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.     

Rapid induction of colonic adenocarcinoma in mice exposed to benzo[a]pyrene and dextran sulfate sodium

Previously, we reported that the mutation frequency was markedly increased in the colon after the oral treatment of mice with an environmental mutagen/carcinogen, benzo[a]pyrene (BP); however this was not followed by tumor development. The reasons for this are as yet unresolved. The purpose of the present study is to explore the mechanisms why a high frequency of mutations induced by BP in the colon is not associated with subsequent tumor development. We show in this study that oral administration of BP to CD2F₁ mice at 125 mg/kg/day for 5 days can lead to adenocarcinomas in the mouse colon both at Weeks 4 (5/8 mice) and 11 (100% of mice), but only in the presence of inflammation induced by 4% dextran sulfate sodium (DSS) in the drinking water for up to 2 weeks. These data indicate that, in this DSS model, BP induced mutagenic events lead to tumors in the mouse colon, a tissue which is not a BP target organ. DSS-induced inflammation in a tissue primed with mutagenic risk is a key to the induction of tumors in this model. This study provides a novel, rapid and useful colon carcinogenesis model (BP/DSS model).     

Benzo[a]pyrene induces complex H2AX phosphorylation patterns by multiple kinases including ATM, ATR, and DNA-PK

H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. While it has been reported that benzo[a]pyrene (BaP) cannot induce γH2AX alone in several cell lines, we have shown that BaP alone could induce γH2AX in human amnion FL cells. Thus, we further examined the ability of BaP to induce γH2AX in different cell systems. It was shown that BaP-induced γH2AX in HeLa cells in a time- and dose-dependent manner. BaP also induced γH2AX in ATM^−/− mouse fibroblasts, DNA-PKcs^−/− mouse fibroblasts, and a genetically modified human osteosarcoma U2OS cell line. PI3K inhibitors caffeine and wortmannin were then used in an effort to identify the kinase(s) responsible for BaP-induced γH2AX. Unexpectedly, in BaP-treated HeLa cells, caffeine pretreatment did not inhibit but rather increased γH2AX level. On the other hand, caffeine or wortmannin can inhibit BaP-induced γH2AX in either U2OS, DNA-PKcs^−/− or ATM^−/− cells. Taken together, these data suggest that BaP alone can induce H2AX phosphorylation in certain cell systems, and that members of the PI3K family, including ATM, ATR, and DNA-PK can participate in the phosphorylation of H2AX in the various cell types.     

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.     

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.     

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.     

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.     

Synthesis of halogen derivatives of benzo[h]chromene and benzo[a]anthracene with promising antimicrobial activities

The synthesis of novel 7-(4-halophenyl)-8,9-dihydro-7H-12-oxa-9,11-diaza-benzo[a]anthracene derivatives has been reported. The key intermediate 3-amino-9-chloro-1-(4-halophenyl)-1H-benzo[h]chromene-2-carbonitrile (3) was obtained by treating 4-halobenzylidenmalononitriles (1a-c) and ethyl 4-halobenzylidenmalonates (1d-f) with 4-chloro-1-naphthol (2) in ethanolic piperidine solution. Antimicrobial activity was shown for most of the synthesized compounds.     

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.     

Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene

Tissue-specific induction of mRNA of cytochrome P450 (P450 or CYP) 1A1 and 1B1 by polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) was investigated in wild and arylhydrocarbon receptor (AhR)-deficient C57BL/6J mice. Ratios of mRNA expression of CYP1A1 or CYP1B1 over beta-actin were determined and used to compare levels of expression and induction of these P450s by PAHs and PCBs in various organs. CYP1A1 mRNA was detected in control mice at very low levels in liver, lung, heart, kidney, intestine, thymus, testis, uterus, ovary, and brain and was highly induced in these organs by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. In AhR(+/+) and AhR(-/-) mice, CYP1B1 mRNA was found to be constitutively expressed at significant levels in heart (the ratio of mRNAs of CYP1B1 to beta-actin was approximately 0.6), kidney ( approximately 0.8), intestine ( approximately 0.3), testis ( approximately 0.9), thymus ( approximately 0.4), uterus ( approximately 0.3), ovary ( approximately 1.4), and brain ( approximately 0.4), whereas it was low in liver and lung (the mRNA ratio to beta-actin was <0.2 in these cases). CYP1B1 in the latter two organs was highly induced by PAHs and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. The induction of CYP1B1 by PAHs and PCBs was more extensive in organs in which the constitutive expression of CYP1B1 was low. For example, CYP1B1 was induced 9-fold and 10-fold by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in livers of male and female mice, respectively, whereas in testis and ovary, the fold induction of CYP1B1 by two inducers was only 1.1 and 1.4, respectively. Liver microsomal xenobiotic oxidation activities were induced by these PAHs and PCBs in male and female AhR(+/+) mice. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in their expression in extrahepatic organs of mice and could be induced by PAHs and PCBs with different extents of induction depending on the inducers used and the organs examined in AhR(+/+) mice. The findings of significant levels of constitutive expression of CYP1B1 in AhR(-/-) mice as well as AhR(+/+) mice in several organs including heart, kidney, thymus, testis, ovary, and brain in AhR(-/-) mice as well as AhR(+/+) mice are of importance in understanding the basis of toxicity and carcinogenesis by chemicals that are metabolized by CYP1B1.     

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.     

In vitro metabolism of benzo[a]pyrene and dibenzo[def,p]chrysene in rodent and human hepatic microsomes

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and often carcinogenic contaminants released into the environment during natural and anthropogenic combustion processes. Benzo[a]pyrene (B[a]P) is the prototypical carcinogenic PAH, and dibenzo[def,p]chrysene (DBC) is a less prevalent, but highly potent transplacental carcinogenic PAH. Both are metabolically activated by isoforms of the cytochrome P450 enzyme superfamily to form reactive carcinogenic and cytotoxic metabolites. Metabolism of B[a]P and DBC was studied in hepatic microsomes of male Sprague-Dawley rats, naïve and pregnant female B6129SF1/J mice, and female humans, corresponding to available pharmacokinetic data. Michaelis–Menten saturation kinetic parameters including maximum rates of metabolism (V_MAX, nmol/min/mg microsomal protein), affinity constants (K_M, μM), and rates of intrinsic clearance (CL_INT, ml/min/kg body weight) were calculated from substrate depletion data. CL_INT was also estimated from substrate depletion data using the alternative in vitro half-life method. V_MAX and CL_INT were higher for B[a]P than DBC, regardless of species. Clearance for both B[a]P and DBC was highest in naïve female mice and lowest in female humans. Clearance rates of B[a]P and DBC in male rat were more similar to female human than to female mice. Clearance of DBC in liver microsomes from pregnant mice was reduced compared to naïve mice, consistent with reduced active P450 protein levels and elevated tissue concentrations and residence times for DBC observed in previous in vivo pharmacokinetic studies. These findings suggest that rats are a more appropriate model organism for human PAH metabolism, and that pregnancy's effects on metabolism should be further explored.     

Identification and expression profile of a new cytochrome P450 isoform (CYP414A1) in the hepatopancreas of Venerupis (Ruditapes) philippinarum exposed to benzo[a]pyrene, cadmium and copper

With the objective to identify promising molecular biomarkers for marine pollution monitoring, a new cytochrome P450 gene was identified from Venerupis (Ruditapes) philippinarum and classified as a member of a new subfamily, CYP414A1. Phylogenetic analysis showed that CYP414A1 was closely related to members of the CYP2 family. CYP414A1 mRNA expression was significantly induced by 50 μg/L B[a]P at 96 h, while no significant change was found in 5 μg/L B[a]P-exposed samples. For heavy metals exposure, the expression of CYP414A1 was significantly up-regulated by Cd but sharply depressed by Cu exposure. These results suggested that CYP414A1 responded to various xenobiotics stresses, and could be used as a candidate biomarker of heavy metals and B[a]P.     

CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how “human-like” can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.     

Gaseous nitrogen oxide repressed benzo[a]pyrene-induced human lung fibroblast cell apoptosis via inhibiting JNK1 signals

Benzo[a]pyrene (B[a]P) is present in environmental pollution and cigarette smoke. B[a]P has been shown to induce apoptosis in hepatoma cells, human B cells, human ectocervical cells, macrophages, and rat lungs. Nitrogen oxides (NOx) are the other important indoor and outdoor air pollutants. Many studies have indicated that NO gas causes lung tissue damage both by its oxidative properties and free radicals. In our previous study we demonstrated that NO gas induced proliferation of human lung fibroblast MRC-5 cells. In this study we showed that NO gas inhibits B[a]P-induced MRC-5 cells apoptosis by cell cycle analysis. Western blot data revealed that NO gas increased the expressions of anti-apoptosis proteins (Bcl-2 and Mcl-1) and decreased the expression of apoptosis proteins (Bax, t-Bid, cytochrome c, FasL, and caspases) after B[a]P treatment. We further clarified that B[a]P-induced MRC-5 cell apoptosis via JNK1/FasL and JNK1/p53 signals. In conclusion, NO gas inhibited B[a]P-induced MRC-5 cells apoptosis via inhibition of JNK1 apoptosis pathway and induction of Bcl-2 and Mcl-1 anti-apoptosis pathway.     

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.     

Aldehyde dehydrogenase 1A1 up-regulates stem cell markers in benzo[a]pyrene-induced malignant transformation of BEAS-2B cells

Recently, Aldehyde dehydrogenase 1A1 (ALDH1A1) has been proposed to be a common marker of cancer stem cells and can be induced by benzo[a]pyrene (B[a]P) exposure. However, the underlying mechanism of how ALDH1A1 contributes to B[a]P-induced carcinogenesis in human bronchial epithelial cells remains unclear. Here, we found that B[a]P up-regulated expression levels of stem cell markers (ABCG2, SOX2, c-Myc and Klf4), epithelial-mesenchymal transition (EMT) associated genes (SNAIL1, ZEB1, TWIST and β-CATENIN) and cancer-related long non-coding RNAs (lncRNAs; HOTAIR and MALAT-1) in malignant B[a]P-transformed human bronchial epithelial cells (BEAS-2B-T cells), and these up-regulations were dependent on increased expression of ALDH1A1. The inhibition of endogenous ALDH1A1 expression down-regulated expression levels of stem cell markers and reversed the malignant phenotype as well as reduced the chemoresistance of BEAS-2B-T cells. In contrast, the overexpression of ALDH1A1 in BEAS-2B cells increased the expression of stem cell markers, facilitated cell transformation, promoted migratory ability and enhanced the drug resistance of BEAS-2B cells. Overall, our data indicates that ALDH1A1 promotes a stemness phenotype and plays a critical role in the BEAS-2B cell malignant transformation induced by B[a]P.     

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.     

Activity of rat UGT1A1 towards benzo[a]pyrene phenols and dihydrodiols

Four UDP-glucuronosyltransferases from the rat UGT1A family were tested for activity towards benzo[a]pyrene phenols and dihydrodiols. UGT1A1 and UGT1A7 were found to be broadly active towards BaP metabolites. Antisera recognizing rat UGT1A1 and UGT1A7 were used to assess UGT levels in relation to UGT activity towards benzo[a]pyrene-7,8-dihydrodiol (BPD). The rank BPD UGT activities were liver = intestine  kidney, whereas UGT1A1 was highest in liver and UGT1A7 was highest in intestine. Phenobarbital, an inducer of hepatic UGT1A1, only slightly increased BPD UGT activity, whereas UGT1A7 inducers more potently increased the activity. Inhibition studies using the differential UGT1A1 inhibitor, bilirubin, suggest that UGT1A1 is not a major contributor to the constitutive BPD glucuronidating activity of control rat liver microsomes. These data suggest that multiple UGT1A enzymes contribute to glucuronidation of BPD and other BaP metabolites, and that their relative contributions depend on tissue- and environmental-specific factors.