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.     

Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke

Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3 h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose–response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34 ng/ml for TPA, 0.050 mg/ml for the 2R4F, 0.044 mg/ml for the Bright cigarette, and 0.060 mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P < 0.0001), and between the single-tobacco cigarettes and the 2R4F (P = 0.0008, 2R4F vs. Burley and P < 0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.     

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.     

Enhancements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism and carcinogenic risk via NNK/arsenic interaction

Epidemiological studies indicated an enhancement of cigarette smoke-induced carcinogenicity, including hepatocellular carcinoma, by arsenic. We believe that arsenic will enhance the expression of hepatic CYP2A enzyme and NNK metabolism (a cigarette smoke component), thus its metabolites, and carcinogenic DNA adducts. Male ICR mice were exposed to NNK (0.5 mg/mouse) and sodium arsenite (0, 10, or 20 mg/kg) daily via gavaging for 10 days and their urine was collected at day 10 for NNK metabolite analysis. Liver samples were also obtained for CYP2A enzyme and DNA adducts evaluations. Both the cyp2a4/5 mRNA levels and the CYP2A enzyme activity were significantly elevated in arsenic-treated mice liver. Furthermore, urinary NNK metabolites in NNK/arsenic co-treated mice also increased compared to those treated with NNK alone. Concomitantly, DNA adducts (N(7)-methylguanine and O(6)-methylguanine) were significantly elevated in the livers of mice co-treated with NNK and arsenic. Our findings provide clear evidence that arsenic increased NNK metabolism by up-regulation of CYP2A expression and activity leading to an increased NNK metabolism and DNA adducts (N(7)-methylguanine and O(6)-methylguanine). These findings suggest that in the presence of arsenic, NNK could induce greater DNA adducts formation in hepatic tissues resulting in higher carcinogenic potential.     

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.     

Mass spectrometric analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung

An improved analytical method was developed for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung samples of patients undergoing surgery for lung cancer. HPB-releasing adducts can be formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, and have been reported to play an important role in tobacco carcinogenesis. [2,2,3,3-D(4)]HPB (D(4)-HPB) was used as an internal standard, and HPB released by acid hydrolysis of DNA was determined by gas chromatography/mass spectrometry in the negative ion chemical ionisation mode. The method is sensitive with a limit of detection of 5.9 fmol HPB and a limit of quantification of 15.2 fmol HBP/mg DNA. The recovery of HPB was 82+/-17% and the background response was 10.1+/-1.8 fmol HPB/sample. The concentration of HPB-releasing lung DNA adducts was significantly higher (p<0.0001) in 21 self-reported smokers compared to in 11 self-reported nonsmokers (404+/-258 fmol versus 59+/-56 fmol HPB/mg DNA, respectively). HPB-releasing hemoglobin adduct concentrations were only marginally higher in a subset of 12 smokers compared to in 7 nonsmokers (63+/-53 fmol versus 42+/-34 fmol HPB/g hemoglobin; p=0.36). No correlation was found between HPB-releasing adducts in DNA and hemoglobin (p=0.074).     

Overexpression of SLURP-1 and -2 alleviates the tumorigenic action of tobacco-derived nitrosamine on immortalized oral epithelial cells

Recent research has demonstrated that mucocutaneous epithelial cells express functional nicotinic acetylcholine receptors (nAChRs) and that tobacco-derived carcinogenic nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein)-1 and -2 can act as non-canonical ligands of these receptors. It was found that recombinant SLURP-1 and -2 can lessen tumorigenic activity of nitrosamines. The immortalized esophageal keratinocytes (Het-1A cells) exhibit low SLURP-1 and -2 mRNA levels that decrease further after treatment with NNK. Based on these observations, we hypothesized that overexpression of full length SLURP proteins may protect Het-1A cells from malignant transformation by NNK. The Het-1A cells transfected with either SLURP-1 or -2 vector produced the highest amounts of respective proteins between 24 and 48 h, at which point they were exposed to 1 microM NNK for 24 h and their tumorigenic activities were subsequently evaluated by plating in soft agar and injecting subcutaneously to Nu/Nu mice. Transfection with either SLURP-1 or -2 cDNA in both cases significantly (p<0.05) diminished the number of colonies produced by NNK exposed cells. SLURP-1 was more efficient than SLURP-2 in abolishing the tumorigenic effect in nude mice. Thus, the anti-tumorigenic activities of SLURP-1 and -2 were demonstrated both in vitro and in vivo. The obtained results suggest that SLURP-like proteins may become useful for developing novel anti-cancer therapies.     

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.     

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and beta-adrenergic receptor signaling pathways

Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor (α7 nAChR) and β-adrenergic receptors. Treatment of cells with α-bungarotoxin (α-BTX, α7nAChR antagonist) or propranolol (β-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE₂ and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE₂ induction can only be suppressed by propranolol, but not α-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.     

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.     

Evaluation of the mutagenic effects of myosmine in human lymphocytes using the HPRT gene mutation assay

The minor tobacco alkaloid myosmine is implicated in DNA damage through pyridyloxobutylation similar to the tobacco-specific nitrosamines (TSNA). In contrast to TSNA, occurrence of myosmine is not restricted to tobacco. Myosmine is genotoxic to human cells in the comet assay. In this study, the mutagenic effect of myosmine was evaluated using the cloning hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. Four hour exposure of isolated peripheral blood lymphocytes from 14 subjects homozygous for the Leu84 wild-type of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene to 1 mM of myosmine increased mutant frequency from 0.73 ± 0.58 × 10⁻⁶ in control to 1.14 ± 0.89 × 10⁻⁶ lymphocytes (P < 0.05). These new data further confirm the mutagenic effects of myosmine.     

Single and concerted effects of benzo[a]pyrene and flavonoids on the AhR and Nrf2-pathway in the human colon carcinoma cell line Caco-2

As phytochemicals have the potential to counteract adverse effects of carcinogens we investigated the influence of the flavonoids quercetin and kaempferol on benzo[a]pyrene (BaP) mediated effects on human colon cancer cells, Caco-2. We focused on concerted effects on the expression of AhR and Nrf2 pathway components. In contrast to kaempferol, BaP and quercetin efficiently induced CYP1A1, CYP1A2 and CYP1B1-mRNA in Caco-2 cells. BaP not only acted via AhR activation but sustainably also by increasing AhR and by down-regulating AhRR mRNA. The flavonoids did not affect AhR expression but counteracted the BaP mediated AhRR repression. Only quercetin was found to induce AhRR mRNA. ARNT mRNA appeared to be slightly but significantly down-regulated by BaP as well as by flavonoids while expression of AIP was not or only slightly modulated. The Nrf2 pathway was activated by BaP and by the flavonoids shown by induction of Nrf2 and several of its target genes such as NQO1, GSTP1, GSTA1 and GCLC. Induction effects of 10 μm BaP on Nrf2, GSTP1 and NQO1 were abolished by the flavonoids. In summary, we show that quercetin supports AhR mediated effects. Both flavonoids, however, may counteract the effects of BaP on expression of AhR, AhRR, Nrf2, GSTP1 and NQO1. In conclusion, quercetin appears to have two faces, a flavonoid-like one and a PAH-like one which supports Ahr-mediated effects while kaempferol acts “just like a flavonoid”. Thus, flavonoids have to be treated individually with respect to their anti-adverse activity.     

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.     

Effect of a flue-curing process that reduces tobacco specific nitrosamines on the tumor promotion in SENCAR mice by cigarette smoke condensate.

A 30-week dermal tumor promotion study was conducted to evaluate the dermal tumor-promoting potential of cigarette smoke condensate (CSC) collected from cigarettes containing flue-cured tobacco cured by a heat-exchange process (HE) relative to that of cigarettes containing flue-cured tobacco cured by the traditional direct-fire process (DF). Heat-exchange process cured tobacco contains significantly lower concentrations of tobacco specific nitrosamines (TSNAs) compared to traditional direct-fire cured tobacco. Mainstream CSCs were collected by cold trap from smoke generators using the Federal Trade Commission puffing regimen. Groups of 40 female SENCAR mice were initiated by a single application of 75 micro g 7,12-dimethylbenz[a]anthracene (DMBA) to the shaved dorsal skin. CSCs were then applied to the skin three times/week for 29 weeks at 9, 18, or 36mg tar/application. End-points included body weights, clinical observations, organ weights, dermal tumor development and histopathology. The numbers of dermal tumors and the numbers of tumor-bearing mice for each CSC were statistically different from the DMBA/acetone control group and increased with increasing dose. When corresponding doses of each CSC were compared, only the DMBA/mid-dose HE CSC group was statistically significantly different (lower) from the corresponding DMBA/mid-dose DF CSC group. In this assay, the dermal tumor-promotion potential of CSC from heat-exchange flue-cured tobacco did not differ from that of traditional direct-fire flue-cured tobacco CSC.     

A new lactoferrin- and iron-dependent lysosomal death pathway is induced by benzo[a]pyrene in hepatic epithelial cells

While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation and cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.     

Toxicogenomic outcomes predictive of forestomach carcinogenesis following exposure to benzo(a)pyrene: Relevance to human cancer risk

Forestomach tumors are observed in mice exposed to environmental carcinogens. However, the relevance of this data to humans is controversial because humans lack a forestomach. We hypothesize that an understanding of early molecular changes after exposure to a carcinogen in the forestomach will provide mode-of-action information to evaluate the applicability of forestomach cancers to human cancer risk assessment. In the present study we exposed mice to benzo(a)pyrene (BaP), an environmental carcinogen commonly associated with tumors of the rodent forestomach. Toxicogenomic tools were used to profile gene expression response in the forestomach. Adult Muta™Mouse males were orally exposed to 25, 50, and 75 mg BaP/kg-body-weight/day for 28 consecutive days. The forestomach was collected three days post-exposure. DNA microarrays, real-time RT-qPCR arrays, and protein analyses were employed to characterize responses in the forestomach. Microarray results showed altered expression of 414 genes across all treatment groups (± 1.5 fold; false discovery rate adjusted P ≤ 0.05). Significant downregulation of genes associated with phase II xenobiotic metabolism and increased expression of genes implicated in antigen processing and presentation, immune response, chemotaxis, and keratinocyte differentiation were observed in treated groups in a dose-dependent manner. A systematic comparison of the differentially expressed genes in the forestomach from the present study to differentially expressed genes identified in human diseases including human gastrointestinal tract cancers using the NextBio Human Disease Atlas showed significant commonalities between the two models. Our results provide molecular evidence supporting the use of the mouse forestomach model to evaluate chemically-induced gastrointestinal carcinogenesis in humans.     

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.     

Differences in benzo(a)pyrene metabolism between cultured human and murine bronchial cells after pre-treatment with benz(a)anthracene

Primary cultures of human and murine (strain C3Hz) bronchial epithelial cells were pretreated with benz(a)anthracene (BA) (10 microM). 16 h later the formation of phenolic as well as dihydrodiol metabolites of benzo(a)pyrene (BP) was measured. Whereas murine cultures showed enhanced metabolism towards both phenolic and dihydrodiol compounds, in the human cultures only phenolic BP-metabolites were increased. In view of their precursor role in the formation of biologically active diol-epoxides, formation of dihydrodiol-derivatives can be considered as a key factor in determining susceptibility to polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. Therefore the observations of this study indicate that animal model systems for PAH carcinogenesis in man have to be selected on the basis of comparable metabolite patterns.