Identification of human CYP2C8 as a retinoid-related orphan nuclear receptor target gene.

Retinoid-related orphan nuclear receptors (RORs) alpha and gamma (NR1F1, -3) are highly expressed in liver, adipose tissue, thymus, and brain and are involved in many physiological processes, such as circadian rhythm and immune function. Enzymes in the cytochrome P450 2C subfamily metabolize many clinically important drugs and endogenous compounds, such as the anticancer drug paclitaxel and arachidonic acid, and are highly expressed in liver. Here, we present the first evidence that RORs regulate the transcription of human CYP2C8. Overexpression of RORalpha and RORgamma in HepG2 cells significantly enhanced the activity of the CYP2C8 promoter but not that of the CYP2C9 or CYP2C19 promoters. Computer analyses, promoter deletion studies, gel shift assays, and mutational analysis identified an essential ROR-responsive element at -2045 base pairs in the CYP2C8 promoter that mediates ROR transactivation. Adenoviral overexpression of RORalpha and -gamma significantly induced endogenous CYP2C8 transcripts in both HepG2 cells and human primary hepatocytes. Knockdown of endogenous RORalpha and -gamma expression in HepG2 cells by RNA interference decreased the expression of endogenous CYP2C8 mRNA by approximately 50%. These data indicate that RORs transcriptionally up-regulate CYP2C8 in human liver and, therefore, may be important modulators of the metabolism of drugs and physiologically active endogenous compounds by this enzyme in liver and possibly extrahepatic tissues where RORs are expressed.

VKORC1 polymorphisms, haplotypes and haplotype groups on warfarin dose among African-Americans and European-Americans.

BACKGROUND: Although the influence of VKORC1 and CYP2C9 polymorphisms on warfarin response has been studied, variability in dose explained by CYP2C9 and VKORC1 is lower among African-Americans compared with European-Americans. This has lead investigators to hypothesize that assessment of VKORC1 haplotypes may help capture a greater proportion of the variability in dose for this under-represented group. However, the inadequate representation of African-Americans and the assessment of a few VKORC1 polymorphisms have hindered this effort. METHODS: To determine if VKORC1 haplotypes or haplotype groups explain a higher variability in warfarin dose, we comprehensively assessed VKORC1 polymorphisms in 273 African-Americans and 302 European-Americans. The influence of VKORC1 polymorphisms, race-specific haplotypes and haplotype groups on warfarin dose was evaluated in race-stratified multivariable analyses after accounting for CYP2C9 (*2, *3, *5, *6 and *11) and clinical covariates. RESULTS: VKORC1 explained 18% (30% with CYP2C9) variability in warfarin dose among European-Americans and 5% (8% with CYP2C9) among African-Americans. Four common haplotypes in European-Americans and twelve in African-Americans were identified. In each race VKORC1 haplotypes emerged into two groups: low-dose (Group A) and high-dose (Group B). African-Americans had a lower frequency of Group A haplotype (10.6%) compared with European-Americans (35%, p < 0.0001).The variability in dose explained by VKORC1 haplotype or haplotype groups was similar to that of a single informative polymorphism. CONCLUSIONS: Our findings support the use of CYP2C9, VKORC1 polymorphisms (rs9934438 or rs9923231) and clinical covariates to predict warfarin dose in both African- and European-Americans. A uniform set of common polymorphisms in CYP2C9 and VKORC1, and limited clinical covariates can be used to improve warfarin dose prediction for a racially diverse population.

The discovery of new coding alleles of human CYP26A1 that are potentially defective in the metabolism of all-trans retinoic acid and their assessment in a recombinant cDNA expression system.

OBJECTIVES: Retinoic acid (RA) is a critical regulator of gene expression during embryonic development and in the maintenance of adult epithelial tissues. This study was undertaken to identify genetic polymorphisms of CYP26A1 which might affect these processes. We sequenced CYP26A1 in racially diverse individuals and assessed the metabolism of retinoic acid by newly identified coding alleles of CYP26A1 in a recombinant system. METHODS: CYP26A1 was sequenced in 24 Caucasians, 24 African-Americans, 24 Asians, and 20 individuals of unknown racial origin. cDNA constructs for wild-type and coding alleles of CYP26A1 were constructed in a pcDNA3.1 expression vector and expressed in Cos-1 cells. A FLAG tag at the C-terminal end of the cDNA was used to quantitate the recombinant CYP26A1 proteins. RESULTS: A total of 13 single nucleotide polymorphisms (SNPs) were identified in CYP26A1. Three SNPs produced coding changes: R173S, F186L, and C358R. These alleles were termed as CYP26A1*2, CYP26A1*3, and CYP26A1*4, respectively, by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee at http://www.cypalleles.ki.se/. Wild type CYP26A1 protein metabolized all-trans-retinoic acid (at-RA) to 4-oxo-RA, 4-OH-RA as well as water-soluble metabolites. CYP26A1.3 (F186L) and CYP26A1.4 (C358R) allelic proteins exhibited significantly lower metabolism (40-80%) of at-RA than wild-type CYP26A1.1 protein. CONCLUSION: This is the first study to identify coding alleles of CYP26A1. Two coding alleles, CYP26A1*3 and CYP26A1*4, are predicted to be defective based on the metabolism of at-RA by the recombinant proteins. These studies suggest the need for future clinical studies of polymorphisms of CYP26A1 in embryonic development.

Cloning, expression, and characterization of three new mouse cytochrome p450 enzymes and partial characterization of their fatty acid oxidation activities.

The mammalian CYP2C subfamily is one of the largest and most complicated in the cytochrome P450 superfamily. In this report, we describe the organization of the mouse Cyp2c locus, which contains 15 genes and four pseudogenes, all of which are located in a 5.5-megabase region on chromosome 19. We cloned three novel mouse CYP2C cDNAs (designated CYP2C50, CYP2C54, and CYP2C55) from mouse heart, liver, and colon, respectively. All three cDNAs contain open reading frames that encode 490 amino acid polypeptides that are 57 to 95% identical to other CYP2Cs. The recombinant CYP2C proteins were expressed in Escherichia coli after N-terminal modification, partially purified, and shown to be active in the metabolism of both arachidonic acid (AA) and linoleic acid, albeit with different catalytic efficiencies and profiles. CYP2C50 and CYP2C54 metabolize AA to epoxyeicosatrienoic acids (EETs) primarily, and linoleic acid to epoxyoctadecenoic acids (EOAs) primarily, whereas CYP2C55 metabolizes AA to EETs and hydroxyeicosatetraenoic acids and linoleic acid to EOAs and hydroxyoctadecadienoic acids. Northern blotting and reverse transcription-polymerase chain reaction analysis reveal that CYP2C50 transcripts are abundant in liver and heart; CYP2C54 transcripts are present in liver, kidney, and stomach; and CYP2C55 transcripts are abundant in liver, colon, and kidney. Immunoblotting studies demonstrate that CYP2C50 protein is expressed in liver and heart, CYP2C54 protein is detected primarily in liver, and CYP2C55 protein is present primarily in colon. Immunohistochemistry reveals that CYP2C55 is most abundant in surface columnar epithelium in the cecum. We conclude that these new CYP2C enzymes are probably involved in AA and linoleic acid metabolism in mouse hepatic and extrahepatic tissues.

CYP2C44, a new murine CYP2C that metabolizes arachidonic acid to unique stereospecific products.

The human CYP2Cs have been studied extensively with respect to the metabolism of clinically important drugs and endogenous chemicals such as arachidonic acid (AA). Five members of the mouse CYP2C family have previously been described that metabolize arachidonic acid into regio- and stereospecific epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids, which have many important physiological roles. Herein, we describe the cloning and characterization of a new mouse cytochrome P450 (P450), CYP2C44, which has the lowest homology with other known mouse CYP2Cs. Western blotting and real-time polymerase chain reaction detected CYP2C44 mRNA and protein in liver >> kidney > adrenals. Kidney contained approximately 10% of the CYP2C44 mRNA content of liver. CYP2C44 metabolized AA to unique stereospecific products, 11R,12S-EET and 8R, 9S-EET, which are similar to those produced by rat CYP2C23. CY2C23 is highly expressed in rat kidney and has been suggested to be important in producing compensatory renal artery vasodilation in response to salt-loading in this species. Immunohistochemistry showed the presence of CYP2C44 in hepatocytes, biliary cells of the liver, and the proximal tubules of the kidney. Unlike mouse CYP2C29, CYP2C38, and CYP2C39, CYP2C44 did not metabolize the common CYP2C substrate tolbutamide. CYP2C44 was not induced by phenobarbital or pregnenolone-16alpha-carbonitrile, two prototypical inducers of hepatic P450s. The presence of CYP2C44 in mouse liver, kidney, and adrenals and the unique stereospecificity of its arachidonic acid metabolites are consistent with the possibility that it may have unique physiological roles within these tissues, such as modulation of electrolyte transport or vascular tone.

Mouse liver CYP2C39 is a novel retinoic acid 4-hydroxylase. Its down-regulation offers a molecular basis for liver retinoid accumulation and fibrosis in aryl hydrocarbon receptor-null mice.

Livers of aryl hydrocarbon receptor (AHR)-null mice have high levels of retinoic acid (RA), retinol, and retinyl palmitate. Hepatic accumulation of RA in these mice may be responsible in part for the hepatic phenotype characterized by small liver size and fibrosis. The increased levels of hepatic RA may be due to decreased metabolism of RA to 4-hydroxyretinoic acid. To identify the P450 isoform(s) involved in RA metabolism, liver microsomes from AHR-null and wild-type mice were subjected to Western blotting and probed with antibodies to rat P450s that cross-react with murine forms. Signal intensity in Western blots probed with anti-rat CYP2C6 antibodies correlated with levels of RA 4-hydroxylation. Furthermore, this anti-rat CYP2C6 antibody inhibited RA 4-hydroxylase activity of wild-type mouse liver microsomes to the levels of AHR-null mouse liver. When used to screen a mouse liver cDNA expression library, this antibody exclusively recognized the murine P450 CYP2C39. Catalytic assays of five recombinant mouse CYP2Cs expressed in Escherichia coli revealed that only CYP2C39 was competent for RA 4-hydroxylation (K(m) = 812.3 nm and V(max) 47.85 (fmol/min/pmol P450)). Real time reverse transcriptase-PCR used to assess the Cyp2C39 mRNA expression showed decreased levels (30%) of this transcript in AHR-null compared with wild-type liver, consistent with decreased protein levels observed by Western blot analysis using an antibody to a CYP2C39-specific peptide. These data show that CYP2C39 catalyzes RA catabolism and thus possibly controls RA levels in mouse liver. Down-regulation of Cyp2C39 is hypothesized to be responsible for the liver phenotype in the AHR-null mouse.