Differentiation of pluripotent C3H10T1/2 cells rapidly elevates CYP1B1 through a novel process that overcomes a loss of Ah Receptor

Stimulation of C3H10T1/2 cells by an adipogenic hormonal mixture (IDM) consisting of insulin (I), dexamethasone (D), and methylisobutylxanthine (M) substantially induces cytochrome P450 (CYP) 1B1 expression. This stimulation represents up to 40% of the level produced by maximum activation of the arylhydrocarbon receptor (AhR) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dexamethasone and methylisobutylxanthine in combination produced near maximum elevation of CYP1B1 along with a subsequent decline in AhR that paralleled the rise in peroxisome proliferator-activated receptorgamma1 (PPARgamma1). Inhibitors of AhR activity, which block TCDD induction, did not affect this increase of CYP1B1 expression, which was, therefore, independent of AhR activity. These responses were unaffected by inhibition of DNA synthesis, which was required for PPARgamma1 induction and terminal differentiation. Induction of CYP1B1 mRNA was paralleled by increased CYP1B1 promoter-luciferase reporter activity. The initial 0.8kb of promoter region, which was sufficient for 24h near maximum stimulation, did not contain either the key AhR-responsive elements that mediate the TCDD response or CREB and SF1 elements that mediate cAMP stimulation of rat CYP1B1 in steroidogenic cells. This reporter response to IDM stimulation, but not to TCDD, was maintained in AhR-null fibroblasts. CYP1B1 expression, unlike TCDD induction, was stimulated by IDM in only about half the cells. CYP1B1 expression partially overlapped with PPARgamma expression, which was also inversely related in clonal sub-lines. CYP1B1 expression may, therefore, represent an early stage of differentiation that requires factors associated with DNA synthesis to subsequently generate PPARgamma1.     

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome p450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome p450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-beta) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.     

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.     

Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.     

Is CYP1B1 involved in the metabolism of dioxins in the pig?

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most difficult to biodegradate and the most toxic dioxin congener. Previously, we demonstrated in silico the ability of pig CYP1A1 to hydroxylate 2,7-dichlorodibenzo-p-dioxin (DiCDD), but not TCDD. To increase our knowledge concerning the low effectiveness of TCDD biodegradability, we analyzed in silico the binding selectivity and affinity between pig CYP1B1 and the two dioxins by means of molecular modeling. We also compared the effects of TCDD and DiCDD on CYP1B1 gene expression (qRT-PCR) and catalytic (EROD) activity in porcine granulosa cells. It was found that DiCDD and TCDD were stabilized within the pig CYP1B1 active site by hydrophobic interactions. The analysis of substrate channel availability revealed that both dioxins opened the exit channel S, allowing metabolites to leave the enzyme active site. Moreover, DiCDD and TCDD increased the CYP1B1 gene expression and catalytic activity in porcine granulosa cells. On the other hand, TCDD demonstrated higher than DiCDD calculated affinity to pig CYP1B1, hindering TCDD exit from the active site. The great distance between CYP1B1's heme and TCDD also might contribute to the lower hydroxylation effectiveness of TCDD compared to that of DiCDD. Moreover, the narrow active site of pig CYP1B1 may immobilize TCDD molecule, inhibiting its hydroxylation. The results of the access channel analysis and the distance from pig CYP1B1's heme to TCDD suggest that the metabolizing potential of pig CYP1B1 is higher than that of pig CYP1A1. However, this potential is probably not sufficiently high to considerably improve the slow TCDD biodegradation.     

The human CYP1A1 gene is regulated in a developmental and tissue-specific fashion in transgenic mice

Regulation and expression of human CYP1A1 is demonstrated in transgenic mice. We have developed two transgenic mouse lines. One mouse strain (CYPLucR) carries a functional human CYP1A1 promoter (-1612 to +293)-luciferase reporter gene, and the other strain (CYP1A1N) expresses CYP1A1 under control of the full-length human CYP1A1 gene and 9 kb of flanking regulatory DNA. With CYPLucR(+/-) mice, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and several other aryl hydrocarbon receptor ligands induced hepatocyte-specific luciferase activity. When other tissues were examined, TCDD induced luciferase activity in brain with limited induction in lung and no detectable luciferase activity in kidney. Treatment of CYP1A1N(+/-) mice with TCDD resulted in induction of human CYP1A1 in liver and lung, while mouse Cyp1a1 was induced in liver, lung, and kidney. Although induced CYP1A1/Cyp1a1 could not be detected by Western blot analysis in brains from CYP1A1N(+/-) mice, induction in brain was verified by detection of CYP1A1/Cyp1a1 RNA. The administration of TCDD to nursing mothers to examine the effect of lactational exposure via milk demonstrated prominent induction of luciferase activity in livers of CYPLucR(+/-) newborn pups with limited induction in brain. However, TCDD treatment of adult CYPLucR(+/-) mice led to a 7-10-fold induction of brain luciferase activity. Combined these results indicate that tissue-specific and developmental factors are controlling aryl hydrocarbon receptor-mediated induction of human CYP1A1.     

Tumor necrosis factor-alpha potentiates genotoxic effects of benzo[a]pyrene in rat liver epithelial cells through upregulation of cytochrome P450 1B1 expression

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant, which may contribute to the development of human cancer. The ultimate carcinogenic BaP metabolite produced by cytochrome P450 enzymes (CYP), such as CYP1A1 and CYP1B1, anti-BaP-7,8-diol-9,10-epoxide, binds covalently to DNA and causes mutations. The levels of various CYP isoforms can be significantly modulated under inflammatory conditions. As the chronic inflammation is known to contribute to carcinogenesis, we investigated interactions of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), and BaP in regulation of the expression of CYP1A1/1B1 and induction of DNA damage in rat liver epithelial WB-F344 cells. TNF-alpha enhanced induction of CYP1B1, while it simultaneously suppressed the BaP-induced CYP1A1 expression. The observed deregulation of CYP1 induction was found to be associated with a significantly enhanced formation of DNA adducts. The elevated DNA damage corresponded with increased phosphorylation of p53 tumor suppressor at Ser-15 residue, enhanced accumulation of cells in the S-phase of cell cycle and potentiation of BaP-induced apoptosis. Inhibition of CYP1B1 by fluoranthene significantly decreased both the formation of DNA adducts and the induction of apoptosis in WB-F344 cells treated with BaP and TNF-alpha, thus suggesting that this isoform might be responsible for genotoxic effects of BaP in nonparenchymal liver cells. Our results seem to indicate that inflammatory conditions might enhance genotoxic effects of carcinogenic polycyclic aromatic hydrocarbons through upregulation of CYP1B1 expression.     

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.     

Characterization of human CYP1A1/1A2 induction by DNA microarray and alpha-naphthoflavone

DNA microarrays and real time PCR were used to analyze the mechanism of gene induction by CYP1A1 inducers, beta-naphthoflavone, and omeprazole, in the human hepatocellular carcinoma HepG2 cells. Reproducible and significant inductions were observed in a limited number of genes including CYP1A1 and CYP1A2. Genes induced by omeprazole included several protein tyrosine kinase targets. This result confirmed that omeprazole could modulate gene expressions through protein tyrosine kinase-mediated pathway. Induction ratios were considerably different from CYP1A1 and CYP1A2 (>10-fold) to other induced genes (<5-fold). alpha-Naphthoflavone, which is known as an antagonist to 2,3,7,8-tetrachlorodibenzo-p-dioxin, inhibited the inductions of heme oxygenase 1, glutamate-cysteine ligase (modifier unit), and thioredoxin reductase by beta-naphthoflavone but not those of CYP1A1 and CYP1A2. It unexpectedly enhanced the beta-naphthoflavone-mediated CYP1A1 and CYP1A2 induction. These results suggest that the CYP1A1 and CYP1A2 genes, which share their 5(') enhancer regions, are regulated differently from the other genes.     

Cell selective cAMP induction of rat CYP1B1 in adrenal and testis cells. Identification of a novel cAMP-responsive far upstream enhancer and a second Ah receptor-dependent mechanism

CYP1B1 is unique among P450 cytochromes in exhibiting inductive responses mediated by both the Ah receptor (AhR) and cAMP. cAMP induction was mediated either by a 189bp far upstream enhancer region (FUER, -5110 to -5298) or by a 230bp AhR-responsive enhancer region (AhER) (-797 to -1026). CYP1B1 luciferase reporters respond selectively to cAMP and TCDD in adrenal Y-1 cells (only cAMP), testis MA10 cells (cAMP>TCDD), and C3H10T1/2 mouse embryo fibroblasts (only TCDD). In Y-1 cells, which lack AhR, cAMP induction is totally dependent on the FUER, including absolute requirements for upstream and downstream halves of this region, and for CREB activity at a CRE sequence located at the 3(')-end. cAMP stimulation of the FUER was remarkably high (27-fold) and equally effective when linked to an HSV-TK promoter, indicating direct cAMP activation of the FUER. Binding of CREB to the essential CRE was demonstrated along with dominant negative effects of functionally impaired mutants. cAMP induction in MA10 cells was partially mediated by the FUER mechanism but was regulated additionally by AhER through AhR activity. MA10 cells also exhibit cAMP-dependent AhR down-regulation and AhR/Arnt complex formation. Mutations in AhER including XRE5 were similarly inhibitory to cAMP stimulation in MA10 cells and to TCDD stimulation in C3H10T1/2 cells. Transfection of AhR into the AhR-deficient Y-1 cells did not introduce this second mechanism, which indicated a need for additional components that are present in MA10 cells.     

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.     

Dioxin-induced adseverin expression in the mouse thymus is strictly regulated and dependent on the aryl hydrocarbon receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ligand for the ubiquitous, intracellular aryl hydrocarbon receptor (AhR), up-regulates the actin-modulating protein adseverin in mouse lymphoid tissues, a response that may be correlated to the immunotoxicity of TCDD. Here, by using chimeric mice with TCDD-responsive (AhR(+/+)) hematopoietic cells and TCDD-unresponsive (AhR(minus sign/minus sign)) thymic stroma, or the reverse, we show that TCDD-induced expression of adseverin in thymus is dependent on AhR expression in hematopoietic cells but not in stroma. The use of fetal thymic organ cultures also indicates that TCDD-induced expression of adseverin is confined to the thymocytes. The thymic stroma showed no induction of adseverin expression after TCDD exposure, although TCDD clearly activated the AhR in these cells, as indicated by the induction of CYP1A1. Adseverin was not induced in the thymus of normal adult C57BL/6 mice exposed to beta-estradiol or dexamethasone, two other agents, which also cause thymic atrophy. This further supports that adseverin induction is a specific gene regulatory effect by TCDD on thymocytes.     

AH receptor antagonist inhibits constitutive CYP1A1 and CYP1B1 expression in rat BP8 cells

The BP8 variant of the 5L rat hepatoma cell line is completely devoid of aryl hydrocarbon receptor (AHR) and is a useful model to examine AHR function. Previous studies showed that BP8 cells, when transfected with mouse AHR, exhibit induction of a plasmid-based reporter even in the absence of exogenous ligands. We transfected BP8 cells with full-length human AHR and found that presence of the AHR alone was sufficient to induce substantial CYP1A1 and CYP1B1 mRNA without any exogenous AHR ligand. An AHR antagonist, 3,4-dimethoxyflavone, inhibited CYP1A1 and CYP1B1 expression in a dose-dependent manner. When we transfected BP8 cells with a mutated human AHR that is defective in ligand binding, expression of CYP1A1 and CYP1B1 was diminished but not abolished. Inhibition by the AHR antagonist along with the diminished response to the mutated AHR indicates that BP8 cells contain some agent that acts as an agonist ligand for the AHR.