Curcumin suppresses the transformation of an aryl hydrocarbon receptor through its phosphorylation

Halogenated and polycyclic aromatic hydrocarbons induce diverse biochemical responses through the transformation of a cytosolic aryl hydrocarbon receptor (AhR). In mouse hepatoma Hepa-1c1c7 cells, curcumin, a yellow pigment of Curcuma longa, did not inhibit the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced translocation of the AhR into the nucleus, but rather accelerated it. In the nucleus, curcumin inhibited the TCDD-induced heterodimerization of the AhR with an AhR nuclear translocator (Arnt), an essential partner for the transformation, and also dose-dependently inhibited the TCDD-evoked phosphorylation of both the AhR and Arnt. Moreover, curcumin significantly inhibited the TCDD-induced activation of protein kinase C (PKC), which is involved in the transformation, decreased the TCDD-induced DNA-binding activity of the AhR/Arnt heterodimer, and downregulated CYP1A1 expression. In a cell-free system, curcumin inhibited the binding of 3-methylcholanthrene, an AhR agonist, to the receptor. These results indicate that curcumin is able to bind to the AhR as a ligand, but suppresses its transformation by inhibiting the phosphorylation of AhR and Arnt, probably by PKC.     

Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes

The orphan nuclear constitutive androstane receptor (CAR) is proposed to play a central role in the response to xenochemical stress. Identification of CAR target genes in humans has been limited by the lack of a selective CAR agonist. We report the identification of 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) as a novel human CAR agonist with the following characteristics: (a) potent activity in an in vitro fluorescence-based CAR activation assay; (b) selectivity for CAR over other nuclear receptors, including the xenobiotic pregnane X receptor (PXR); (c) the ability to induce human CAR nuclear translocation; and (d) the ability to induce the prototypical CAR target gene CYP2B6 in primary human hepatocytes. Using primary cultures of human hepatocytes, the effects of CITCO on gene expression were compared with those of the PXR ligand rifampicin. The relative expression of a number of genes encoding proteins involved in various aspects of steroid and xenobiotic metabolism was analyzed. Notably, CAR and PXR activators differentially regulated the expression of several genes, demonstrating that these two nuclear receptors subserve overlapping but distinct biological functions in human hepatocytes.     

Antagonistic and agonistic effects of indigoids on the transformation of an aryl hydrocarbon receptor

Halogenated and polycyclic aromatic hydrocarbons, exogenous ligands of the aryl hydrocarbon receptor (AhR), cause various toxicological effects through the transformation of the AhR. In this study, we investigated the antagonistic effects of indigoids on the transformation in addition to their agonistic ones. In a cell-free system, indigoids induced the transformation dose-dependently, but suppressed the transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the binding of 3-methylcholanthrene to the AhR. In mouse hepatoma Hepa-1c1c7 cells, indigoids, especially indirubin, suppressed the transformation and expression of CYP1A1 by inhibiting the translocation of AhR into the nucleus. When orally administered to mice at 10 mg/kg BW/day for three successive days, indigoids did not induce AhR transformation and expression of the CYP1A subfamily in the liver, while indirubin and indigo upregulated quinone reductase activity. These results indicate that indigoids are able to bind to the AhR as ligands and exhibit antagonistic effects at lower concentrations in mammalian cells.     

Enantiospecific Effects of Ketoconazole on Aryl Hydrocarbon Receptor

Azole antifungal ketoconazole (KET) was demonstrated to activate aryl hydrocarbon receptor (AhR). Since clinically used KET is a racemic mixture of two cis-enantiomers (2R,4S)-(+)-KET and (2S,4R)-(−)-KET, we examined the effects of KET enantiomers on AhR signaling pathway. (+)-KET dose-dependently activated AhR in human gene reporter cell line AZ-AHR, and displayed 5–20× higher agonist activity (efficacy), as compared to (−)-KET; both enantiomers were AhR antagonists with equal potency (IC₅₀). Consistently, (+)-KET strongly induced CYP1A1 mRNA and protein in human HepG2 cells, while (−)-KET exerted less than 10% of (+)-KET activity. In primary human hepatocytes, both enantiomers preferentially induced CYP1A2 over CYP1A1 mRNA and protein, and the potency of (+)-KET was slightly higher as compared to (−)-KET. Ligand binding assay with guinea pig liver cytosols revealed that both (+)-KET and (−)-KET are weak ligands of AhR that displaced [³H]-TCDD with comparable potency. Similarly, both enantiomers weakly transformed AhR to DNA-binding form with similar potency, as showed by EMSA, in guinea pig liver cytosolic extracts and nuclear extracts from mouse Hepa-1 cells. We also examined effects of KET on glucocorticoid receptor (GR), a regulator of AhR activity. Both KET enantiomers antagonized GR with similar potency, as revealed by gene reporter assay in AZ-GR cell line and down-regulation of tyrosine aminotransferase mRNA in human hepatocytes. Finally, we demonstrate enantiospecific antifungal activities of KET enantiomers in six Candida spp. strains. In conclusion, the significance of current study is providing the first evidence of enatiospecific effects of cis-enantiomers of ketoconazole on AhR-CYP1A pathway.     

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.     

The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics

Although they have several important limitations primary human hepatocytes still represent the in vitro gold standard model for xenobiotic metabolism and toxicity studies. The large use of human liver cell lines either from tumoral origin or obtained by oncogenic immortalisation is prevented by the loss of various liver-specific functions, especially many cytochrome P450 (CYP)-related enzyme activities. We review here recent results obtained with a new human hepatoma cell line, named HepaRG, derived from a human hepatocellular carcinoma. These cells exhibit unique features: when seeded at low density they acquire an elongated undifferentiated morphology, actively divided and after having reached confluency formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. Moreover contrary to other human hepatoma cell lines including HepG2 cells, HepaRG cells express various CYPs (CYP1A2, 2B6, 2C9, 2E1, 3A4) and the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) at levels comparable to those found in cultured primary human hepatocytes. They also express various other functions such phase 2 enzymes, apical and canalicular ABC transporters and basolateral solute carrier transporters, albumin, haptoglobin as well as aldolase B that is a specific marker of adult hepatocytes. HepaRG cells could represent a surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies and even more, a unique model system for analysing genotoxic compounds.     

Characterization of the molecular and structural properties of the transformed and nuclear aryl hydrocarbon (Ah) receptor complexes by proteolytic digestion

Ligand-dependent differences in the molecular properties of the transformed cytosolic and nuclear aryl hydrocarbon receptor (AhR) were investigated using the proteolytic clipping band shift assay. AhR complexes were incubated with [³²P]dioxin responsive element (DRE) (26-mer) or bromodeoxyuridine (BrdU)-DRE and the resulting protein-DNA or crosslinked protein-DNA complexes were treated with trypsin or V8 protease and analyzed by electrophoresis. The results showed that for several different AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran, 1,2,7,8-tetrachlorodibenzofuran and -naphthoflavone, the pattern of degraded protein-DNA products were similar using transformed cytosolic or nuclear AhR complexes. In contrast, the proteolytic clipping band shift assay showed that there were significant differences in the pattern of degraded protein-DNA products using nuclear AhR complexes derived from mouse Hepa 1c1c7 cells treated with TCDD or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF). The differences detected in this in vitro assay parallel the in vivo and in vitro activities of these compounds in which TCDD is a potent AhR agonist whereas MCDF is a partial AhR agonist and antagonist.     

The small heterodimer partner interacts with the pregnane X receptor and represses its transcriptional activity

SHP (small heterodimer partner, NR1I0) is an atypical orphan member of the nuclear receptor subfamily in that it lacks a DNA-binding domain. It is mostly expressed in the liver, where it binds to and inhibits the function of nuclear receptors. SHP is up-regulated by primary bile acids, through the activation of their receptor farnesoid X receptor, leading to the repression of cholesterol 7alpha-hydroxylase (CYP7alpha) expression, the rate-limiting enzyme in bile acid production from cholesterol. PXR (pregnane X receptor, NR1I2) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs as well as endogenous compounds such as bile acid precursors. Upon activation, PXR induces CYP3A and inhibits CYP7alpha, suggesting that PXR can act on both bile acid synthesis and elimination. Indeed, CYP7alpha and CYP3A are involved in biochemical pathways leading to cholesterol conversion into primary bile acids, whereas CYP3A is also involved in the detoxification of toxic secondary bile acid derivatives. Here, we show that PXR is a target for SHP. Using pull-down assays, we show that SHP interacts with both murine and human PXR in a ligand-dependent manner. From transient transfection assays, SHP is shown to be a potent repressor of PXR transactivation. Furthermore, we report that chenodeoxycholic acid and cholic acid, two farnesoid X receptor ligands, induce up-regulation of SHP and provoke a repression of PXR-mediated CYP3A induction in human hepatocytes as well as in vivo in mice. These results reveal an elaborate regulatory cascade, tightly controlled by SHP, for both the maintenance of bile acid production and detoxification in the liver.     

Regulatory factors involved in species-specific modulation of arylhydrocarbon receptor (AhR)-dependent gene expression in humans and mice

The arylhydrocarbon receptor (AhR) mediates toxicities of dioxins, including the most potent congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), by being translocated to the nucleus upon ligand-binding and inducing expression of target genes. Although the species-specific activity of the AhR is primarily attributable to species-specific AhR-ligand affinity, the precise mechanism has not been clarified. We investigated the modulation mechanisms of AhR in Hepa1c1c7 and HepG2 hepatoma cells, which were derived from high-affinity-AhR-expressing C57BL/6 mice and low-affinity-AhR-expressing humans, respectively. Although, consistent with their AhR affinities, TCDD induced a greater amount of cytochrome P450 1A1 (CYP1A1) mRNA, one of the most sensitive AhR-targets, in Hepa1c1c7 cells than in HepG2 cells immediately after exposure, both cells expressed a similar level of CYP1A1 mRNA from 4 h onward. A rapid decrease in the AhR protein after nuclear translocation in Hepa1c1c7 cells was suggested to contribute to suppression of CYP1A1 induction to the same level as in HepG2 cells. Different profiles of histone deacetylase 1 (HDAC1)-binding to the CYP1A1 promoter and histone acetylation between both cell lines and lower degradation rate of CYP1A1 mRNA in HepG2 cells were also implicated in regulating their target gene expression. These factors have been highly suggested to be involved in the species-specific modulation mechanism of AhR function.     

Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation

The arylamide 2-acetylaminofluorene (AAF) is a powerful carcinogen displaying a marked promoting activity, also known to regulate expression of liver detoxifying proteins. In this study we identified CYP3A23, a major inducible cytochrome P-450 (CYP) isoform, as an AAF target in hepatocytes. Indeed, exposure to AAF of primary rat hepatocytes resulted in a marked up-regulation of CYP3A23 expression at both mRNA and protein levels. Using CYP3A23 reporter gene constructs, we further demonstrated that AAF activated the CYP3A23 Direct Repeat 3 (DR3) promoter element interacting with the nuclear pregnane X receptor (PXR). Moreover, the PXR antagonist ecteinascidin-743 fully suppressed AAF-related CYP3A23 induction. Low doses of AAF inhibiting DNA synthesis in hepatocytes however failed to trigger PXR-related CYP3A23 induction and PXR-negative epithelial liver cells remained sensitive to the mito-inhibitory effects of AAF. Such data indicate that AAF up-regulates CYP3A23 through PXR activation but does not require PXR for exerting its carcinogenic promoting properties based on inhibition of cell growth.