Evidence for the involvement of the fatty acid and peroxisomal β-oxidation pathways in the inhibition by dehydroepiandrosterone (DHEA) and induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benz(a)anthracene (BA) of cytochrome P4501B1 (CYP1Bl) in mouse embryo fibroblasts (C3H10T1/2 cells)

Treatment of intact C3H10T1/2 cells or microsomes therefrom with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzanthracene (BA) enhanced CYP1B1 activity and CYP1B1 expression as revealed by elevations of CYP1B1-catalyzed DMBA metabolism, CYP1B1 apoprotein level and CYP1B1 gene expression. One hundred µM DHEA caused an 80-90% inhibition of cellular DMBA metabolism without inflicting cell death. Cytosolic glucose-6-phosphate dehydrogenase (G6PDH) was also inhibited in DHEA-treated cells, presumably due to the inhibition of NADP reduction. In contrast, neither DMBA metabolism nor CYP1B1 apoprotein was inhibited by DHEA in the microsomes isolated from these cells. DHEA (100 M), TCDD (10 nM) and BA (10 M) stimulated the activities and increased the apoprotein levels of two peroxisomal enzymes, namely, acyl CoA oxidase (ACOX) and acyl CoA hydrolase (ACH₂) and also induced the expression of CYP1B1 and ACOX genes. Cytosolic fatty acyl-CoA -oxidation was also stimulated by DHEA, TCDD and BA. In corroboratory experiments, it was found that concomittant with the stimulation of the activity of a key enzyme regulator of fatty acid homeostasis, namely, glycerol-3-phosphate dehydrogenase (G3PDH), these agents enhanced arachidonic acid (AA) metabolism as judged by the release of [³H] from AA into the culture medium. Collectively, these data suggest that DHEA mediates the regulation of CYP1B1 and inhibits BA and TCDD-induced CYP1B1-catalyzed carcinogen (DMBA) activation in 10T1/2 cells through metabolic interactions that involve the activation of the peroxisomal and fatty acid -oxidation signaling pathways. These results also present evidence for the first time, for the possible peroxisomal effects of TCDD and BA which are similar to those of DHEA in this mouse embryo fibroblast cell line.     

Mechanism of action of aryl hydrocarbon receptor antagonists: inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 gene expression as determined by increased CYP1A1 mRNA levels and ethoxyresorufin O-deethylase (EROD) activity in mouse Hepa 1c1c7, rat hepatoma H-4II E and human Hep G2 cancer cell lines. In contrast, treatment of these cell lines with either alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) at concentrations as high as 10(-6) M resulted in only minimal induction of CYP1A1 mRNA levels or EROD activity. Cotreatment of the cells with 10(-9) M TCDD plus different concentrations (10(-8)-10(-6) M) of MCDF or alpha NF resulted in a concentration-dependent decrease in TCDD-induced CYP1A1 mRNA levels and EROD activity in the three cell lines. Moreover, using 10(-9) M [3H]TCDD, it was shown that the alpha NF- and MCDF-mediated antagonism of TCDD-induced CYP1A1 gene expression was paralleled by a decrease in levels of the nuclear [3H]TCDD-Ah receptor complex as determined by velocity sedimentation analysis of the nuclear extracts. The binding of nuclear extracts from the treated cells to a synthetic consensus dioxin responsive element (DRE) (a 26-mer) was determined by gel retardation studies using 32P-DRE. In cells treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M alpha NF, the concentration-dependent decrease in TCDD-induced CYP1A1 gene expression by alpha NF was also paralleled by decreased levels of a retarded band associated with the nuclear Ah receptor-DRE complex. In contrast, the results of the gel shift assay of nuclear extracts treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M MCDF indicated that there were relatively high levels of nuclear MCDF-Ah receptor complex in the cells co-treated with TCDD plus the antagonist but this was not accompanied by induced CYP1A1 gene expression. The results suggest that alpha NF and possibly MCDF compete with TCDD for cytosolic Ah receptor binding sites; however, MCDF may also inhibit the induction response by competing for and/or partially inactivating genomic binding sites.     

Trans-resveratrol modulates the catalytic activity and mRNA expression of the procarcinogen-activating human cytochrome P450 1B1

The present study was performed to determine if trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) modulates the catalytic activity and gene expression of cytochrome P450 1B1 (CYP1B1). In vitro, trans-resveratrol decreased human recombinant CYP1B1-catalyzed 7-ethoxyresorufin O-dealkylation activity, with an IC50 value of 1.4 +/- 0.2 microM (mean +/- SEM). Enzyme kinetic analysis indicated that trans-resveratrol inhibited CYP1B1 enzyme activity by a mixed-type inhibition and the apparent Ki was 0.75 +/- 0.06 microM. To determine if trans-resveratrol modulates constitutive CYP1B1 gene expression, cultured MCF-7 human breast carcinoma cells were treated with trans-resveratrol. As indicated by RT-PCR analysis, treatment of MCF-7 cells with 10 microM trans-resveratrol decreased relative CYP1B1 mRNA levels after 5 h, but not after 1.5 or 3 h, of exposure. trans-Resveratrol treatment at 5, 7.5, 10, or 20 microM for 5 h produced a concentration-dependent decrease in CYP1B1 mRNA levels. The extent of suppression was approximately 50% at 20 microM concentration. The suppressive effect was not a consequence of a toxic response to the compound as assessed by a cell proliferation assay. Overall, our novel finding that trans-resveratrol inhibits the catalytic activity and suppresses the constitutive gene expression of CYP1B1 leads to the possibility that this nutraceutical confers protection against toxicity and carcinogenicity induced by compounds that undergo CYP1B1-catalyzed bioactivation.     

Eugenol inhibit 7,12-dimethylbenz[a]anthracene-induced genotoxicity in MCF-7 cells: Bifunctional effects on CYP1 and NAD(P)H:quinone oxidoreductase

Typically, chemopreventive agents either inhibit the cytochrome P450s (CYPs) that are essential for the metabolism of carcinogens or induce phase II detoxifying enzymes. This study examined the chemopreventive effect of eugenol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells. Eugenol inhibited the formation of the DMBA-DNA adduct in a dose dependent manner. CYP1A1 and CYP1B1 activity, which catalyze the biotransformation of DMBA, were strongly inhibited by eugenol. Eugenol also suppressed the CYP1A induction by DMBA through decreased aryl hydrocarbon receptor activation and subsequent DNA binding. Furthermore, eugenol increased the expression and activity of NAD(P)H:quinone oxidoreductase (QR), a major detoxifying enzyme for DMBA, through NF-E2 related factor2 binding to antioxidant response element in QR gene. Therefore, eugenol has a potent protective effect against DMBA-induced genotoxicity, presumably through the suppression of the DMBA activation and the induction of its detoxification. These results suggest that eugenol has potential as a chemopreventive.     

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β-estradiol (E₂) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERα, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E₂ metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998. © 1998 Wiley-Liss, Inc.     

Effects of co-exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and perfluorooctane sulfonate or perfluorooctanoic acid on expression of cytochrome P450 isoforms in chicken (Gallus gallus) embryo hepatocyte cultures

In this study we investigated the effect of a single-compound exposure or two compound co-exposure to tetrachlorodibenzo-p-dioxin (TCDD) plus perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA) on the mRNA expression of cytochromes P450 (CYP) 1A4, 4V2 and 3A37, ethoxyresorufin-O-deethylase (EROD) activity and cell viability in chicken (Gallus gallus domesticus) embryo primary hepatocyte cultures. Cell viability after 24 h of incubation was significantly decreased in cells exposed to PFOS at concentrations between 30 µM and 60 µM with or without co-exposure to TCDD (0.3 nM at maximum). PFOA did not decrease cell viability even at maximum concentrations of 60 µM. TCDD induced CYP1A4 mRNA and EROD activity substantially as reported previously. PFOS also increased CYP1A4 mRNA in a concentration-dependent manner. Co-exposure of cells to PFOS plus TCDD did not change CYP1A4 mRNA levels compared to cells treated with TCDD alone. PFOS alone did not induce CYP4V2 mRNA, however 40–50 µM PFOS plus TCDD (0.3 nM) induced CYP4V2 mRNA compared to TCDD alone (P < 0.05). This trend was similar to that observed with co-exposure to TCDD plus PFOA, suggesting that PFOA alone did not induce CYP4V2 mRNA, whereas co-exposure to TCDD plus PFOA induced the expression levels. PFOS alone decreased CYP3A37 mRNA by a maximum of 45%, however after co-exposure to TCDD, recovery of mRNA expression to levels measured in DMSO-treated cells was observed. Our data suggest a complex gene response to mixtures of dioxin-like and perfluorinated compounds.     

Effects of suberoylanilide hydroxamic acid and trichostatin A on induction of cytochrome P450 enzymes and benzo[a]pyrene DNA adduct formation in human cells

In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on the metabolism of polycyclic aromatic hydrocarbons (PAH) in human mammary carcinoma derived MCF-7 cells in culture. Benzo[a]pyrene (B[a]P) induces cytochrome P450 (CYP) 1A1, CYP1B1 and other xenobiotic metabolizing enzymes. Results from our study indicated a significant increase in CYP activity in comparison to vehicle control in cells treated with SAHA or TSA as measured by ethoxyresorufin-O-deethylase assay. However, co-treatment with 1.0 microM SAHA and BP, reduced the mRNA levels of CYP1B1 relative to B[a]P alone. When co-treated with 1.0 microM TSA and BP, a reduction in the mRNA levels of both CYP1A1 and CYP1B1 was observed relative to BP alone. We further investigated to ascertain if the differential expression and activity of CYP1A1 and CYP1B1 influenced levels of B[a]P DNA adduct formation. MCF-7 cells co-treated with B[a]P and SAHA or TSA formed DNA adducts, although no significant differences in levels of DNA binding were revealed. These results suggest that while CYP enzyme activity and gene expression were affected by the HDAC inhibitors SAHA and TSA, they had no apparent influence on B[a]P DNA binding.     

Cell-specific differences in the susceptibility of potential cellular targets of human origin derived from blood and lung following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The induction of cytochrome P4501A (CYP1A1) enzyme activity is one of the best-studied direct effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds and has been shown to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons (PAH) in different experimental animal species as well as in humans. TCDD has also been shown to modulate cytokine gene expression in human keratinocytes, including IL-1, TGF- and TFG-₂. In the present studies, the aim was to determine whether different cellular targets of human origin differed in susceptibility to TCDD as measured by CYP1A1 activity and mRNA expression, and whether cytokine gene induction/suppression correlated with TCDD susceptibility. Human airway epithelial cells, alveolar macrophages (AM), peripheral blood monocytes and lymphocytes (PBL) were exposed to 10^-10–10^-7 mol/L TCDD. CYP1A1 enzyme activity was determined by ethoxyresorufin-O-deethylase (EROD) activity, mRNA expression of CYP1A1 was measured by semiquantitative PCR assay. The secretion and/or gene expression of specific cytokines, including IL-6, IL-8, and IL-1 were also examined. Overall, there was a clear correlation between TCDD-induced enzyme activity and CYP1A1 mRNA levels, which were dose-dependently increased in the bronchoepithelial cells and PBL. The human airway epithelial cells (BEAS-S6 cell line and primary cells) appeared to be the most inducible cellular target, with up to 50-fold increases at 10^-8 mol/L TCDD with an EC₅₀ of 3×10^-11 mol/L TCDD. The pokeweed mitogen-activated peripheral blood lymphocytes revealed approximately 5-fold less capacity in CYP1A1 activity, with high interindividual variabilities (EC₅₀ 3×10^-9 mol/L TCDD). In contrast, CYP1A1 enzyme activity in both AM and purified peripheral blood monocytes, which were costimulated with LPS and/or GM-CSF, could not be detected. CYP1A1 mRNA levels, however, were detectable and only marginally enhanced in response to TCDD. The ability of all these cells to express and produce the proinflammatory cytokines IL-6 and IL-8 was neither enhanced nor impaired by TCDD. These results indicate that cell types found in human lung and peripheral blood vary in susceptibility to TCDD, with the lung epithelium being highly susceptible and the alveolar macrophage being nonsusceptible. However, expression and production of specific cytokines such as IL-6 and IL-8, which may potentiate inflammatory processes and/or work as mitogens, does not appear to be influenced by TCDD.     

Negative regulation of the murine cytosolic aldehyde dehydrogenase-3 (Aldh-3c) gene by functional CYP1A1 and CYP1A2 proteins.

We have examined enzyme activities and mRNA levels corresponding to aldehyde dehydrogenase-3 genes encoding cytosolic (ALDH3c) and microsomal (ALDH3m) forms. In contrast to negligible activities in the intact mouse liver, both ALDH3c and ALDH3m enzyme activities are inducible by benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mouse hepatoma Hepa-1c1c7 cell cultures. Constitutive mRNA levels of ALDH3c are virtually absent, whereas those of ALDH3m are substantial; using Hepa-1 mutant lines, we show that both ALDH3c and ALDH3m are TCDD-inducible by an Ah receptor-dependent mechanism. Basal mRNA levels of ALDH3c, but not those of ALDH3m, are strikingly elevated in untreated mutant cells lacking a functional CYP1A1 enzyme; low ALDH3c basal mRNA levels can be restored by introduction of a functional murine CYP1A1 or human CYP1A2 enzyme into these mutant cells. These data suggest that the TCDD induction process is distinct from the CYP1A1/CYP1A2 metabolism-dependent repression of constitutive gene expression; we suggest that this latter property classifies the Aldh-3c gene, but not the Aldh-3m gene, as a member of the murine [Ah] battery.     

The effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a novel 17β-estradiol 4-hydroxylase

Rates of microsomal 17β-estradiol (E₂) hydroxylation at the C-2, -4, -6, and -15 positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 and -15 positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low K_m 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E₂ catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low K_m hydroxylation of E₂ at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE₂) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE₂ formation predominantly over 4-MeOE₂. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E₂ 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.     

Preferential inducibility of CYP1A1 and CYP1A2 by TCDD: differential regulation in primary human hepatocytes versus transformed human cells

Cytochrome P4501A1 (CYP1A1) induction, a marker of aryl hydrocarbon (Ah) receptor activation, has been associated with carcinogenicity of the environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Consistently, we show that TCDD treatment led to induction of CYP1A1 in responsive human cancer cell lines including HepG2, LS174T, and MCF-7, as determined by Western blotting and CYP1A form-selective R-warfarin 6- and 8-hydroxylation. TCDD, however, preferably induced CYP1A2, not CYP1A1, in primary human hepatocytes. Such CYP1A form-preferred induction at the protein level was apparently uncorrelated with non-preferred mRNA induction in any cells studied. Moreover, while both genes were up-regulated by TCDD in primary hepatocytes and HepG2 cells, the induction of CYP1A1 and CYP1A2 at the mRNA level was distinguishable, indicated by the marked differences in activation kinetics and the response to the protein synthesis inhibitors, anisomycin and cycloheximide. Furthermore, formation of total benzo(a)pyrene (BaP)-DNA adducts was not altered following BaP exposure in TCDD-treated primary hepatocytes, whereas significantly elevated, in a CYP1A1-dependent manner, in the treated HepG2 cells. Taken together, our findings, demonstrating the complexities of TCDD-associated human Ah receptor function and differential regulations of CYP 1A enzymes, suggest clearly the need for caution when extrapolating data obtained in cell-based models.     

Estrogen-mediated regulation of CYP7B1: a possible role for controlling DHEA levels in human tissues

The current study examines regulation of CYP7B1, a DHEA 7alpha-hydroxylase, by sex hormones. Transfection with estrogen receptor alpha and treatment with 17beta-estradiol in human embryonic kidney 293 cells significantly increased CYP7B1 catalytic activity and mRNA, and stimulated a human CYP7B1 reporter gene. Transfection with estrogen receptor beta showed similar but less significant effects. In the absence of receptors, 17beta-estradiol suppressed CYP7B1 activity, suggesting that estrogenic effects may be different in cells not expressing receptors. Quantitation of CYP7B1 mRNA in adult and fetal human tissues showed markedly higher CYP7B1 mRNA levels in fetal tissues compared with the corresponding adult ones, except in the liver. This indicates a tissue-specific, developmental regulation of CYP7B1 and suggests an important function for this enzyme in fetal life. DHEA secreted by fetal adrenals is an essential precursor for placental estrogen formation. Since CYP7B1 diverts DHEA from the sex hormone biosynthetic pathway, estrogen receptor-mediated up-regulation of CYP7B1 should lead to less DHEA available for sex hormone synthesis and may help to maintain normal levels of estrogens and androgens in human tissues, especially during fetal development. Regulation by estrogens may also be of importance in other processes where CYP7B1 is involved, including cholesterol homeostasis, cellular proliferation, and CNS function.     

Altered regulation of P-450IA1 expression in a multidrug-resistant MCF-7 human breast cancer cell line

MCF-7 human breast cancer cells, selected for resistance to adriamycin (AdrR), exhibit the phenotype of multidrug resistance (MDR). Previous studies have shown that resistance in AdrR MCF-7 cells is associated with several biochemical changes that are similar to those induced in rat hyperplastic nodules, preneoplastic liver lesions which display broad spectrum resistance to carcinogens and hepatotoxins. In this report, we show that these changes in the AdrR MCF-7 cells are also associated with the development of cross-resistance to the procarcinogen benzo(a)pyrene (BP) and are associated with a marked defect in the conversion of BP to its cytotoxic, carcinogenic metabolites by AdrR cells. Since aryl hydrocarbon hydroxylase is the principle enzyme activity which converts benzo(a)pyrene to toxic hydroxylated forms, the regulation of cytochrome P-450IA1 expression, the gene encoding this enzyme activity in MCF-7 cells, was examined. Incubation with 100 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h results in a marked increase in aryl hydrocarbon hydroxylase activity in wild type (WT) but not AdrR MCF-7 cells. The alteration in aryl hydrocarbon hydroxylase expression in the AdrR cells is not overcome by incubation either with higher concentrations of TCDD (1 microM) or for longer periods of time (4 days). Northern blot analysis indicates that this defect in AdrR MCF-7 cells involves a regulatory defect at the level of P-450IA1 RNA. Following transfection of a construct containing the normal mouse P-450IA1 promoter fused to a reporter gene (bacterial chloramphenicol acetyltransferase) into WT and AdrR MCF-7 cells, TCDD induced chloramphenicol acetyltransferase activity in WT MCF-7 cells only. Furthermore, TCDD also induces both DT-diaphorase and UDP-glucuronyltransferase activities in WT, but not AdrR cells. These data suggest that the defect in the AdrR MCF-7 cells is not due to a structural P-450IA1 gene mutation, but rather involves a product regulating the polycyclic hydrocarbon-inducible expression of several drug-metabolizing enzyme activities. This defect in the AdrR MCF-7 cells is also associated with the development of resistance to ellipticine, an anticancer agent which is converted to more toxic hydroxylated species by aryl hydrocarbon hydroxylase or a similar mixed function oxidase. The WT and AdrR MCF-7 cells represent a useful model to study the regulation of the P-450IA1 gene in human cells.