Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and (32)P-postlabelling

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kgbody weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.     

Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines

Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants classified as carcinogens in humans and rodents. The cytochromes P4501A1 and 1B1 have both shown capacity to carry out bioactivation of the prototype PAH, benzo[a]pyrene (B[a]P) to its ultimate carcinogenic B[a]P-diol-epoxide-I-1 form. The part played by each enzyme in human lung cells, however, has not been clarified. To get further insight into their individual role in the metabolic activation of B[a]P, RNA-interference was used to down-regulate CYP1A1 and/or CYP1B1 gene expression in the human lung cell lines BEP2D and NCIH2009. Fluorescence-HPLC analysis revealed that formation of B[a]P-tetrol-I-1 (hydrolyzed form of the corresponding diol-epoxide) was dependent primarily on CYP1A1. In cells without down-regulation of CYP1A1, the B[a]P-tetrol-I-1 was the major tested isomer formed. In contrast, the B[a]P-cis- and trans-7,8-dihydrodiol isomers were readily formed in cells expressing high levels of either CYP-gene. Simultaneous down-regulation of CYP1A1 and CYP1B1 mRNA resulted in low levels of metabolites overall. Residual unmetabolized B[a]P levels followed the expression of CYP1A1 in an inverse manner. In conclusion, these results indicate a major role of CYP1A1 in the bioactivation of B[a]P to carcinogenic B[a]P-diol-epoxides and in overall metabolism of B[a]P in human lung cell lines. In contrast, both CYP1A1 and CYP1B1 contribute significantly to the formation of the B[a]P-cis- and trans-7,8-dihydrodiol isomers.     

Proteomic analysis of MCF-7 cells treated with benzo[a]pyrene, dibenzo[a,l]pyrene, coal tar extract, and diesel exhaust extract

Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.     

Benzo(a)pyrene-induced metabolic responses in Manila clam Ruditapes philippinarum by proton nuclear magnetic resonance ((1)H NMR) based metabolomics

Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) which causes carcinogenic, teratogenic and mutagenic effects in various species and the level of contamination of this toxic agent in the marine environment is of great concern. In this study, metabolic responses induced by two doses (0.02 and 0.2 μM) of BaP were characterized in the gill tissues of Manila clam Ruditapes philippinarum after exposure for 24, 48 and 96 h. The high dose (0.2 μM) of BaP induced the disturbances in energy metabolism and osmotic regulation based on the metabolic biomarkers such as succinate, alanine, glucose, glycogen, branched chain amino acids, betaine, taurine, homarine, and dimethylamine in clam gills after 24 h of exposure. In addition, hormesis induced by BaP was found in clams exposed to both doses of BaP. Overall, our results demonstrated the applicability of metabolomics for the elucidation of toxicological effects of marine environmental contaminants in a selected bioindicator species such as the Manila clam.     

Benzo[a]pyrene-induced immunotoxicity in Japanese medaka (Oryzias latipes): relationship between lymphoid CYP1A activity and humoral immune suppression

Exposure to the environmental contaminant benzo[a]pyrene (BaP) results in suppression of immune function in both mammalian and fish species. This laboratory has previously demonstrated that a single intraperitoneal (IP) injection of BaP reduced lymphocyte proliferation, phagocyte-mediated superoxide generation, and antibody-forming cell (AFC) numbers in Japanese medaka (Oryzias latipes). The objective of the current study was to determine the role of BaP metabolism in the observed immunosuppression. Results from rodent studies have suggested that BaP elicits its immunotoxic effects via upregulation of cytochrome P4501A1 (CYP1A1) and the subsequent production of immunosuppressive BaP metabolites. In this study, exposure of medaka to 200 microg BaP/g BW significantly induced CYP1A expression or activity within lymphoid tissue 48 h post-IP injection; induction was observed specifically within distinct subpopulations of kidney mononuclear cells. Concurrent injection of fish with BaP and the CYP1A1 inhibitors alpha-naphthoflavone (ANF) or dehydroepiandrosterone (DHEA) resulted in inhibition of renal EROD activity and amelioration of BaP-induced suppression of medaka AFC numbers. Results of this study suggest that (1) BaP-induced suppression of medaka humoral immunity relies upon the CYP1A-catalyzed production of immunotoxic BaP metabolites and (2) BaP metabolites may be created in situ, directly by specific cells within kidney lymphoid tissue. Thus, apparently, mechanisms involved in BaP-induced immunosuppression have been phylogenetically conserved from fish to mammals.     

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.     

Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site

Benzo[a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (King's Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to King's Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC–MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure.     

Fluoranthene enhances p53 expression and decreases mutagenesis induced by benzo[a]pyrene

Fluoranthene (Fla) is the most abundant polycyclic aromatic hydrocarbon (PAH) in diesel particulate extracts. Benzo[a]pyrene (BaP) is genotoxic and is a prototype of PAH carcinogens. Fla's toxicity and mutagenicity are minor relative to BaP's. Our data showed that Fla enhanced BaP-induced p53 expression and promoted BaP-induced cell death. In contrast, Fla decreased BaP-induced mutagenesis. Fla had almost no influence on the cell cycle. However, the effect of cotreatment with BaP (1 μM) and Fla (10 μM) in regulating the cell cycle was greater than that of BaP (2 μM) alone. It is known that BaP activates the aryl hydrocarbon receptor (AhR), and, in turn, the AhR induces cytochrome P450 (Cyp)1a1 expression. The expression of Cyp1a1 corresponds well with the induction of apoptosis and mutagenesis by BaP. Fla did not activate the AhR or antagonize BaP's induction of AhR activity and Cyp1a1 expression. Therefore, the actions of Fla on BaP's toxicity were independent of the AhR signal and Cyp1a1. In summary, results indicated that Fla directs BaP-treated cells into death rather than mutagenesis, consequently preventing cells from being transformed. The novel cooperation between Fla and BaP provides valuable information for how to increase expression of the p53 tumor suppressor.     

Benzo(a)pyrene decreases brain and ovarian aromatase mRNA expression in Fundulus heteroclitus

The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100 microg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3-month-old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100 microg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development.     

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimuriumumu strains expressing human cytochrome P450 and N-acetyltransferase

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1 nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.     

All-trans retinoic acid enhances the transport of phase II metabolites of benzo[a]pyrene by inducing the Breast Cancer Resistance Protein expression in Caco-2 cells

All-trans retinoic acid (atRA) is the most active metabolite of vitamin A. It is a ligand of retinoic acid receptors (RAR) as well as of retinoid X receptors (RXR) and effectively stimulates the RAR/RXR signalling pathway.     

The critical DNA damage by benzo(a)pyrene in lung tissues of smokers and approaches to preventing its formation

Benzo(a)pyrene (BP) and cadmium are environmental pollutants found in foodstuffs, cigarette smoke, and polluted air. BP is converted in liver and lung to benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) by the enzymes of the cytochrome P450 (CYP) superfamily, namely CYP1A1/1A2, and CYP1B1. BPDE reacts with DNA primarily at the N²-position of guanine, producing benzo(a)pyrene-7,8-diol-9,10-epoxide-N²-deoxyguanosine (BPDE-dG) adduct. BPDE reacts with DNA also at N⁶ position of adenine, producing the minor N⁶-deoxyadenosine adduct, but BPDE-dG adduct is a well-established risk factor for lung cancer. We thus argue that BPDE-dG adduct could be used as a model biomarker in searching and validating of approaches to reducing lung cancer risk. If the formation of BPDE-dG adduct were to be inhibited or blocked in bronchial epithelial cells, so could lung cancer development. The best way to lower BPDE-dG formation in the lung is to stop smoking. However, the following approaches could also be considered for highly addicted smokers: (a) decrease BP and cadmium intake from food, cigarettes and other environmental sources; (b) avoid meat and other food high in BP and cadmium; (c) decrease the CYP-mediated conversion of BP in liver and lung; (d) lower free radicals and cadmium in cigarette smoke; and (e) increase BPDE detoxification.     

Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites

The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 microM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17beta-estradiol (E(2)) metabolism, whereas BKF levels greater than 1 muM inhibited E(2) metabolism. Time course studies showed that induction of CYP1-catalyzed E(2) metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.     

Carotenoids as regulators for inter-species difference in cytochrome P450 1A expression and activity in ungulates and rats

Ungulates (deer, cattle and horses) are reported as animal species which show extreme-accelerated metabolism of CYP1A substrates, such as ethoxyresorufin compared to rats. This study was undertaken to investigate whether accumulation of carotenoids is a possible cause for inter-species difference in CYP1A-dependent activity in this group of animals. The relationship between inter-species differences in CYP1A-dependent activity and the accumulated carotenoids and retinoids as candidates of dietary CYP1A inducers in ungulate species was clarified. Interestingly, there were positive correlations between the accumulated carotenoids, such as β-carotene, with both EROD activity and CYP1A protein expression. These correlations were negative with the accumulated retinoids, such as retinol. The β-carotene was major component of carotenoids in ungulates, and known as an inducer of CYP1A. On the other hand, the retinol is reported as the inhibitor of CYP1A. Other factors which affect CYP1A1 expression, such as polycyclic aromatic hydrocarbons, were also analyzed. To cancel the effects of inter-species difference in CYP1A induction signal cascade among these animals, the rat cell line (H4-II-cells) was treated with the extracted carotenoids from the examined animals. In conclusion, carotenoids and retinoids may have direct effects on the inter-species differences in CYP1A-dependent activity and protein expression.     

3-Methylcholanthrene and benzo(a)pyrene modulate cardiac cytochrome P450 gene expression and arachidonic acid metabolism in male Sprague Dawley rats

Background and purpose:

There is a strong correlation between cytochrome P450 (P450)-dependent arachidonic acid metabolism and the pathogenesis of cardiac hypertrophy. Several aryl hydrocarbon receptor (AhR) ligands were found to alter P450-dependent arachidonic acid metabolism. Here, we have investigated the effect of 3-methylcholanthrene (3-MC) and benzo(a)pyrene (BaP), two AhR ligands, on the development of cardiac hypertrophy.

Experimental approach:

Male Sprague Dawley rats were injected (i.p.) daily with either 3-MC (10 mg·kg⁻¹) or BaP (20 mg·kg⁻¹) for 7 days. Then hearts were removed, and the heart to body weight ratio and the gene expression of the hypertrophic markers and P450 genes were determined. Levels of arachidonic acid metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry.

Key results:

Both 3-MC and BaP increased the heart to body weight ratio as well as the hypertrophic markers, atrial natriuretic peptide and brain natriuretic peptide. 3-MC and BaP treatment increased the gene expression of CYP1A1, CYP1B1, CYP2E1, CYP4F4, CYP4F5 and soluble epoxide hydrolase. Both 3-MC and BaP treatments increased the dihydroxyeicosatrienoic acids (DHETs) : epoxyeicosatrienoic acids (EETs) ratio and the 20-hydroxyeicosatetraenoic acid (20-HETE) : total EETs ratio. Treatment with benzo(e)pyrene, an isomer of BaP that is a poor ligand for the AhR, did not induce cardiac hypertrophy in rats, confirming the role of AhR in the development of cardiac hypertrophy. Treatment with the ω-hydroxylase inhibitor, HET0016, significantly reversed BaP-induced cardiac hypertrophy.

Conclusions and implications:

3-MC and BaP induce cardiac hypertrophy by increasing the ratio of DHETs : EETs and/or the ratio of 20-HETE : total EETs, through increasing soluble epoxide hydrolase activity.     

Effect of polymeric black tea polyphenols on benzo(a)pyrene [B(a)P]-induced cytochrome P4501A1 and 1A2 in mice

The chemopreventive activity of green tea polyphenols (GTPs) is, in part, due to modulation of cytochrome P450s (CYPs). To investigate the enzyme modulatory properties of major black tea polyphenols, the effect of decaffeinated black tea extract (DBTE) or polymeric black tea polyphenol (PBP) mix was studied on CYP1A1 and CYP1A2 in mouse tissues. Animals receiving 2.5% DBTE or 1% PBP mix or drinking water (15 days) were challenged with single oral benzo(a)pyrene (B(a)P) (1?mg/mouse) treatment on the 14th day. Liver and lung microsomes isolated after 24?h were analysed for CYP1A1 and CYP1A2, using biochemical substrate(s) and Western blot analysis. Treatment with 2.5% DBTE or 1% PBP mix did not significantly alter the basal activity and level of CYP1A1 and CYP1A2, whereas pretreatment with 2.5% DBTE or 1% PBP mix resulted in a significant decrease in both the activity and the level of B(a)P-induced CYP1A1 and CYP1A2 in liver and lungs. The PBP mix possesses enzyme modulatory properties exhibited by monomeric GTPs.     

Synergistic enhancement of H2O2 production in human epidermoid carcinoma cells by Benzo[a]pyrene and ultraviolet A radiation

Benzo[a]pyrene (BaP) is an ubiquitous environmental pollutant with potential carcinogenecity. It was shown that BaP, upon irradiation by UV A, enhanced the formation of 8-hydroxy-2'-deoxyguanosine in purified DNA and in cultured cells. The purpose of this present study was to determine whether BaP and UV radiation synergistically generate reactive oxygen species (ROS) that consequently result in the oxidation of DNA bases. In this study, the levels of H(2)O(2) were measured as an indicator of ROS in A431 cells and primary human keratinocytes treated with BaP plus UV radiation. Production of H(2)O(2) significantly increased from cells treated with BaP plus UVB or UVA, with the latter having a much greater effect. The responses of A431 cells and primary human keratinocytes to BaP and UVA irradiation were similar in generation of extracellular H(2)O(2). Also, H(2)O(2) production proportionally correlated with UVA and UVB dose, but was independent of time or BaP concentration. Treatment with catalase and general ROS scavengers significantly decreased H(2)O(2) production from cells treated with BaP plus UVA, whereas scavengers of *O2-, *OH, and (1)O(2) had minimal effects. These results demonstrate that BaP synergistically enhances the production of H(2)O(2) from cultured cells by UVA and, to a lesser extent, by UVB, supporting the hypothesis that interaction of BaP and UVA can generate ROS and further substantiate oxidative DNA damage that may lead to carcinogenesis.     

Reduction of androgen receptor expression by benzo[alpha]pyrene and 7,8-dihydro-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene in human lung cells

5Alpha-dihydrotestosterone significantly increased cell growth of lung adenocarcinoma cell line H1355. Benzo[alpha]pyrene (BaP) was a pulmonary carcinogen found in cigarette smoke. Treatment with 1microM BaP tremendously reduced constitutive androgen receptor (AR) expression, as determined with Western immunoblotting and the real-time RT-PCR assay, as well as testosterone-induced AR protein levels in H1355 cells. Similarly, 1microM BaP significantly reduced AR mRNA levels in human bronchial epithelial cells BEAS-2B. Although BaP, 2,3,7,8-tetrachlorodibenzo-p-dixin and polychlorinated biphenyl 126 activated aryl hydrocarbon receptor (AhR), which subsequently induced cytochrome P4501A1 (CYP1A1) and P4501B1 (CYP1B1) expression in H1355 cells, unexpectedly, neither TCDD nor PCB126 reduced AR expression. Antagonizing AhR activation and cytochrome P4501 activity with alpha-naphthoflavone, or inhibiting CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene, however, prevented BaP-induced AR reduction. Furthermore, 7,8-dihydro-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene, a BaP carcinogenic metabolite catalyzed by CYP1A1 and CYP1B1, significantly reduced AR expression in H1355 cells and human lung fibroblasts WI-38. This was the first study that reports that BaP and BPDE reduced endogenous AR expression. These data suggest that metabolically activated BaP may disrupt androgen function by reducing AR levels in androgen-responsive organs.     

Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (−)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2′-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (−)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.     

Comparative effects of cadmium, copper, paraquat and benzo[a]pyrene on the actin cytoskeleton and production of reactive oxygen species (ROS) in mussel haemocytes.

The immune defence of mussels is comprised of cell-mediated and humoral mechanisms, in which haemocytes or blood cells play a key role. Environmental pollutants such as metallic and organic xenobiotics exert immunotoxical effects on aquatic organisms. Some of these xenobiotics are known to give rise to highly reactive oxygen species (ROS), thereby leading to oxidative damage to tissue macromolecules including DNA, proteins and lipids. Previously we have detected enhancement of ROS production together with severe alterations in the actin cytoskeleton after exposure of mussel haemocytes to the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (b[a]p). In a similar way, cadmium is also known to cause disruption of the actin cytoskeleton in mussel haemocytes, however it is not known whether this effect occurs by direct action or through ROS production. The aim of the present study was to decipher whether cytoskeletal alterations caused by Cd in mussel haemocytes are linked to increased ROS production. ROS-producing model compounds copper (Cu), paraquat and b[a]p were used in parallel experiments for comparative purposes. In all contaminant exposure experiments actin cytoskeleton appeared damaged. On the other hand, ROS production was increased in paraquat and b[a]p exposure experiments but decreased in haemocytes exposed to Cu while no significant effects were detected in Cd exposure experiments. In conclusion, it appears that deleterious effects of Cu and Cd on the integrity of the actin cytoskeleton of haemocytes are not directly linked to ROS production, at least at the exposure conditions used in the present study.