Down-regulation of murine Cyp1a-1 in mouse hepatoma Hepa-1c1c7 cells by bisphenol A

Cultured mouse hepatoma Hepa-1c1c7 cells were treated with either bisphenol A or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or in combination to assess the role of bisphenol A in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cyp1a-1, as determined by analysis of 7-ethoxyresorufin O-deethylase (EROD) activities. Bisphenol A alone did not affect the activity of Cyp1a-1-specific EROD; in contrast, TCDD-induced EROD activities were markedly reduced in the concomitant treatment of TCDD and bisphenol A in a dose-dependent manner. Treatment with tamoxifen, an antiestrogen that acts through the estrogen receptor, did not affect the suppressive effects of bisphenol A on TCDD-induced EROD activity. TCDD-induced Cyp1a-1 mRNA levels were markedly suppressed in the concomitant treatment of TCDD and bisphenol A consistent with their effects on EROD activity. Transient transfection assay using dioxin-response element (DRE)-linked luciferase revealed that bisphenol A reduced transformation of the aryl hydrocarbons (Ah) receptor to a form capable of specifically binding to the DRE sequence in the promoter of the Cyp1a-1 gene. These results suggest the down-regulation of the Cyp1a-1 gene expression by bisphenol A in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear Ah receptor but not mediated through estradiol receptor.     

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.     

Induction of class 3 aldehyde dehydrogenase in the mouse hepatoma cell line Hepa-1 by various chemicals.

The mouse hepatoma cell line Hepa-1 was shown to express an aldehyde dehydrogenase (ALDH) isozyme which was inducible by TCDD and carcinogenic polycyclic aromatic hydrocarbons. The induced activity could be detected with benzaldehyde as substrate and NADP as cofactor (B/NADP ALDH). As compared with rat liver and hepatoma cell lines, the response was moderate (maximally 5-fold). There was an apparent correlation between this specific form of ALDH and aryl hydrocarbon hydroxylase (AHH) in the Hepa-1 wild-type cell line--in terms of inducibility by several chemicals. However, the magnitude of the response was clearly smaller for ALDH than for AHH. Southern blot analysis showed that a homologous gene (class 3 ALDH) was present in the rat and mouse genome. The gene was also expressed in Hepa-1 and there was a good correlation between the increase of class 3 ALDH-specific mRNA and B/NADP ALDH enzyme activity after exposure of the Hepa-1 cells to TCDD. It is concluded that class 3 ALDH is inducible by certain chemicals in the mouse hepatoma cell line, although the respective enzyme is not inducible in mouse liver in vivo.     

Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction.

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.     

Presence on human chromosome 10 of omeprazole-sensitivity gene whose product mediates CYP1A1 induction

Previously, we showed that CYP1A1 expression can be induced by omeprazole (OP) in the human cell line HepG2, but not in the mouse cell line Hepa-1. Now we show induction of CYP1A1 by alpha-naphthoflavone (alphaNF) in Hepa-1 cells. This induction was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by the aromatic hydrocarbon (Ah)-receptor antagonist PD98059, suggesting the presence of a ligand-independent signal-transduction pathway in the mouse cell line too. We utilized the lack of CYP1A1 induction by OP in Hepa-1 cells to map a putative human gene for OP-respon- siveness in cell hybrids produced by fusion of Hepa-1 and HepG2 cells. OP-induced CYP1A1 expression was detected in four out of the 32 Hepa-1 x HepG2 cell hybrids analyzed. To help identify the gene locus, a radiation-hybrid cell (E11) was constructed. Use of reverse-fluorescence in situ hybridization revealed that these five cell lines commonly retained human chromosome 10p. These results suggest that the human gene for OP-responsiveness is present on chromosome 10p.     

Defective binding of 3-methylcholanthrene to the Ah receptor within C3H/1OT1/2 clone 8 mouse fibroblasts in culture

C3H/1OT1/2 clone 8 mouse fibroblasts (C3H/1OT1/2 cells) exhibit induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) when exposed in culture to benzo(a)pyrene, benz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but do not display the induction response when treated with 3-methylcholanthrene (MCA), the classical inducer of cytochrome P1-450. Induction of cytochrome P1-450 is regulated by the Ah receptor which initially binds inducing chemicals in the cytoplasm, after which the inducer x receptor complex translocates into the nucleus. Cytosolic and nuclear forms of the Ah receptor can be detected in C3H/1OT1/2 cells using [3H]TCDD as the radioligand in culture, but specific Ah receptor binding is not detectable within C3H/1OT1/2 cells incubated with [3H]MCA. In contrast, in Hepa-1c1 cells, which exhibit cytochrome P1-450 induction when treated with MCA, cytosolic and nuclear Ah receptor can be detected by incubation of the cells either with [3H]MCA or with [3H]TCDD. Nonradioactive MCA is able to compete with [3H]TCDD for Ah receptor sites in C3H/1OT1/2 cells, but the relative potency of MCA as a competitor is lower within C3H/1OT1/2 cells than in C3H/1OT1/2 cytosol during extracellular incubation. Specific binding of [3H]MCA to Ah receptor can be detected by incubation of [3H]MCA with C3H/1OT1/2 cytosol outside the cell. The selective loss of response to MCA as a cytochrome P1-450 inducer (while retaining response to other inducers) appears to be due to defective interaction of MCA with the Ah receptor within the intracellular environment. The specific molecular alteration which makes the MCA x receptor complex ineffective within C3H/1OT1/2 cells is unknown. Some fibroblast lines other than C3H/1OT1/2 also selectively fail to respond to MCA; thus, this variation in Ah receptor function may not be due to a mutational change in the Ah regulatory gene which codes for the Ah receptor.