New insights into BaP-induced toxicity: role of major metabolites in transcriptomics and contribution to hepatocarcinogenesis.

Benzo(a)pyrene (BaP) is a ubiquitous carcinogen resulting from incomplete combustion of organic compounds and also present at high levels in cigarette smoke. A wide range of biological effects has been attributed to BaP and its genotoxic metabolite BPDE, but the contribution to BaP toxicity of intermediary metabolites generated along the detoxification path remains unknown. Here, we report for the first time how 3-OH-BaP, 9,10-diol and BPDE, three major BaP metabolites, temporally relate to BaP-induced transcriptomic alterations in HepG2 cells. Since BaP is also known to induce AhR activation, we additionally evaluated TCDD to source the expression of non-genotoxic AhR-mediated patterns. 9,10-Diol was shown to activate several transcription factor networks related to BaP metabolism (AhR), oxidative stress (Nrf2) and cell proliferation (HIF-1α, AP-1) in particular at early time points, while BPDE influenced expression of genes involved in cell energetics, DNA repair and apoptotic pathways. Also, in order to grasp the role of BaP and its metabolites in chemical hepatocarcinogenesis, we compared expression patterns from BaP(-metabolites) and TCDD to a signature set of approximately nine thousand gene expressions derived from hepatocellular carcinoma (HCC) patients. While transcriptome modulation by TCDD appeared not significantly related to HCC, BaP and BPDE were shown to deregulate metastatic markers via non-genotoxic and genotoxic mechanisms and activate inflammatory pathways (NF-κß signaling, cytokine-cytokine receptor interaction). BaP also showed strong repression of genes involved in cholesterol and fatty acid biosynthesis. Altogether, this study provides new insights into BaP-induced toxicity and sheds new light onto its mechanism of action as a hepatocarcinogen.

Aldo-keto reductases protect lung adenocarcinoma cells from the acute toxicity of B[a]P-7,8-trans-dihydrodiol.

Tobacco smoke exposure stimulates the expression of genes that are likely to be involved in the metabolism of its combustion products such as polycyclic aromatic hydrocarbons (PAH). Four of the smoke induced genes are aldo-keto reductases (AKR), enzymes that metabolically activate PAH to PAH o-quinones. Alternatively, PAHs are metabolized to (±)-anti-diol epoxides, such as (±)-anti-benzo[a]pyrene diol epoxide ((±)-anti-BPDE)), by the combined action of P4501A1/1B1 and epoxide hydrolase. (±)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause DNA damage by oxidative stress through a futile redox cycle. To address the role of AKRs in PAH cytotoxicity, we compared the cytotoxicity of PAH metabolites and the effects of overexpressing AKR1A1 in lung cells. (±)-anti-BPDE and B[a]P-7,8-trans-dihydrodiol, an intermediate in (±)-anti-BPDE metabolism, are toxic to A549 cells at concentrations with an IC(50) of ∼2 µM. In contrast, the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC(50) > 20 µM. Similar differences in cytoxicity were observed with two other PAH o-quinones (benz[a]anthracene-3,4-dione and 7,12-dimethylbenz[a]anthracene-3,4-dione) compared with their respective diol-epoxide counterparts (BA-3,4-diol-1,2-epoxide and DMBA-3,4-diol-1,2-epoxide). In addition, both anti-BPDE and B[a]P-7,8-trans-dihydrodiol induced p53 expression ∼6 h post-treatment at concentrations as low as 1 µM consistent with extensive DNA damage. B[a]P-7,8-dione treatment did not induce p53 but generated reactive oxygen species (ROS) in A549 cells and induced the expression of oxidative response genes in H358 cells. We also observed that overexpression of AKR1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2-10-fold from the toxic effects of B[a]P-7,8-trans-dihydrodiol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH.

Protective efficacy of hGSTM1-1 against B[a]P and (+)- or (-)-B[a]P-7,8-dihydrodiol cytotoxicity, mutagenicity, and macromolecular adducts in V79 cells coexpressing hCYP1A1.

Transgenic cell lines were constructed to study the dynamics of competition between activation versus detoxification of benzo[a]pyrene (B[a]P) or B[a]P-7,8-dihydrodiol metabolites. Stably transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone or in combination with human glutathione-S-transferase M1 (hGSTM1) were used to determine how effectively this GST isozyme protects against cytotoxic, genotoxic, and mutagenic effects of B[a]P or the enantiomeric dihydrodiol metabolites (+)-benzo[a]pyrene-7,8-dihydrodiol ((+)-B[a]P-7,8-diol) and (-)-benzo[a]pyrene-7,8-dihydrodiol ((-)-B[a]P-7,8-diol). Expression of hGSTM1 in the presence of hCYP1A1 conferred significant 8.5-fold protection against B[a]P-induced cytotoxicity, but protection against cytotoxicity of either B[a]P-7,8-diol enantiomer was not significant. Mutagenicity of B[a]P at the hprt locus was dose and time dependent in cells that expressed hCYP1A1. Mutagenicity of B[a]P was reduced by 21-32% and mutagenicity induced by the B[a]P-7,8-diols was reduced 20-58% in cells further modified to coexpress hGSTM1-1 compared to cells expressing hCYP1A1 alone. Expression of hGSTM1-1 reduced adducts in total cellular macromolecules by twofold, in good correlation with the reduction in B[a]P mutagenicity. These results indicate that while hGSTM1-1 effectively protects against hCYP1A1-mediated cytotoxicity of B[a]P, a significant fraction of the mutagenicity that results from activation of B[a]P and its 7,8-dihydrodiol metabolites by hCYP1A1 is derived from B[a]P metabolites that are not detoxified by hGSTM1.

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (+/-)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1.

We have used V79MZ hamster lung fibroblasts stably transfected with human cytochrome P450-1A1 (hCYP1A1; cell line designated V79MZh1A1) or P450-1B1 (hCYP1B1; cell line designated V79MZh1B1) alone, or in combination with human glutathione-S-transferase (GST) alpha-1 (hGSTA1), in order to examine GST protection against cytotoxicity and mutagenicity of dibenzo[a,l]pyrene (DBP) and the intermediate dihydrodiol metabolite (+/-)-DBP-11,12-dihydrodiol (DBPD). At comparable expression levels of hCYP1A1 and hCYP1B1, both DBP and DBPD were more cytotoxic in V79MZ1A1 (IC(50)=2.7 and 0.7nM, respectively) than in V79MZh1B1 (IC(50)=6.0 and 4.8nM, respectively). In contrast, both DBP and DBPD were two- to four-fold more mutagenic in V79MZh1B1 than in V79MZ1A1. Co-expression of hGSTA1 with hCYP1A1 decreased DBP cytotoxicity two-fold compared to V79MZh1A1 with hCYP1A1 alone, and provided a small, yet still statistically significant, 1.3-fold protection against DBPD. Protection against mutagenicity of these compounds was comparable to that for cytotoxicity in cells expressing hCYP1A1. In V79MZh1B1 cells, co-expression of hGSTA1 conferred up to five-fold protection against DBP cytotoxicity, and up to nine-fold protection against the (+/-)-DBP-dihydrodiol cytotoxicity relative to the cells expressing hCYP1B1 alone. Co-expression of hGSTA1 also reduced mutagenicity of DBP or its dihydrodiol to a lesser extent (1.3-1.8-fold) than the protection against cytotoxicity in cells expressing hCYP1B1. These findings demonstrate that the protective efficacy of hGSTA1 against DBP and DBPD toxicity is variable, depending on the compound or metabolite present, the specific cytochrome P450 isozyme expressed, and the specific cellular damage endpoint examined.

Cytoplasmic suppression of tumorigenicity in reconstructed mouse cells.

Previous cybrid studies aimed at demonstrating cytoplasmic suppression of tumorigenicity have been generally inconclusive because of (a) the use of mutagens or carcinogens to introduce nuclear-coded and cytoplasmic-coded genetic markers and (b) dilution of putative cytoplasmic suppressors with tumorigenic cytoplasm of whole cells used in the cybrid construction. We have circumvented these potential problems by examining tumorigenicity in reconstructed cells made from tumorigenic karyoplasts and nontumorigenic cytoplasts and by using a ricin-antiricin selection to obtain the reconstructed cells. Karyoplasts from tumorigenic NIH/3T3 cells that were derived from a clone that had survived incubation with benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxy (anti) and been passaged 17 times were fused to NIH/3T3 cytoplasts derived from nontumorigenic cells. The cytoplasts were loaded with antiricin antibody prior to fusion. Ten clones which survived ricin selection were not tumorigenic in nude mice. These findings offer support for the presence of cytoplasmic factors in nontumorigenic mouse cells that suppress benzo(a)pyrene epoxide-induced tumorigenicity.

Modulation of oxidant formation in mouse skin in vivo by tumor-promoting phorbol esters.

The pathways of oxidant generation in mouse epidermis were investigated by 32P-postlabeling analysis of diastereomeric DNA adducts derived from oxidation of (7S,8S)-dihydroxy-7,8-dihydrobenzo(a)pyrene ((+)-BP-7,8-diol). The pattern of deoxynucleoside-3'-5'-bis-phosphate adducts in epidermal scrapings from female CD-1 mice indicated that cytochrome P-450 was the major oxidant. When animals were pretreated with the tumor-promoting phorbol ester, tetradecanoyl phorbol acetate (TPA), 24 h before coadministration of TPA and (+)-BP-7,8-diol, the pattern of DNA adducts indicated that peroxyl radicals made a major contribution to (+)-BP-7,8-diol epoxidation. Peroxy radical-dependent epoxidation was maximal when the time between the 2 TPA administrations was 24-72 h. No increase in radical-derived adducts was observed when the non-tumor-promoting phorbol ester 4-O-methyl-TPA was substituted for TPA. The calcium ionophore A23187 stimulated radical generation when substituted for the first, but not the second, TPA treatment. The antiinflammatory steroid fluocinolone acetonide inhibited (-)-anti-BPDE-DNA adduct formation when coadministered with the first but not the second TPA treatment. In contrast, all-trans-retinoic acid inhibited (-)-anti-BPDE-DNA adduct formation when coadministered with the second but not the first TPA treatment. These findings demonstrate that tumor promoting phorbol esters stimulate oxygen radical generation in mouse skin and that radical generation is blocked by inhibitors of tumor promotion.

Nuclear respiratory factor 1 overexpression attenuates anti-benzopyrene­7,8-diol-9,10-epoxide-induced S-phase arrest of bronchial epithelial cells.

Nuclear respiratory factor 1 (NRF-1) has important roles in the regulation of several key metabolic genes required for cellular growth and respiration. A previous study by our group indicated that NRF­1 is involved in mitochondrial dysfunction induced by the environmental pollutant benzo[a]pyrene in the 16HBE human bronchial epithelial cell line. In the present study, it was observed that its genotoxic metabolite, anti­benzopyrene­7,8­diol­9,10­epoxide (BPDE), triggered cell cycle arrest in S­phase in 16HBE cells by activating ataxia-telangiectasia (ATM)/checkpoint kinase (Chk)2 and ATM and Rad3 related (ATR)/Chk1 signaling pathways. NRF­1 expression was suppressed by BPDE after treatment for 6 h. Flow cytometric analysis revealed that NRF­1 overexpression attenuated cell cycle arrest in S­phase induced by BPDE. In line with this result, DNA­damage checkpoints were activated following NRF­1 overexpression, as demonstrated by increased phosphorylation of ATM, Chk2 and γH2AX, but not ATR and Chk1, according to western blot analysis. It was therefore indicated that NRF­1 overexpression attenuated BPDE­induced S­phase arrest via the ATM/Chk2 signaling pathway.

Hepatocarcinogenic potency of mixed and pure enantiomers of trans-7,8-dihydrobenzo[a]pyrene-7,8-diol in trout.

The hepatocarcinogenic potency of pure and racemic trans-7,8-dihydrobenzo[a]pyrene-7,8-diol was investigated in embryos and sac-fry rainbow trout. Embryos microinjected with (+/-)-trans-7,8-dihydrobenzo[a]pyrene-7,8-diol ((+/-) BP-7,8-DHD) developed liver tumors 9 months after hatching. However, this exposure protocol resulted in high mortalities. Microinjection of newly hatched sac-fry with 0.01-1.0 microgram of (+/-) BP-7,8-DHD resulted in a dose-dependent production of liver tumors (0-13%) similar to the results with embryos but without the problem of high mortalities. Co-injection of sac-fry with (+/-) BP-7,8-DHD and either beta-naphthoflavone or carbon tetrachloride significantly enhanced the tumor response (approx. 3-fold). The relative carcinogenic potencies of the pure (+) and (-) enantiomers of BP-7,8-DHD were evaluated by microinjection into sac-fry at doses of 0.5-5.0 micrograms. The results demonstrated that the (-) enantiomer was 4-18 times more potent than the (+). Mixed carcinomas were the most prevalent liver tumors observed. These results demonstrate that trout embryos and sac-fry are both responsive to hepatocarcinogenesis initiation by injection with BP-7,8-DHD. The marked enhancement seen with co-injection of sac-fry with beta-naphthoflavone or carbon tetrachloride suggests that both cytochrome P-450-dependent and lipid peroxidation-dependent pathways could be involved in bioactivation of this compound, presumably through epoxidation at the 9,10-position. As is the case with mammals, the (-) enantiomer of BP-7,8-DHD is a more potent carcinogen than the (+) enantiomer.

Inhibition of the skin tumorigenicity of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene by tannic acid, green tea polyphenols and quercetin in Sencar mice.

The effect of pretreatment of skin of Sencar mice with topically applied tannic acid, quercetin and green tea polyphenols (GTP) on the skin tumor initiating activity of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-2) has been evaluated. The animals were pretreated with the plant phenols (tannic acid and quercetin (3000 nmol) or GTP 24 mg/mouse) for 7 days after which they received a single topical application of 200 nmol of BPDE-2 as the initiating agent. Beginning 7 days following initiation animals received twice weekly applications of 3.24 nmol of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Tannic acid and GTP afforded significant protection against skin tumor induction. These inhibitory effects were verified both by prolongation of the latency period and subsequent development of tumors. Quercetin, on the other hand, afforded only moderate protection. Each phenolic compound was found to be highly effective in accelerating the disappearance of BPDE-2 from aqueous medium. Our results suggest that tannic acid and GTP have substantial potential for protecting against the skin tumorigenic response to BPDE-2 and the mechanism of inhibition may involve inactivation of the reactive carcinogenic moiety.

Inhibition of liver microsome-mediated mutagenesis, metabolism and DNA-binding of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in the rat following glucose administration.

Aroclor 1254-induced rat liver microsomes prepared from control and glucose-treated rats (30% glucose in drinking water 48 h prior to sacrifice) were used in studies of benzo[a]pyrene (BaP) and BaP 7,8-dihydrodiol (BaP 7,8-DHD)-induced mutagenesis in Salmonella typhimurium TA100. Microsome-dependent metabolism and metabolite binding of BaP and BaP 7,8-DHD to calf thymus DNA was also investigated. BaP-induced mutagenesis in TA100 was inhibited 27% and BaP 7,8-DHD-induced mutagenesis was inhibited 55% by microsomes from glucose-treated rats. [3H]BaP and [3H]BaP 7,8-DHD metabolite binding to DNA was inhibited 17% and 20%, respectively. High performance liquid chromatographic (hplc) analysis of enzyme-hydrolyzed DNA yielded 7R and 7S-diol epoxide-1 deoxyguanosine (BPDE-1:dG) adducts and BPDE-2:dG adducts of [3H]BaP and [3H]BaP 7,8-DHD. These adducts were inhibited 38% and 50%, respectively, by microsomes from glucose-treated rats. Hplc analysis of organosoluble metabolites of [3H]BaP and [3H]BaP 7,8-DHD showed an inhibition of metabolism of 28% and 50%, respectively, by microsomes from glucose-treated rats. The inhibition of metabolism correlated with the effect of glucose treatment on inhibition of BaP and BaP 7,8-DHD-induced mutagenesis and adduct formation. These results suggest that the mechanism by which glucose produces its effects on mutagenesis, DNA-binding and adduct formation is by an inhibition of microsome-mediated metabolism of BaP and BaP 7,8-DHD.

Tumorigenicity and liver tumor ras-protooncogene mutations in CD-1 mice treated neonatally with 1- and 3-nitrobenzo[a]pyrene and their trans-7,8-dihydrodiol and aminobenzo[a]pyrene metabolites.

The environmental pollutants 1- and 3-nitrobenzo[a]pyrene (1- and 3-NBaP) are metabolized by mammalian microsomes through ring oxidation to 1-NBaP trans-7,8-dihydrodiol and 3-NBaP trans-7,8-dihydrodiol, and by nitroreduction to 1- and 3-aminobenzo[a]pyrene. To determine if these compounds are tumorigenic, 1- and 3-NBaP, along with several of their metabolites and the parent benzo[a]pyrene (BaP) and its trans-7,8-dihydrodiol metabolite, were tested in the neonatal CD-1 mouse bioassay. Male mice were administered i.p. injections at a total dose of 100 or 400 nmol per mouse on 1, 8 and 15 days after birth. While the liver tumor incidences for BaP, BaP trans-7,8-dihydrodiol, and the positive control 6-nitrochrysene (6-NC) were significantly higher than in the solvent control animals, all the other tested compounds exhibited no tumorigenicity. The frequency of Ha- and Ki-ras mutations in liver tumors of mice treated with BaP, BaP trans-7,8-dihydrodiol, and 6-NC were higher than in the few liver tumors isolated from control mice or mice treated with the NBaPs or their metabolites. Since 1- and 3-NBaP and their metabolites are potent mutagens in the Salmonella assay and moderate mutagens in the Chinese hamster ovary (CHO) mammalian mutagenicity assay, our results indicate that the in vitro mutagenicity of these compounds does not correlate with their tumorigenicity.

Covalent binding of (+) 7S-trans-7,8-dihydrobenzo [a]pyrene-7,8-diol to trout DNA: P-450- and peroxidation-dependent pathways.

Bioactivation in vivo of pure (+) 7S-trans-7,8-dihydrobenzo[a]pyrene- 7,8-diol ((+) BP-7,8-DHD) was investigated in rainbow trout. Embryos, microinjected with 0.01-1.0 microgram of [3H]-(-)-7S-trans-7,8-dihydrobenzo[a]-pyrene-7,8-diol-anti-9,10-epoxide ((-) anti-BPDE), exhibited a dose-dependent increase in DNA adduction. Subsequently, microinjection of trout embryos with [14C] (+) BP-7,8-DHD also demonstrated a dose-dependent increase in DNA adduction. To determine the relative contribution of P-450-dependent versus peroxidation-dependent epoxygenation of (+)-BP-7,8-DHD, trout embryos were co-injected with [14C]-(+)-BP-7,8-DHD and either beta-naphthoflavone (BNF) (CYP1A1 inducer) or carbon tetrachloride (CCl4) (lipid peroxidation enhancer). Co-injection with BNF tended to enhance covalent binding to DNA, which was consistent with rapid induction of CYP1A1. Co-injection with CCl4, significantly increased covalent binding of [14C]-(+)-BP-7,8-DHD to DNA, suggesting a contribution from non-enzymic cooxidation. 32P-Postlabeling analysis of liver DNA adducts following i.p. injections of (+) BP-7,8-DHD did not detect appreciable amounts of (-) anti-BPDE-dG from juvenile trout fed control diets or diets containing hydrogen peroxide or BNF. On the contrary, BNF pre-feeding markedly enhanced the levels of an adduct which co-chromatographed with authentic (+) syn-BPDE-dG. These results confirm that trout are capable of metabolically activating BP-DHD to the ultimate carcinogen BPDE and that BNF stimulates CYP1A1-dependent epoxygenation, but peroxidation-dependent activation may not contribute significantly to the bioactivation of BP-7,8-DHD in vivo.

Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts. Analysis of cellular protooncogenes.

Treatment of diploid human fibroblasts with stereoisomeric benzo[alpha]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5-12) X 10(-4)] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1-8) X 10(-4)] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (less than 1.6 X 10(-7) frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.

Peroxisome proliferators increase the formation of BPDE-DNA adducts in isolated rat hepatocytes.

Peroxisome proliferators are known to modulate the activity of xenobiotic-metabolising enzymes, including glutathione S-transferase (GST) and cytochrome P-450 (CYP). In this study the effect of peroxisome proliferators silvex and di(2-ethylhexyl)phthalate (DEHP) on the formation of (+)-anti-benzo(a)pyrene -7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts from a proximate mutagen and carcinogen (-)-transbenzo(a)pyrene-7,8-dihydrodiol (BPDD) has been investigated. Rat CYP1A1 metabolises BPDD to mutagenic BPDE, which may form DNA adducts or, alternatively, be detoxified by hydrolysis or glutathione conjugation. In this experiment the formation of BPDE-DNA adducts was significantly increased in hepatocytes isolated from all silvex treated rats and two out of four DEHP treated rats (14 day treatment). The activity of CYP1A1 was increased whereas GST was reduced by the peroxisome proliferator silvex. These changes were more significant than those induced by DEHP. We have hypothesised that the formation of BPDE-DNA adducts was primarily due to the increased BPDD activation to BPDE versus reduced detoxication of BPDE. Other hepatic changes induced by the peroxisome proliferators, e.g. peroxisome proliferation per se and increased mitotic activity of the liver could have an effect on the outcome of BPDD exposure.

The cytotoxicity of mitomycin C and adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes.

The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and beta-naphthoflavone (beta NF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and alpha-naphthoflavone (alpha NF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by alpha NF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or beta NF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in beta NF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas alpha NF only partially inhibited the cytotoxicity of MMC in beta NF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells.

Depression of hepatic cytochrome P450 monooxygenases after chronic environmental tobacco smoke exposure of young ferrets.

Six-week-old ferrets were exposed head-only to clean air or environmental tobacco smoke (ETS) for 2 h/day, 5 days a week for a total of 8 weeks. Exposure to ETS caused a significant reduction in the levels of hepatic microsomal cytochrome P450 and P450 reductase activity in both the male and female ferrets. The content of cytochrome b5 and the activity of its reductase were significantly reduced in the hepatic microsomes of female ferrets. 7-Ethoxycoumarin O-deethylase activity and cytochrome P450 (CYP) 1A protein were markedly decreased in the hepatic microsomes of both the male and female ferrets after ETS exposure. In accord with the downregulation of P450, total metabolites formed from benzo[a]pyrene were significantly reduced in the liver homogenates of ETS-exposed animals. Similarly, sum total of free (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, glutathione conjugates and DNA-bound metabolites formed from precursor (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol showed marked reduction in both the male and female ferrets after ETS exposure in a dose-response manner. This is the first report showing downregulation of hepatic P450 and accompanying benzo[a]pyrene metabolism after tobacco smoke exposure which apparently occurred after an initial upregulation of these parameters (Rasmussen et al. (1994) FASEB J. 8, A122).

Metabolism of (+)-trans-benzo[a]pyrene-7,8-dihydrodiol by 3-methylcholanthrene-induced rat liver homogenates.

Using a new sensitive reverse-phase HPLC assay with on-line radioactivity detector, metabolism of (+)-trans-benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol) to the ultimate carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) was studied using 3-methylcholanthrene-induced rat liver homogenates. The results demonstrate that the stereoselectivity of B[a]PDE formation is a function of the concentration of the cellular constituents in the incubation media. At more dilute concentrations of the homogenate, the ratio of anti- to syn-B[a]PDE was the highest and decreased as the homogenate protein was increased in the incubation medium. However, there was a marked and parallel decrease of free B[a]PDE and DNA-bound radioactivity with increasing concentrations of cellular constituents in the incubation medium. The decreased DNA-bound radioactivity appears to be due to the preferential binding of B[a]PDE to glutathione and to proteins as the homogenate concentration was increased in the incubation media. These results indicate that liver homogenates, while apparently preserving the function of microsomes, present additional opportunities to study the interrelationship among cytochrome P450 monooxygenase activity, water-soluble conjugates, and binding of B[a]P diol metabolites to macromolecules in the study of benzo[a]pyrene-induced carcinogenesis.

Metabolism of polycyclic aromatic hydrocarbons to mutagenic species by rat and porcine ovarian granulosa cells: detection by cocultivation with V79 Chinese hamster cells.

The capacity of ovarian granulosa cells from rat and pig to release reactive metabolites produced from polycyclic aromatic hydrocarbons with the ability to cause mutations in neighbouring cells has been studied. For this purpose we have used cocultivation with V79 Chinese hamster cells as a detection system. The cells were treated with two different polycyclic aromatic hydrocarbons (PAHs), 7,12-dimethylbenz(a)anthracene (DMBA) or (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol). The resulting mutation frequency in the V79 cells after cocultivation, as a function of granulosa cell number, DMBA, or BP-7,8-diol concentration and time, was determined. The progesterone concentration in the medium after cocultivation was also analyzed as a measure of the differentiation of the granulosa cells. These studies demonstrate an increasing frequency of mutations in the V79 cells with an increasing number of granulosa cells. The maximal number of mutations were achieved with a DMBA or BP-7,8-diol concentration of 5 or 2 microM, respectively. The optimal cocultivation time was 24 h. These results clearly show that the granulosa cells can bioactivate PAHs to reactive metabolites with the capacity to migrate into surrounding cells and cause mutations in these cells. Compounds metabolized to mutagenic products by granulosa cells might thus cause mutations in the neighbouring germ cells, with possible consequences for the offspring.

Differential efficiency of mutagenesis at three genetic loci in CHO cells by a benzo[a]pyrene diol epoxide.

The formation of DNA adducts by the ultimate carcinogen 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (BPDE-I) has been implicated in the process of carcinogenesis. In a line of Chinese hamster ovary (CHO) cells designated AT3-2 and in two derivative mutant lines, UVL-1 and UVL-10, originally selected for hypersensitivity to UV-irradiation, we have measured the formation of BPDE-I: DNA adducts and the production of biological damage. The quantity and quality of BPDE-I: DNA adducts formed initially in the 3 cell lines are identical over a wide range of BPDE-I doses. However, the UVL lines are unable to remove adducts from their DNA, while the AT3-2 cells remove about 50% of the BPDE-I: DNA adducts in a 24-h incubation. Correlated with this, the UVL lines are more sensitive to the lethal effects of BPDE-I than are the AT3-2 cells. Mutant frequencies were measured at the aprt, hprt and oua loci and were found to increase linearly with BPDE-I: DNA adduct formation at doses which gave greater than 50% survival. At the hprt and oua loci, the efficiency of mutation induction was similar for AT3-2 and UVL-10 cells. UVL-1 cells showed slightly higher (within a factor of 2-3) mutant frequencies in response to BPDE-I compared to AT3-2 at these two loci. However, at the aprt locus the repair-deficient cells were much more highly mutable (9-15-fold) than the repair-proficient AT3-2 cells. Based on the measured average level of adduct formation, it is calculated that 15% of the BPDE-I: DNA adducts in the aprt gene are converted into mutations. However, the possibility exists that the aprt locus is subject to higher levels of modification by BPDE-I than is the bulk DNA, which would lead to an artifactually high apparent conversion frequency.

Induction of cytotoxicity and mutagenesis is facilitated by fatty acid hydroperoxidase activity in Chinese hamster lung fibroblasts (V79 cells).

The metabolic activation of benzo[a]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was studied in V79 Chinese hamster fibroblasts after supplementations with arachidonic acid or treatments with linoleic acid hydroperoxide. The extent of metabolic activation was estimated using cytotoxicity and mutagenesis as endpoints. Pretreatment of cells with arachidonic acid for 24 h resulted in significant elevations in the content of this fatty acid in cell phospholipids and increased prostaglandin synthesis. Arachidonic acid and linoleic acid hydroperoxide facilitated 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene cytotoxicity and mutagenesis, and to a lesser extent increased the cytotoxicity and mutagenicity of benzo[a]pyrene. No other compounds tested were mutagenic under these conditions, however, linoleic acid hydroperoxide markedly increased their cytotoxicity. Arachidonic acid-facilitated toxicity and mutagenesis was inhibited by indomethacin, whereas no inhibition was seen when linoleic acid hydroperoxide was used. Nordihyroquairaretic acid abolished the cytotoxicity and mutagenesis facilitated by arachidonic acid and linoleic acid hydroperoxide. Our findings demonstrate that induction of cytotoxicity and mutagenesis following treatment of V79 cells with carcinogens may be limited by low levels of arachidonic acid in these cells. A peroxidatic mechanism is proposed, with limited substrate specificity, for the metabolic activation of chemicals in V79 cells.