Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines.
This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.
The pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR) transcriptionally activates cytochrome P4503A4 (CYP3A4) when ligand activated by endobiotics and xenobiotics. We cloned the human PXR gene and analysed the sequence in DNAs of individuals whose CYP3A phenotype was known. The PXR gene spans 35 kb, contains nine exons, and mapped to chromosome 13q11-13. Thirty-eight single nucleotide polymorphisms (SNPs) were identified including six SNPs in the coding region. Three of the coding SNPs are non-synonymous creating new PXR alleles [PXR*2, P27S (79C to T); PXR*3, G36R (106G to A); and PXR*4, R122Q (4321G to A)]. The frequency of PXR*2 was 0.20 in African Americans and was never found in Caucasians. Hepatic expression of CYP3A4 protein was not significantly different between African Americans homozygous for PXR*1 compared to those with one PXR*2 allele. PXR*4 was a rare variant found in only one Caucasian person. Homology modelling suggested that R122Q, (PXR*4) is a direct DNA contact site variation in the third alpha-helix in the DNA binding domain. Compared with PXR*1, and variants PXR*2 and PXR*3, only the variant PXR*4 protein had significantly decreased affinity for the PXR binding sequence in electromobility shift assays and attenuated ligand activation of the CYP3A4 reporter plasmids in transient transfection assays. However, the person heterozygous for PXR*4 is normal for CYP3A4 metabolism phenotype. The relevance of each of the 38 PXR SNPs identified in DNA of individuals whose CYP3A basal and rifampin-inducible CYP3A4 expression was determined in vivo and/or in vitro was demonstrated by univariate statistical analysis. Because ligand activation of PXR and upregulation of a system of drug detoxification genes are major determinants of drug interactions, it will now be useful to extend this work to determine the association of these common PXR SNPs to human variation in induction of other drug detoxification gene targets.
The fully active dihydroxylated metabolite of vitamin D 3 induces the expression of CYP3A4 and, to a lesser extent, CYP2B6 and CYP2C9 genes in normal differentiated primary human hepatocytes. Electrophoretic mobility shift assays and cotransfection in HepG2 cells using wild-type and mutated oligonucleotides revealed that the vitamin D receptor (VDR) binds and transactivates those xenobiotic-responsive elements (ER6, DR3, and DR4) previously identified in CYP3A4, CYP2B6, and CYP2C9 promoters and shown to be targeted by the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR). Full VDR response of various CYP3A4 heterologous/homologous promoter-reporter constructs requires both the proximal ER6 and the distal DR3 motifs, as observed previously with rifampicinactivated PXR. Cotransfection of a CYP3A4 homologous promoter-reporter construct (including distal and proximal PXR-binding motifs) and of PXR or CAR expression vectors in HepG2 cells revealed the ability of these receptors to compete with VDR for transcriptional regulation of CYP3A4. In conclusion, this work suggests that VDR, PXR, and CAR control the basal and inducible expression of several CYP genes through competitive interaction with the same battery of responsive elements.Cytochrome P450 (CYP) 1 enzymes are mainly expressed in the liver and catalyze the metabolic conversion of xenobiotics, including environmental pollutants and drugs, to more polar and easily disposable derivatives (2, 3). CYP genes from the CYP2 and CYP3 families are inducible by many xenobiotics, notably including barbiturates and rifampicin. Two nuclear receptors, the pregnane X receptor (PXR; NR1I2) and the constitutive androstane receptor (CAR; NR1I3), have recently been shown to mediate CYP2 and CYP3 gene induction in animals and man (4 -6). Both PXR and CAR form heterodimers with the retinoid X receptor (RXR; NR2B1). PXR is activated by a wide spectrum of xenobiotics and steroids (4, 7, 8) and controls CYP3A4 and CYP3A7 induction by targeting two specific responsive elements present in the regulatory region of these genes (4, 7-12). The first of these is the proximal PXR-responsive element, located at -160. It consists of an everted repeat of the nuclear receptor half-site AGGTCA separated by 6 nucleotides (ER6); this element is necessary but not sufficient for full transactivation of the CYP3A4 promoter. Indeed, full PXRmediated induction requires the presence of a second distal xenobiotic-responsive element (dPXRE), located between -7800 and Ϫ7200 (9). This element is composite and consists of two direct repeats separated by 3 nucleotides (DR3), encompassing an ER6 motif. In contrast to PXR, CAR is sequestered in the cytoplasm and translocates into the nucleus upon activation, notably in response to phenobarbital (6, 13). Several groups have identified a complex phenobarbital-responsive element module that consists of two nuclear receptor-binding sites (termed NR1 and NR2) and one nuclear factor 1 binding site (12,14). Both NR1 and NR2 are imperfect DR4...
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