Mechanism of benzo[a]pyrene-induced Cyp1a-1 gene expression in mouse Hepa 1c1c7 cells: role of the nuclear 6 s and 4 s proteins.

Treatment of wild-type (wt) aryl hydrocarbon (Ah)-responsive mouse Hepa 1c1c7 cells with benzo[a]pyrene (B[a]P) caused a concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast, B[a]P was inactive as an inducer in Ah nonresponsive class 1 and class 2 mutant cell lines. In parallel experiments, the nuclear fractions from wt cells treated with 10(-7) M [3H]B[a]P contained both the 4 s carcinogen binding protein and the 6 s (Ah receptor) complexes, whereas only the 4 s complex was present in the nuclear fraction of the class 2 mutant cells. The results obtained from cotreatment of wt Hepa 1c1c7 cells with 10(-6) or 10(-7) M B[a]P and 5 x 10(-7) or 10(-7) M 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) showed that MCDF inhibited the induction of EROD activity and Cyp1a-1 mRNA levels by B[a]P. Moreover, using 10(-7) M [3H]B[a]P and unlabeled MCDF, it was shown that MCDF not only inhibited the induction response but also caused a concentration-dependent decrease in levels of the nuclear 6 s complex but not the 4 s complex. In contrast, in situ competition studies with unlabeled 10(-6) M benzo[ghi]-perylene (B[ghi]P) resulted in the elimination of the nuclear [3H]B[a]P 4 s complex (but not the 6 s complex); however, the EROD activity and Cyp1a-1 mRNA levels in cells treated with 10(-7) M B[a]P in the presence or absence of 10(-6) M B[ghi]P were not significantly different. These results indicate that the 4 s binding protein is not required for the induction of Cyp1a-1 gene expression in Hepa 1c1c7 cells and suggest that B[a]P and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce Cyp1a-1 gene expression via a common mechanism which involves binding to the Ah receptor.     

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.     

2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes

We report here a novel observation that 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) induced predominantly cytochrome P4501A1 (CYP1A1) in rat hepatocytes and predominantly CYP1A2 in human hepatocytes. As part of our research program to evaluate species-differences in response to CYP inducers, we studied the effects of TCDD on CYP1A activity, protein, and gene expression in primary cultures of rat and human hepatocytes. TCDD was found to induce CYP1A activity, measured as ethoxyresorufin-O-deethylase (EROD) activity, in both rat and human hepatocytes. TCDD induction of EROD activity in human hepatocytes (2-5 fold of concurrent solvent control), was significantly lower than that found in rat hepatocytes ( 20-fold of concurrent solvent control). Two structural analogs of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF), were also evaluated. As observed for TCDD, human hepatocytes consistently showed a lower response than rat hepatocytes. As most TCDD-related effects are believed to be mediated via binding of the TCDD-Ah receptor (AhR) complex to DNA, nuclear AhR levels were measured in rat and human hepatocytes after TCDD treatment. We found that the nuclear AhR levels in TCDD-treated rat hepatocytes were approximately 4 times higher than found in TCDD-treated human hepatocytes. However, the estimated binding affinity of [3H]TCDD to nuclear AhR from rat hepatocytes was similar. The species difference in response to TCDD was further evaluated by analysis of CYP1A1 and CYP1A2 mRNA levels using Northern analysis, and P4501A1 and 1A2 protein levels using Western immunoblotting. Results showed that, at both gene expression and protein levels, TCDD induced predominantly CYP1A1 in rat hepatocytes and CYP1A2 in human hepatocytes.     

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.     

Induction of cytochrome P450 1A and DNA damage in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes by 2,3,7,8 tetrachlorodibenzo-p-dioxin

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.     

Induction of cytochrome P450 1A and DNA damage in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes by 2,3,7,8 tetrachlorodibenzo-p-dioxin

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.     

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.     

Differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1A1 expression in human breast carcinoma cell lines involve altered trans-acting factors

Differences in expression of the CYP1A1 gene have previously been observed in human breast carcinoma cell lines exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using an expression vector containing the functional 5'-regulatory region of human CYP1A1 (up to -1140) fused to the reporter gene CAT (for chloramphenicol acetyltransferase), the breast carcinoma cell lines, MCF-7, T47-D and ZR-75-1, classified as highly responsive to TCDD, were highly responsive to TCDD in the chloramphenicol acetyltransferase assay as well. Gel mobility shift assays have shown that these cell lines express a nuclear protein that binds the aryl hydrocarbon (Ah) receptor responsive element. The low or non-responsive cell lines, AL-1, BT-20 and CAMA-1, were low or non-responsive to TCDD in the chloramphenicol acetyltransferase assay, suggesting that the low-responsive phenotype is caused by altered trans-acting factors. However, the mechanism appears to differ among the cell lines. Whereas no induction was observed in AL-1, a fivefold induction in activity was observed in BT-20 and CAMA-1. The TCDD concentration giving half-maximum induction differed greatly between CAMA-1 and BT-20. The gel mobility shift assay showed the presence of a protein that bound specifically to the Ah responsive element in the non-responsive cell line AL-1, as well as the low-responsive cell lines, BT-20 and CAMA-1. The high basal activity but low induction observed in CAMA-1 may be due to an Ah receptor constitutively bound to the Ah responsive element.     

Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: characterization of 6-[125I]methyl-8-iodo-1,3-dichlorodibenzofuran-Ah receptor complexes.

6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.     

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.     

Rapid activation of c-Src kinase by dioxin is mediated by the Cdc37-HSP90 complex as part of Ah receptor signaling in MCF10A cells

We investigated the mechanism by which activation of the Ah receptor by dioxin (TCDD) was accompanied by rapid activation of c-Src kinase activity. A Western blotting analysis showed that such action of TCDD in MCF10A cells could effectively be suppressed by treatment with a specific inhibitor of Src family kinase, PP-2, as judged by Western blot detection of the active form of Src protein, indicating that Src kinase is directly activated by TCDD. Such an event, occurring within 10-30 min of the addition of TCDD, is also accompanied by simultaneous translocation of both Src and cdc37 proteins from cytosol into the 100,000 x g membrane fraction containing the plasma membrane. By dissociating the cytosolic Src-cdc37-HSP90 complex with 17 nM geldanamycin, an optimum concentration for affecting this cytosolic cdc37 complex, but not the cytosolic Ah receptor complex, we could show that the action of TCDD in activating c-Src and cdc37 was abolished, but not its action on CYP1A1. The important role of cdc37 in the action of TCDD-induced activation of c-Src was also confirmed by blocking cdc37 gene translation with the antisense oligonucleotide treatment as well as the siRNA preparation designed to silence cdc37 expression. To understand the functional meaning of the disruption of the Src-cdc37-HSP90 complex by 17 nM geldanamycin at the cellular level, we investigated its effect on TCDD-induced anti-apoptotic action. The results showed that geldanamycin at this concentration could also abolish this cellular effect of TCDD. Interestingly, such a role of cdc37 in mediating the action of TCDD appears to be limited to activation of c-Src kinase, but not kinases associated with activation of NFkB, C/EBPalpha, or ERK. Together, these observations support the hypothesis that there is a specific coordination between the activation of the cytosolic Ah receptor and the c-Src- and cdc37-containing HSP90 complex.     

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.