a]P-metabolite BPDE-I supported the previous suggestion that p53 has a role in the pro-apoptotic signaling pathway induced by B[ a]P. Furthermore, these studies also show that the reactive metabolites of B[ a]P induce the anti-apoptotic signals, Akt and ERK. Neither the induction nor the activity of p38 MAPK and JNK seems to be of major importance for the B[ a]P-induced apoptosis.