Phenobarbital (PB) induces various gene encoding drug/steroid-metabolizing enzymes such as cytochromes P450 (P450s) and transferases. Although the nuclear orphan constitutive active receptor (CAR) has been identified as a key transcription factor that regulates the induction of CYP2B, the full scope of CAR-regulated genes still remains a major question. To this end, reverse transcriptase-polymerase chain reaction and cDNA microarray techniques were employed to examine gene expression in wild-type and CAR-null mice. The results show that a total of 138 genes were detected to be either induced or repressed in response to PB treatment, of which about half were under CAR regulation. Including CYP2B10, CYP3A11, and NADPH-CYP reductase, CAR regulated a group of the PB-induced drug/steroid-metabolizing enzymes. Enzymes such as amino levulinate synthase 1 and squalene epoxidase displayed CAR-independent induction by PB. Cyp4a10 and Cyp4a14 represented the group of genes induced by PB only in CAR-null mice, indicating that CAR may be a transcription blocker that prevents these genes from being induced by PB. Additionally, the group of genes encoding enzymes and proteins involved in basic biological processes such as energy metabolism underwent the CAR-dependent repression by PB. Thus, CAR seems to have diverse roles, both as a positive and negative regulator, in the regulation of hepatic genes in response to PB beyond drug/steroid metabolism.

Diverse Roles of the Nuclear Orphan Receptor CAR in Regulating Hepatic Genes in Response to Phenobarbital

Induction of drug metabolism was described more than 40 years ago. Progress in understanding the molecular mechanism of induction of drug-metabolizing enzymes was made recently when the important roles of the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), two members of the nuclear receptor superfamily of transcription factors, were discovered to act as sensors for lipophilic xenobiotics, including drugs. CAR and PXR bind as heterodimeric complexes with the retinoid X receptor to response elements in the regulatory regions of the induced genes. PXR is directly activated by xenobiotic ligands, whereas CAR is involved in a more complex and less well understood mechanism of signal transduction triggered by drugs. Most recently, analysis of these xenobiotic-sensing nuclear receptors and their nonmammalian precursors such as the chicken xenobiotic receptor suggests an important role of PXR and CAR also in endogenous pathways, such as cholesterol and bile acid biosynthesis and metabolism. In this review, recent findings regarding xenosensors and their target genes are summarized and are put into an evolutionary perspective in regard to how a living organism has derived a system that is able to deal with potentially toxic compounds it has not encountered before.

Induction of Drug Metabolism: The Role of Nuclear Receptors

Phenobarbital (PB) response elements are composed of various nuclear receptor (NR)-binding sites A 51-bp distal element PB-responsive enhancer module (PBREM) conserved in the PB-inducible CYP2B genes contains two NR-binding direct repeat (DR)-4 motifs Responding to PB exposure in liver, the NR constitutive active receptor (CAR) translocates to the nucleus, forms a dimer with the retinoid X receptor (RXR), and activates PBREM via binding to DR-4 motifs For CYP3A genes, a common NR site [DR-3 or everted repeat (ER)-6] is present in proximal promoter regions In addition, the distal element called the xenobiotic responsive module (XREM) is found in human CYP3A4 genes, which contain both DR-3 and ER-6 motifs Pregnane X receptor (PXR) could bind to all of these sites and, upon PB induction, a PXR:RXR heterodimer could transactivate XREM These response elements and NRs are functionally versatile, and capable of responding to distinct but overlapping groups of xenochemicals

Phenobarbital response elements of cytochrome P450 genes and nuclear receptors.

The nuclear receptor PXR (pregnane X receptor) protects the body from hepatotoxicity of secondary bile acids such as lithocholic acid (LCA) by inducing expression of the hydroxylating cytochrome P450 enzyme CYP3A and promoting detoxification. We found that activation of PXR also increases the activity and gene expression of the phase II conjugating enzyme dehydroepiandrosterone sulfotransferase (STD) known to sulfate LCA to facilitate its elimination. This activation is direct and appears to extend to other xenobiotic sulfotransferases as well as to 3′-phosphoadenosine 5′-phosphosulfate synthetase 2 (PAPSS2), an enzyme that generates the donor cofactor for the reaction. Because sulfation plays an important role in the metabolism of many xenobiotics, prescription drugs, and toxins, we propose that PXR serves as a master regulator of the phase I and II responses to facilitate rapid and efficient detoxification and elimination of foreign chemicals.

https://www.pnas.org/content/pnas/99/21/13801.full.pdf

Regulation of a xenobiotic sulfonation cascade by nuclear pregnane X receptor (PXR)

Cytosolic sulfotransferase (SULT)-mediated sulfation plays an essential role in the detoxification of bile acids and is necessary to avoid pathological conditions, such as cholestasis, liver damage, and colon cancer. In this study, using transgenic mice bearing conditional expression of the activated constitutive androstane receptor (CAR), we demonstrate that activation of CAR is both necessary and sufficient to confer resistance to the hepatotoxicity of lithocholic acid (LCA). Surprisingly, the CAR-mediated protection is not attributable to the expected and previously characterized CYP3A pathway; rather, it is associated with a robust induction of SULT gene expression and increased LCA sulfation. We have also provided direct evidence that CAR regulates SULT expression by binding to the CAR response elements found within the SULT gene promoters. Interestingly, activation of CAR was also associated with an increased expression of the 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2), an enzyme responsible for generating the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. Analysis of gene knockout mice revealed that CAR is also indispensable for ligand-dependent activation of SULT and PAPSS2 in vivo. Therefore, we establish an essential and unique role of CAR in controlling the mammalian sulfation system and its implication in the detoxification of bile acids.

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A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification.

The molecular basis of how rodent nongenotoxic hepatocarcinogens such as phenobarbitone cause liver-tumor formation is poorly understood. An early effect of phenobarbitone exposure is to induce hepatocyte proliferation transiently, and there is evidence that this may be important for subsequent tumor development. In this investigation, we have used the differential display reverse transcriptase polymerase chain reaction technique to analyze differential gene expression in male C57Bl/10J mouse liver during the mitogenic phase of the phenobarbitone response. Seventy-seven putative differentially expressed cDNAs were isolated by differential display, and 13 of them were subsequently confirmed as being differentially expressed (both increased and decreased by phenobarbitone). Seven of the cDNAs were homologous to known mouse or human genes (carboxylesterase, coagulation factor X, amine N-sulphotransferase, human protein disulphide isomerase-related protein, cytochrome c oxidase subunit IV, golgin-245, thioredoxin reductase, betaine-homocysteine methyl transferase) and the remainder were novel. The expression pattern of the sulphotransferase was further characterized, and in mouse liver it was found to be significantly induced by phenobarbitone and not by five other rodent nongenotoxic hepatocarcinogens. In summary, the technique has enabled the identification of previously uncharacterized genes whose expression patterns are differentially altered by phenobarbitone in the mouse liver. © 2000 John Wiley & Sons, Inc. J Biochem Toxicol 14: 65–72, 2000

Identification of phenobarbitone-modulated genes in mouse liver by differential display.

The high incidence and profile of ras gene mutations reported in spontaneous and chemically induced liver tumours of the B6C3F1 mouse provides a potential means of determining in vivo genotoxicity and its relevance to carcinogenicity. We analysed spontaneous and chemically induced [with 4-amino-biphenyl (ABP), 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN)] hepatocellular tumours of the C57Bl/10J mouse for H-ras, K-ras and N-ras gene mutations to see if mutational analysis of the ras genes could be useful for such a determination in this strain. Regions of DNA spanning codons 12, 13 and 61 of the ras genes were amplified from formalin fixed liver tumour sections using the polymerase chain reaction. Mutations were detected using allele specific oligonucleotide probing and confirmed by sequencing. We have found that there are few ras mutations in either spontaneous or chemically induced liver tumours in the C57Bl/10J mouse. Out of 25 spontaneous tumours two contained an A to T transversion and one contained an A to G transition in base 2 of H-ras codon 61 and two contained a G to A transition in base 2 of K-ras codon 13 (the K-ras mutations were only faintly detectable and may be present in a subpopulation of the tumour cells). In the case of the 18 ABP induced tumours one contained a C to A transversion in base 1 of H-ras codon 61, and one contained an A to T transversion in base 2 of H-ras codon 61 and one contained a G to C transversion in base 1 of K-ras codon 13. One C to A transversion in base 1 of H-ras codon 61 was detected out of eight AAF induced tumours. Of the 25 DEN induced tumours, one contained an A to G transition and one contained an A to C transversion in base 2 of H-ras codon 61. The data indicate that at least in hepatocellular tumours of the C57Bl/10J strain and using chronic dosing regimes the ras genes do not represent markers for in vivo genotoxic activity.

Johanna M. Loughlin

Papers

Identification of phenobarbitone-modulated genes in mouse liver by differential display.

Point mutation analysis of ras genes in spontaneous and chemically induced C57Bl/10J mouse liver tumours.