Expression and induction of CYP3As in human fetal hepatocytes

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.     

Regulation of cytochrome P450 2C9 expression in primary cultures of human hepatocytes

Cytochrome P450 2C9 (CYP2C9) expression is regulated by multiple nuclear receptors including the constitutive androstane receptor (CAR) and pregnane X receptor (PXR). We compared coregulation of CYP2C9 with CYP2B6 and CYP3A4, prototypical target genes for human CAR and PXR using human hepatocyte cultures treated for three days with the PXR activators clotrimazole, rifampin, and ritonavir; the CAR/PXR activator phenobarbital (PB); and the CAR‐selective agonists CITCO, (6‐(4‐chlorophenyl)imidazo[2,1‐β][1,3]thiazole‐5‐carbaldehyde‐O‐(3,4‐dichlorobenzyl)oxime) and phenytoin. Clotrimazole, rifampin, ritonavir, phenytoin, and phenobarbital induced CYP2C9 consistent with previous findings for CYP3A4. We observed EC₅₀ values of 519 μM (phenobarbital), 11 μM (phenytoin), and 0.75 μM (rifampin), similar to those for CYP3A4 induction. Avasimibe, a potent PXR activator, produced nearly identical concentration‐dependent CYP2C9 and CYP3A4 activity profiles and EC₅₀ values. In 17 donors, rifampin increased mean basal CYP2C9 activity from 59 ± 43 to 143 ± 68 pmol/mg protein/min; fold induction ranged from 1.4‐ to 6.4‐fold. Enzyme activity and mRNA measurements after rifampin, CITCO and PB treatment demonstrated potency and efficacy consistent with CYP2C9 regulation being analogous to CYP3A4 rather than CYP2B6. We demonstrate that hepatic CYP2C9 is differentially regulated by agonists of CAR and PXR, and despite sharing common regulatory mechanisms with CYP3A4 and CYP2B6; this enzyme exhibits an induction profile more closely aligned with that of CYP3A4. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:43–58, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20264     

Expression and distribution of CYP3A genes, CYP2B22, and MDR1, MRP1, MRP2, LRP efflux transporters in brain of control and rifampicin-treated pigs

The in vivo effect of rifampicin, a potent ligand of PXR, on gene expression of CYP2B22, 3A22, 3A29, 3A46, CAR, PXR and MDR1, MRP1, MRP2, LRP transporters in liver and cortex, cerebellum, midbrain, hippocampus, meninges and brain capillaries of pig was investigated. Animals were treated i.p. with four daily doses of rifampicin (40 mg/kg). The basal mRNA expressions of the individual CYP3As, CYP2B22, CAR, and PXR in various brain regions, except meninges, were about or below 10% of the corresponding hepatic mRNA values, whereas the mRNAs of brain transporters were closer or comparable to those in liver. After pig treatment with rifampicin, the mRNA expression of CYPs and transporters from brain regions did not appear to change, except CYP3A22 and 3A29 in cortex and hippocampus, CYP2B22 in meninges. An enzymatic analysis for CYP3As and CYP2B, in microsomes and mitochondria from liver and brain tissues using the marker activities 7-benzyloxyquinoline O-debenzylase and the anthraldehyde oxidase, showed the lack of rifampicin induction in all the brain regions, unlike liver. Taken together, our results demonstrate that CYP2B22, CYP3As, and MDR1, MRP1, MRP2, and LRP transporters are all expressed, although at different extent, in the brain regions but, despite the presence of PXR and CAR, are resistant to induction indicating that the regulation of these proteins is more complex in brain than in liver. These data obtained in vivo in the brain regions and liver of pig may be of interest to human metabolism in CNS.     

PXR-mediated transcriptional activation of CYP3A4 by cryptotanshinone and tanshinone IIA

Danshen (Radix Salvia miltiorrhiza) is a famous Traditional Chinese Medicine used widely for the treatment of coronary heart disease and cerebrovascular disease. Diterpenoid tanshinones including tanshinone I, tanshinone IIA and cryptotanshinone are the major bioactive components from Danshen herb. Previous reports have demonstrated that Danshen extracts could induce the expression of CYP3A in rodents, however, the constituents responsible for Danshen-mediated CYP3A induction and the underlying molecular mechanisms remain unknown. The discovery of a family of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR) and glucocorticoid receptor (GR) gives insight into the molecular explanation of CYP3A induction by xenobiotics. In the present study, interactions between Danshen constituents and human PXR were evaluated using a reporter gene assay. Our observations showed that Danshen ethanol extract could activate human PXR and induce the CYP3A4 reporter construct in HepG2 cells. Tanshinone IIA and cryptotanshinone were identified as efficacious PXR agonists, and cryptotanshinone activated the CYP3A4 promoter more strongly than tanshinone IIA. Furthermore, CAR and GR were also involved in the induction of CYP3A4 expression by tanshinones, though their roles seemed not as important as PXR. Treatment of LS174T cells with cryptotanshinone or tanshinone IIA resulted in a significant increase of CYP3A4 mRNA, which was consistent with the results from the reporter gene assay. Collectively, activation of PXR and the resultant CYP3A4 induction mediated by cryptotanshinone and tanshinone IIA provide a molecular mechanism for previously observed CYP3A induction by Danshen extracts, and our findings also suggest that caution should be taken when Danshen products are used in combination with therapeutic drugs metabolized by CYP3A4.     

Collaborated regulation of female-specific murine Cyp3a41 gene expression by growth and glucocorticoid hormones

CYP3A41 is a female-specific major CYP3A in mouse livers. Adrenalectomy decreased expression of CYP3A41 as well as CYP3A11, another major CYP3A, and dexamethasone (DEX) restored the decreased expression. Hypophysectomy completely abolished CYP3A41 expression and growth hormone (GH) replacement only slightly restored the expression. Treatment with DEX alone did not induce expression of either CYP3A41 or CYP3A11 in hypophysectomized mice. However, combined treatment with GH and DEX strongly induced expression of CYP3A41 but not CYP3A11. In primary cultured mouse hepatocytes, DEX induced expression of both CYP3A41 and CYP3A11, and DEX-inducible expression of CYP3A41 was suppressed by RU486, a potent antiglucocorticoid. In contrast, RU486 by itself enhanced basal expression of CYP3A11 mRNA, while it showed no inhibitory effect on DEX-inducible expression. These observations indicate that glucocorticoids may participate in the GH-dependent control of the Cyp3a41 gene expression, probably mediated via the glucocorticoid receptor, which may be different from that of the Cyp3a11 gene expression.     

Regulation of the expression of two female-predominant CYP3A mRNAs (CYP3A41 and CYP3A44) in mouse liver by sex and growth hormones

A second female-predominant murine CYP3A, CYP3A44, was isolated from liver and its mRNA expression was compared with that of the previously described CYP3A41. The expression of CYP3A44 was relatively constant after birth in females, whereas it gradually declined in males after 5 weeks of age. The expression of CYP3A41 increased with age in females after 3 weeks of age, whereas it gradually declined in males after 5 weeks of age. Hypophysectomy and growth hormone replacement indicated that expression of both CYP3A mRNAs in females was dependent on the feminine plasma growth hormone profile. Estradiol induced the expression of both mRNAs and the effect was dependent on the presence of the pituitary gland. These observations suggest that endocrine control of expression might be similar, but not identical, for two female-predominant CYP3A mRNAs.     

Functionally conserved xenobiotic responsive enhancer in cytochrome P450 3A7

Nuclear receptors CAR and PXR play a key role in cytochrome P450 gene induction by xenobiotics. Human cytochrome P450 3A7 (CYP3A7) is expressed from early in gestation until the perinatal period, when there is a switch in expression to CYP3A4. Here we demonstrate that a PXR and CAR responsive enhancer is located approximately 8 kb upstream of the proximal CYP3A7 promoter. This distal xenobiotic responsive enhancer module (XREM) is conserved with the XREM of CYP3A4. Interestingly, not only the XREM, but also the entire promoters exhibit 90% sequence identity up to -8.8 kb, indicating a close evolutionary distance. We propose that the promoters have coevolved to functionally conserve P450 gene induction in response to xenobiotics through CAR and PXR. Thus, nuclear receptors for xenobiotics may not only play a role to provide a survival advantage during adulthood, but also to protect the embryo against endogenous and exogenous toxins.     

Peppermint (Mentha piperita L.) extract effectively inhibits cytochrome P450 3A4 (CYP3A4) mRNA induction in rifampicin-treated HepG2 cells

Interaction between foods and drugs is an important consideration in pharmaceutical therapy. Therefore, here, we examined the suppressive effects of the extracts from seven edible herbs on the induction of CYP3A4 gene expression in rifampicin-treated HepG2 cells. We evaluated the structure and suppressive activity of the most effective active compound isolated from dried peppermint (Mentha piperita L.). The structure of the compound was identified as that of pheophorbide a based on spectroscopic data. It suppressed the induction of CYP3A4 mRNA expression by rifampicin in a dose-dependent manner. Quantitative high-performance liquid chromatography showed that 2 g of dry leaves 0.43 mg in one cup of peppermint tea. These findings demonstrate that pheophorbide a suppresses the induction of CYP3A4 mRNA expression in rifampicin-treated HepG2 cells. Pheophorbide is known to cause photosensitivity. However, the effective dose of pheophorbide a that had a suppressive effect was very low, indicating a high safety margin.

Abbreviations: DAD: diode array detector; DMEM: Dulbecco’s modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; HPLC: high-performance liquid chromatography; PCR: polymerase chain reaction; PXR: pregnane X receptor; CAR: constitutive androstane receptor; AHR: aryl hydrocarbon receptor; TLC: thin-layer chromatography     

Cholesterol 25-hydroxylation activity of CYP3A

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4β-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4β-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.     

Amelioration of PXR-mediated CYP3A4 induction by mGluR2 modulators

This work describes the rational amelioration of Cytochrome P450 4/5 (CYP3A4/5) induction through the Pregnane-X Receptor (PXR) pathway in a series of compounds that modulate the metabotropic glutamate Receptor 2 (mGluR2) via an allosteric mechanism. The compounds were initially shown to induce CYP3A4/5 via the gold-standard induction assay measured in primary human hepatocytes. This was followed up by testing the compounds in a PXR assay which correlated well with the assay in primary cells. Further, one of the compounds was crystallized with PXR (pdb code 6DUP). Analysis of this co-crystal structure, together with previously published PXR co-crystal structures, lead to modification ideas. The compounds synthesized based on these ideas were shown not to be CYP3A4/5 inducers. The mGluR2 activity of the resulting compounds was maintained.     

Mechanism of arsenite-mediated decreases in CYP3A23 in rat hepatocytes

In primary cultures of rat hepatocytes, exposure to arsenite causes a major decrease in dexamethasone (DEX)-mediated induction of CYP3A23 hemoprotein, with a minor decrease in CYP3A23 mRNA. Here we show that addition of heme did not prevent the arsenite-mediated decreases in CYP3A23 protein, and arsenite did not decrease intracellular glutathione levels, indicating that heme and glutathione were not limiting for formation of holoCYP3A23. We also investigated whether arsenite decreases CYP3A23 protein by increasing CYP3A23 degradation by the calpain pathway. The calpain inhibitor, calpeptin, caused greater than a 90% inhibition of calpain-mediated proteolysis, but had no effect on DEX-mediated induction of CYP3A23 protein following 24h treatments. However, calpeptin enhanced the effect of arsenite to decrease induction of CYP3A23 protein. In addition, in short-term studies, calpeptin appeared to be a suicidal inhibitor of CYP3A-catalyzed enzyme activity. Our findings suggest that CYP3A23 protein is not degraded by calpain-mediated proteolysis, even in the presence of arsenite.     

Structure–activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers,part I: Core region

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。