Biochemical and Biophysical Research Communications
Regular ArticleSequence of the 5′-Flanking Region of CYP3A5: Comparative Analysis with CYP3A4 and CYP3A7
Abstract
Two genomic clones containing the 3′-end of CYP3A7, the whole 5′-flanking region plus the first exon of CYP3A5 were isolated from two different human genomic banks and sequenced. The 5′-flanking region of CYP3A5, from nucleotide -1 to -1434, was 60% and 59% similar with the corresponding regions of CYP3A4 and CYP3A7, respectively. However, the similarity increased to 74% when regions from nucleotide -1 to -700 were compared and droped to less than 42% for regions upstream from -700. The CYP3A5 promoter contains a CATA box instead of the typical TATA box and a basic transcription element (BTE). Other consensus sequences previously found in CYP3A4 and CYP3A7 were also identified in the 5′-flanking of CYP3A5 from -1 to -700. Both CYP3A5 and CYP3A7 genes appear to be tandemly associated and transcribed in the same direction on chromosome 7.
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Genetic contribution to variable human CYP3A-mediated metabolism
2012, Advanced Drug Delivery ReviewsCitation Excerpt :However, recent publication of the sequence for the entire CYP3A gene locus has revealed the presence of two CYP3A pseudogenes, CYP3AP1 and CYP3AP2 [87]. Importantly, the 5′-flanking and exon-1 sequence for CYP3AP1 is identical to that described by Jounaïdi et al. [86] as the putative CYP3A5 promoter sequence. The true 5′-flanking sequence for CYP3A5*1A (AC005020), is homologous to that of CYP3AP1 (89%), but contains important differences.
The human CYP3A subfamily plays a dominant role in the metabolic elimination of more drugs than any other biotransformation enzyme. CYP3A enzyme is localized in the liver and small intestine and thus contributes to first-pass and systemic metabolism. CYP3A expression varies as much as 40-fold in liver and small intestine donor tissues. CYP3A-dependent in vivo drug clearance appears to be unimodally distributed which suggests multi-genic or complex gene–environment causes of variability. Interindividual differences in enzyme expression may be due to several factors including: variable homeostatic control mechanisms, disease states that alter homeostasis, up- or down-regulation by environmental stimuli (such as smoking, drug intake, or diet), and genetic mutations. This review summarizes the current understanding and implications of genetic variation in the CYP3A enzymes. Unlike other human P450s (CYP2D6, CYP2C19) there is no evidence of a ‘null’ allele for CYP3A4. More than 30 SNPs (single nucleotide polymorphisms) have been identified in the CYP3A4 gene. Generally, variants in the coding regions of CYP3A4 occur at allele frequencies < 5% and appear as heterozygous with the wild-type allele. These coding variants may contribute to but are not likely to be the major cause of inter-individual differences in CYP3A-dependent clearance, because of the low allele frequencies and limited alterations in enzyme expression or catalytic function. The most common variant, CYP3A4*1B, is an A-392G transition in the 5′-flanking region with an allele frequency ranging from 0% (Chinese and Japanese) to 45% (African-Americans). Studies have not linked CYP3A4*1B with alterations in CYP3A substrate metabolism. In contrast, there are several reports about its association with various disease states including prostate cancer, secondary leukemias, and early puberty. Linkage disequilibrium between CYP3A4*1B and another CYP3A allele (CYP3A5*1) may be the true cause of the clinical phenotype. CYP3A5 is polymorphically expressed in adults with readily detectable expression in about 10–20% in Caucasians, 33% in Japanese and 55% in African-Americans. The primary causal mutation for its polymorphic expression (CYP3A5*3) confers low CYP3A5 protein expression as a result of improper mRNA splicing and reduced translation of a functional protein. The CYP3A5*3 allele frequency varies from approximately 50% in African-Americans to 90% in Caucasians. Functionally, microsomes from a CYP3A5*3/*3 liver contain very low CYP3A5 protein and display on average reduced catalytic activity towards midazolam. Additional intronic or exonic mutations (CYP3A5*5, *6, and *7) may alter splicing and result in premature stop codons or exon deletion. Several CYP3A5 coding variants have been described, but occur at relatively low allelic frequencies and their functional significance has not been established. As CYP3A5 is the primary extrahepatic CYP3A isoform, its polymorphic expression may be implicated in disease risk and the metabolism of endogenous steroids or xenobiotics in these tissues (e.g., lung, kidney, prostate, breast, leukocytes). CYP3A7 is considered to be the major fetal liver CYP3A enzyme. Although hepatic CYP3A7 expression appears to be significantly down-regulated after birth, protein and mRNA have been detected in adults. Recently, increased CYP3A7 mRNA expression has been associated with the replacement of a 60-bp segment of the CYP3A7 promoter with a homologous segment in the CYP3A4 promoter (CYP3A7*1C allele). This mutational swap confers increased gene transcription due to an enhanced interaction between activated PXR:RXRα complex and its cognate response element (ER-6). The genetic basis for polymorphic expression of CYP3A5 and CYP3A7 has now been established. Moreover, the substrate specificity and product regioselectivity of these isoforms can differ from that of CYP3A4, such that the impact of CYP3A5 and CYP3A7 polymorphic expression on drug disposition will be drug dependent. In addition to genetic variation, other factors that may also affect CYP3A expression include: tissue-specific splicing (as reported for prostate CYP3A5), variable control of gene transcription by endogenous molecules (circulating hormones) and exogenous molecules (diet or environment), and genetic variations in proteins that may regulate constitutive and inducible CYP3A expression (nuclear hormone receptors). Thus, the complex regulatory pathways, environmentally susceptible milieu of the CYP3A enzymes, and as yet undetermined genetic haplotypes, may confound evaluation of the effect of individual CYP3A genetic variations on drug disposition, efficacy and safety.
Mechanisms of CYP3A induction by glucocorticoids in human fetal liver cells
2012, Drug Metabolism and PharmacokineticsHuman fetal liver (HFL) cells express major drug metabolic enzymes CYP3A4, CYP3A5 and CYP3A7. In the fetal hepatocytes, betamethasone and dexamethasone (DEX) markedly enhanced the expression levels of CYP3A4 and CYP3A7 mRNAs and slightly increased the expression level of CYP3A5 mRNA. Interestingly, a high correlation between the CYP3A induction ability and the intensity of antiinflammatory effect was observed. Human glucocorticoid receptor (GR)–small interfering RNA clearly attenuated the expression level of GR mRNA, and diminished the DEX-stimulated CYP3A4, CYP3A5 and CYP3A7 expression in HFL cells. These findings indicate that GR mediates the induction of CYP3A4 and CYP3A7 expression in human fetal hepatocytes as well as the CYP3A5.
Cytoprotective properties of α-tocopherol are related to gene regulation in cultured d-galactosamine-treated human hepatocytes
2007, Free Radical Biology and MedicineVitamin E (α-tocopherol) has demonstrated antioxidant activity and gene-regulatory properties. d-Galactosamine (D-GalN)-induced cell death is mediated by nitric oxide in hepatocytes, and it is associated with hepatic steatosis. The beneficial properties of α-tocopherol and their relation to oxidative stress and gene regulation were assessed in D-GalN-induced cell death. Hepatocytes were isolated from human liver resections by a collagenase perfusion technique. α-Tocopherol (50 μM) was administered at the advanced stages (10 h) of D-GalN-induced cell death in cultured hepatocytes. Cell death, oxidative stress, α-tocopherol metabolism, and NF-κB-, pregnane X receptor (PXR)-, and peroxisome proliferator-activated receptor (PPAR-α)-associated gene regulation were estimated in the hepatocytes. D-GalN increased cell death and α-tocopherol metabolism. α-Tocopherol exerted a moderate beneficial effect against apoptosis and necrosis induced by D-GalN. Induction (rifampicin) or inhibition (ketoconazole) of α-tocopherol metabolism and overexpression of PXR showed that the increase in PXR-related CYP3A4 expression caused by α-tocopherol enhanced cell death in hepatocytes. Nevertheless, the reduction in NF-κB activation and inducible nitric oxide synthase expression and the enhancement of PPAR-α and carnitine palmitoyl transferase gene expression by α-tocopherol may be relevant for cell survival. In conclusion, the cytoprotective properties of α-tocopherol are mostly related to gene regulation rather than to antioxidant activity in toxin-induced cell death in hepatocytes.
The human cytochrome P450 sub-family: Transcriptional regulation, inter-individual variation and interaction networks
2007, Biochimica et Biophysica Acta - General SubjectsThe Cytochrome P450 super-family is a fundamental requirement for the viability of most life, with Cytochrome P450 proteins having been identified in organisms ranging from bacteria to man. These enzymes may be subdivided into those that metabolise purely endogenous chemicals, and those that are involved in xenobiotic metabolism. Of the latter group it can be argued that CYP3A sub-family members rank as the most important; their high expression in the liver and wide substrate specificity mean that they are clinically important in the metabolism of many therapeutic drugs, and alteration in their activity is central to many clinically-relevant drug–drug interactions. In this review I will examine the human CYP3A enzymes, discussing their genome structure, common allelic variants and, in greatest detail, their transcriptional regulation. Through examination of these characteristics we will see both striking similarities and differences between the four human CYP3A enzymes, which may have important impacts on inter-individual response to chemical exposure. Finally, the role of nuclear receptors in regulating CYP3A gene expression, and indeed that of many other proteins involved in drug metabolism, will be examined: Such an examination will show the need to utilize a systems biology approach to understand fully how the human body responds to chemical exposure.
Up-regulation of the alligator CYP3A77 gene by toxaphene and dexamethasone and its short term effect on plasma testosterone concentrations
2006, Aquatic ToxicologyIn this study we describe an alligator hepatic CYP3A gene, CYP3A77, which is inducible by dexamethasone and toxaphene. CYP3A plays a broad role in biotransforming both exogenous compounds and endogenous hormones such as testosterone and estradiol. Alligators collected from sites in Florida that are contaminated with organochlorine compounds exhibit differences in sex steroid concentrations. Many organochlorine compounds induce CYP3A expression in other vertebrates; hence, CYP3A induction by organochlorine contaminants could increase biotransformation and clearance of sex steroids by CYP3A and provide a plausible mechanism for the lowering of endogenous sex steroid concentrations in alligator plasma. We used real time PCR to examine whether known and suspected CYP3A inducers (dexamethasone, metyrapone, rifampicin, and toxaphene) up-regulate steady state levels of hepatic CYP3A77 transcript to determine if induction patterns in female juvenile alligators are similar to those reported in other vertebrates and whether toxaphene, an organochlorine compound found in high concentrations in Lake Apopka alligators, induces this gene. Estrogen receptor α (ERα), estrogen receptor β (ERβ), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), and steroid-xenobiotic receptor (SXR) transcripts were also measured to determine whether any of these nuclear receptors are also regulated by these compounds in alligators.
Dexamethasone (4.2-fold) and toxaphene (3.5-fold) significantly induced CYP3A77 gene transcript, whereas rifampicin (2.8-fold) and metyrapone (2.1-fold) up-regulated ERβ after 24 h. None of the compounds significantly up-regulated AR, ERα, GR, PR, or SXR over this time period. Plasma testosterone (T) did not change significantly after 24 h in alligators from any of the treatment groups. Dexamethasone treated animals exhibited a strong relationship between the 24 h plasma T concentrations and CYP3A77 (R2 = 0.9, positive) and SXR (R2 = 0.77, negative) transcripts, which suggests that the expression of these genes is related to plasma T in alligators.
In light of our findings, we hypothesized that higher steady state CYP3A77 (and possibly SXR) gene expression would be observed in alligators collected from Lake Apopka, a polluted lake containing organochlorine compounds known to induce CYP3A isoforms in other taxa. Therefore, we measured basal levels of CYP3A77 and SXR gene transcripts in wild juvenile alligators collected from Orange Lake (reference lake), Lake Woodruff (reference lake), and Lake Apopka (contaminated lake). We found that no differences existed in CYP3A77 or SXR gene expression among animals from the lakes sampled suggesting that exposure to organochlorine compounds at concentrations present in Lake Apopka does not lead to variation in the expression of these genes, although capture stress could be interfering with these results since the glucocorticoid dexamethasone induces CYP3A77 transcript in alligators.
The induction of cytochrome P450 3A5 (CYP3A5) in the human liver and intestine is mediated by the xenobiotic sensors pregnane X receptor (PXR) and constitutively activated receptor (CAR)
2004, Journal of Biological ChemistryCitation Excerpt :Previous analyses of the inducibility of hepatic CYP3A5 produced conflicting results (11–14). Thus, the original analyses of the presumptive CYP3A5 promoter (15, 16) were invalid (10, 17) because of confusion between the real CYP3A5 5′-region and an almost identical pseudogene sequence localized ∼20 kb upstream of the CYP3A5 gene. A more recent analysis of the correct gene failed to detect rifampin-induced transcription (18).
Induction of cytochrome P450 3A (CYP3A) by xenobiotics may lead to clinically relevant drug interactions. In contrast with other CYP3A family members, studies on the inducibility of CYP3A5 indicate conflicting results. We report the induction of CYP3A5 mRNA in 13 of 16 hepatocyte preparations exposed to rifampin. Furthermore, induction of CYP3A5 mRNA was observed in intestinal biopsies in three of eight probands following exposure to the antibiotic. The highest absolute levels of CYP3A5 transcripts were found following rifampin treatment in hepatocytes and intestines from carriers of CYP3A5*1 alleles. Elucidation of the mechanism involved in CYP3A5 induction revealed that constitutively activated receptor (CAR) and pregnane X receptor (PXR) transactivated the CYP3A5 promoter (–688 to +49) and that the transactivation was dependent on an everted repeat separated by 6 bp (ER6-dependent). Treatment with the prototypical PXR ligand rifampin led to a 2-fold induction of the CYP3A5 promoter activity. In agreement with these observations, PXR and CAR bound specifically to the ER6 motif. Hepatic expression of PXR correlated with that of CYP3A5 mRNA levels in a bank of liver samples. Taken together, studies here revealed the presence of a functional ER6 motif in the CYP3A5 promoter located –100 bp upstream from the transcription start site, suggesting that CYP3A5 is inducible by mechanisms similar to those involved in CYP3A4 induction. Enhanced expression of CYP3A5 caused by exposure to inducers may phenocopy the effects of the high expression allele CYP3A5*1. In this manner, induction of CYP3A5 may contribute to the overall importance of this P450 in drug metabolism and drug interactions.