DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

doi:10.1016/j.taap.2007.08.009

Toxicology and Applied Pharmacology

Volume 225, Issue 3, 15 December 2007, Pages 329-336

https://doi.org/10.1016/j.taap.2007.08.009 Get rights and content

Abstract

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 μM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.

Introduction

Hepatic cytochrome P450s (CYPs) display diverse functions in the metabolism of biological compounds, including endogenous molecules such as steroid hormones (You, 2004) and xenochemicals such as drugs and environmental pollutants (Iwase et al., 2006, Arlt et al., 2006). Phenobarbital (PB) has been identified as a potent inducer of the CYP 2B subfamily in both human and rodent primary hepatocytes (Wang et al., 2004, Li et al., 2006). Increased CYP 2B activity induced by xenochemicals may result in the accelerated metabolism of co-administered drugs and lead to decreased therapeutic efficacy and increased toxicity (Lin, 2006). PB-responsive enhancer module (PBREM), which responds to numerous PB-type inducers, has been identified in the promoter of human and rat CYP 2B genes (Sueyoshi et al., 1999). PBREM contains a nuclear factor-1 (NF-1) binding site flanked by two DR-4 nuclear receptor-binding motifs, NR-1 and NR-2 (Honkakoski and Negishi, 1998). Specific mutation of these NR motifs results in a complete loss of responsiveness to PB induction (Honkakoski et al., 1998).

The constitutive androstane receptor (CAR), which is present mostly in the cytosol of noninduced mammalian liver and hepatocytes, has been recognized as a transcription factor. It plays an important role not only in the regulation of hepatic fatty acid oxidation, gluconeogenesis (Miao et al., 2006), and the metabolism of steroid hormones and bilirubin (Yamamoto et al., 2003), but also in the modulation of xenochemical-metabolizing gene expression, including that of the phase I enzymes, such as CYPs 2B, 2C, and 3A, and the phase II enzymes, such as glutathione S-transferase alpha 1 (GSTA1) and UDP-glucuronosyltransferase 1A1 (UGT1A1) (Pascussi et al., 2003a, Reschly and Krasowski, 2006). Upon activation by PB-type inducers, CAR translocates from the cytosol to the nucleus and forms a heterodimer with retinoid X receptor (RXR). Subsequently, the heterodimer binds to NR-1 and trans-activates the target genes (Kawamoto et al., 1999, Kim et al., 2001).

N-6 and n-3 polyunsaturated fatty acids (PUFAs) have been identified to play an important role in energy metabolism and in endogenous hormone synthesis (Spector and Yorek, 1985). N-6 and n-3 PUFAs have also been shown to regulate the expression of many genes, such as acetyl-CoA carboxylase (Suchankova et al., 2005), leptin (Reseland et al., 2001), and fatty acid synthetase (Kim et al., 2004). PUFAs have also been studied for their role in the regulation of gene expression involved in the prevention of cardiovascular diseases (Le Jossic-Corcos et al., 2005), tumor development (Trombetta et al., 2007), and neurodegenerative disorders (Kim and Takahashi, 2006). However, studies of the roles of PUFAs in the expression of detoxifying genes are relatively scarce. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 PUFAs, especially docosahexaenoic acid (DHA) (Li et al., 2006). However, the mechanism for this down-regulation by DHA was not fully understood. In the present study, we used the rat primary hepatocyte culture system to study whether CAR plays a crucial role in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression.

Section snippets

Materials

Cell culture medium (RPMI-1640) was purchased from Gibco-BRL (Gaithersburg, MD); collagenase type I was from Worthington Biochemical (Lakewood, NJ); Matrigel and ITS⁺ (insulin, transferrin, selenium, bovine serum albumin, and linoleic acid) were from Collaborative Biomedical Products (Bedford, MA); and arachidonic acid (AA), linoleic acid (LA), eicosapentaenoic acid (EPA), and DHA were from Cayman Chemical (Ann Arbor, MI). Anti-CAR antibody (sc-13065) and anti-RXRα antibody (sc-553) were

Effect of PB on the nuclear accumulation of CAR in rat primary hepatocytes

Transactivation of the nuclear orphan receptor CAR was identified as a crucial event in PB-inducible CYP 2B gene expression in mouse primary hepatocytes (Kawamoto et al., 1999). To determine the effect of PB on the nuclear accumulation of CAR in rat primary hepatocytes, cells were treated with increasing concentrations of PB (5–500 μM) for 1 h. As shown in Fig. 1A, the nuclear accumulation of CAR was increased by PB in a dose-dependent manner. Also, the nuclear accumulation of CAR with 50 μM PB

Discussion

The nuclear orphan receptor CAR activates the transcription of detoxifying genes, such as CYP 2B (Faucette et al., 2006). CAR accumulates in the nucleus in response to PB and PB-type inducers in rat liver (Pustylnyak et al., 2005a) and mouse primary hepatocytes (Hosseinpour et al., 2006). To demonstrate whether PB triggers the nuclear accumulation of CAR in our rat primary hepatocyte culture system, we subjected the cell nuclear extracts to Western blotting. We showed that a basal level of

Acknowledgments

This work was supported by NSC 94-2320-B-040-033 and NSC-95-2320-B-040-029-MY2.

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DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

Abstract

Introduction

Section snippets

Materials

Effect of PB on the nuclear accumulation of CAR in rat primary hepatocytes

Discussion

Acknowledgments

Cancer Lett.

J. Biol. Chem.

J. Nutr.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Nutr. Biochem.

J. Nutr. Biochem.

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Toxicol. Lett.

J. Biol. Chem.

Toxicology

Toxicology

J. Lipid Res.

Methods Biol.

J. Lipid Res.

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Chem. Biol. Interact.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Chem. Biol. Interact.

Transcriptional regulation of CYP2B1 induction in primary rat hepatocyte cultures: repression by epidermal growth factor is mediated via a distal enhancer region

Mol. Pharmacol.

Docosahexaenoic acid induces proteasome-dependent degradation of {beta}-catenin, down-regulation of survivin and apoptosis in human colorectal cancer cells not expressing COX-2.

Carcinogenesis