Comparative studies on the effects of green tea extracts and individual tea catechins on human CYP1A gene expression.

Green tea possesses significant anticancer activity in numerous experimental animal models, including demonstrated protection against aryl hydrocarbon induced cancers. The aryl hydrocarbon receptor (AhR) mediates the transcriptional activation of CYP1A1 and CYP1A2. In the present study, we investigated the effects of commercially available green tea extracts (GTEs) and individual tea catechins on the function of the AhR and on CYP1A gene expression in human hepatoma HepG2 cells and primary cultures of human hepatocytes. GTEs inhibited the transcription of a human CYP1A1 promoter-driven reporter gene induced by the AhR ligand 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in a concentration-dependent manner and inhibited the induced accumulation of both CYP1A1 and CYP1A2 mRNAs. GTEs blocked TCDD-induced binding of the AhR to DNA in HepG2 cells and in vitro in isolated hepatic cytosol. To determine if the observed effects were due to a single green tea component, we examined the four major catechins present in GTEs. Only (-)-epigallocatechin gallate (EGCG), the most abundant catechin in green tea, was able to inhibit TCDD-induced binding of the AhR to DNA and subsequent CYP1A transcription, however EGCG alone was less effective than GTEs. We next examined GTEs and catechins for AhR agonist activity. GTEs caused a concentration-dependent increase in CYP1A1-promoter driven reporter gene activity and caused accumulation of CYP1A1 mRNA and protein, but we found that individual catechins were unable to induce the expression of CYP1A1. Our results demonstrate that GTEs as a whole exert mixed agonist/antagonist activity on the AhR, while EGCG functions as a strict AhR antagonist. Therefore, modulation of human CYP1A expression by green tea extracts can not be attributed to the action of a single tea catechin, but rather is due to the effects of a complex mixture. These findings may be useful in future studies concerning green tea as a cancer preventive agent.

Effects of histone deacetylase inhibitors on the Ah receptor gene promoter.

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent basic helix-loop-helix-PAS-containing transcription factor which is activated by chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Constitutive expression of the AhR gene occurs in a tissue- and developmentally specific manner and appears to be altered by chemicals which affect histone deacetylase (HDAC) activity in cells in culture. Here we have directly characterized the effects of two HDAC inhibitors, n-butyrate and trichostatin A, on the promoter activity of the murine AhR gene. HDAC inhibitors increased the constitutive activity of the AhR gene promoter in a luciferase reporter construct by five- to sevenfold in a dose- and time-dependent manner in several cell lines and was correlated with an increase in endogenous AhR activity in an AhR-deficient cell line. Deletion analysis of the upstream region of the AhR gene localized the HDAC inhibitor effect to a 167-bp region encompassing -77 to +90 of the AhR gene promoter. Cotransfection of an AhR promoter-luciferase reporter plasmid with a vector expressing the E1A(12s) oncoprotein, a negative regulator of p300, a protein with histone acetylase activity, decreased AhR promoter activity fivefold. Overall, our results support a role for histone acetylation in the transcriptional activity of the AhR gene promoter.

The Ah receptor can bind ligand in the absence of receptor-associated heat-shock protein 90.

The Ah receptor (AhR) is a soluble ligand-dependent DNA regulatory protein that mediates many of the biological responses to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. In the absence of ligand, the cytosolic form of the AhR is found complexed with at least two molecules of hsp90, a heat shock protein of 90 kDa. In addition to its role in AhR protein folding and ability to repress the inherent nuclear localization, dimerization, and DNA binding activity of the AhR, it has been reported that hsp90 is absolutely required for maintaining the AhR in its high-affinity ligand binding conformation. The ability of high salt conditions (0. 4 M KCl) to dissociate the multimeric AhR complex into its monomeric form provides us with an avenue to examine the role of hsp90 in AhR ligand binding activity. In contrast to previous reports, we demonstrate that salt-dissociated "hsp90-free" AhR from several species still retains the ability to specifically bind ligand ([3H]TCDD). Although partial inactivation of ligand binding of salt-dissociated rat hepatic AhR was observed (to a maximum of 50% of total AhR binding), the presence of bound ligand protected against this inactivation. Little or no inactivation of the ligand binding ability of salt-dissociated guinea pig or rabbit AhR occurred. Our results not only indicate a significant species-difference in AhR ligand binding stability and/or activity, but also demonstrate that AhR ligand binding activity does not absolutely require the presence of receptor-bound hsp90.

Ortho-substituted polychlorinated biphenyls alter microsomal calcium transport by direct interaction with ryanodine receptors of mammalian brain.

A stringent structure-activity relationship among polychlorinated biphenyls (PCBs) possessing two or more ortho-chlorine substituents is observed for activation of ryanodine receptors in mammalian brain, revealing an arylhydrocarbon receptor-independent mechanism through which non-coplanar PCBs disrupt neuronal Ca2+ signaling. Of the congeners assayed, non-coplanar PCB 95 exhibits the highest potency (EC50 = 12-24 microM) toward activating high affinity [3H]ryanodine-binding in rat hippocampus, cerebellum, and cerebral cortex. Coplanar PCB 66 and PCB 126 have no ryanodine receptor activity in all brain regions examined. PCB 95 enhances [3H]ryanodine-binding affinity and capacity by significantly altering modulation by Ca2+ and Mg2+, thereby stabilizing a high affinity conformation of the ryanodine receptor. Ca2+ transport measurements using cortical microsomes reveal that PCB 95 discriminates between inositol 1,4,5-trisphosphate- and ryanodine-sensitive stores. PCB 95 selectively mobilizes Ca2+ from ryanodine-sensitive stores in a dose-dependent manner (EC50 = 3.5 microM) and is completely inhibited by ryanodine receptor blockers, whereas coplanar PCBs are inactive. These data demonstrate that ortho-substituted PCBs disrupt Ca2+ transport in central neurons by direct interaction with ryanodine receptors, showing high selectivity and specificity. Alteration of Ca2+ signaling mediated by ryanodine receptors in specific regions of the central nervous system may account, at least in part, for the significant impact of these agents toward neurodevelopment and neuroplasticity in mammals.