外源化合物对孕烷X受体和组成型雄烷受体介导的细胞色素P4503A4和2B6转录调节共转染体系的优化

目的优化孕烷X受体(hPXR)和组成型雄烷受体(hCAR)介导的细胞色素P450(CPY)3A4和CYP2B6诱导共转染体系,提高检测系统的灵敏度。方法利用invitrogen脂质体2000共同转染表达质粒hPXR/hCAR、报告基因质粒CPY3A4/CYP2B6和内参质粒pRL-TK到HepG-2细胞中。系统以hPXR的激动剂利福平,hCAR的激动剂CITCO为阳性对照组,以二甲基亚砜(DMSO)为溶剂阴性对照组。通过调整3种质粒的转染比例,以利福平/DMSO和CITCO/DMSO的比活值,即阳性药物的诱导倍数作为优化系统灵敏度的指标,分别获得最大比值以表示系统具有最佳灵敏度。结果当共转染体系比例为hPXR/hCAR表达质粒150ng、CPY3A4/CYP2B6报告基因质粒600ng、PLR-TK内参质50ng时,转染体系的检测灵敏度最高。结论针对所使用的转染细胞系和共转染质粒,通过优化质粒的转染比例可提高系统的灵敏度,优化的共转染系统可用于药物代谢酶诱导机制的研究。     

PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides

OCP are xenobiotics which display various toxic effects on animal and human health. One of their effects is to bind and activate estrogen receptor alpha (ERalpha). We have previously studied the down-regulation of induced CYP1A1 (cytochrome P450) expression by this class of molecules in mammary carcinoma cells and shown the importance of ERalpha in this process. However, an alternative mechanism was suggested by those experiments in hepatoma cells. In this study, we have performed Northern blot and transient transfection assays in various cell lines and shown that OCP activate human pregnane X receptor (PXR) and subsequent CYP3A4 mRNA expression. This effect is mediated by the distal xenobiotic responsive element modulator of the promoter. The induction of CYP3A4 by OCP was dose-dependent within the 1-10 microM range. The data suggest that chronic exposure to OCP could alter a major metabolite pathway in human liver and putatively modify the pharmacokinetics of drugs and pollutants.     

姜黄素对酒精性肝损伤大鼠CYP3A的影响及机制探讨

目的 探究姜黄素对酒精性肝损伤（ALD）大鼠细胞色素P450 3A（CYP3A）的影响及其机制。方法 60只 建模成功的ALD大鼠按随机数字表法分为模型组,姜黄素低、中、高剂量组（分别灌胃40、80、160 mg/kg姜黄素）及阳 性对照组（腹腔注射200 mg/kg腺苷蛋氨酸）,每组12只;对照组12只正常饲养,灌胃等体积生理盐水,连续6周。全 自动生化分析仪检测血清中丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和碱性磷酸酶（ALP）水平;苏木精-伊红 （HE）染色观察肝脏形态变化;荧光定量聚合酶链反应（qPCR）检测肝脏组织中孕烷X受体（PXR）、组成型雄甾烷受 体（CAR）、CYP3A25 mRNA水平;Western blot检测肝脏组织中PXR、CAR蛋白水平。以大鼠原代肝细胞为研究对象, 分别用0、100、200、300、400、500 mmol/L乙醇培养细胞,取细胞增殖抑制率约为50%时的乙醇浓度进行下一步实验; 0、0.5、1.0、2.0、4.0、8.0、16.0 μmol/L姜黄素培养细胞,取最适姜黄素浓度进行后续研究。实验分为对照组、乙醇组、姜 黄素组、姜黄素+siRNA-NC 组及姜黄素+siRNA-CYP3A25 组。CCK-8 检测细胞增殖情况;qPCR 检测细胞中 CYP3A25 mRNA水平;Western blot检测细胞中PXR、CAR蛋白水平。结果 动物实验：与对照组相比,模型组血清中 ALT、AST、ALP水平升高（P＜0.05）;与模型组相比,姜黄素低剂量组血清中ALT、ALP水平降低,肝脏组织中PXR、 CAR mRNA和蛋白,CYP3A25 mRNA水平升高（P＜0.05）,姜黄素中、高剂量组血清中ALT、AST、ALP水平降低,肝脏 组织中PXR、CAR mRNA和蛋白,CYP3A25 mRNA水平升高（P＜0.05）;随着剂量升高,各指标逐渐恢复。细胞实验： 与对照组相比,乙醇组细胞增殖抑制率升高（P＜0.05）,细胞中PXR、CAR蛋白水平降低（P＜0.05）;与乙醇组相比,姜 黄素组细胞增殖抑制率降低（P＜0.05）,细胞中CYP3A25 mRNA,PXR、CAR蛋白水平升高（P＜0.05）;与姜黄素组相 比,姜黄素+siRNA-CYP3A25 组细胞增殖抑制率升高（P＜0.05）,细胞中 CYP3A25 mRNA,PXR、CAR 蛋白水平降低 （P＜0.05）。结论 姜黄素可上调 CYP3A25 水平,促进药物代谢,实现对 ALD 的缓解,这一过程可能与升高 PXR、 CAR表达有关。     

Application and interpretation of hPXR screening data: Validation of reporter signal requirements for prediction of clinically relevant CYP3A4 inducers

A human pregnane X receptor (PXR) reporter-gene assay was established and validated using 19 therapeutic agents known to be clinical CYP3A4 inducers, 5 clinical non-inducers, and 6 known inducers in human hepatocytes. The extent of CYP3A4 induction (measured as RIF ratio in comparison to rifampicin) and EC50 was obtained from the dose-response curve. All of the clinical inducers (19/19) and human hepatocyte inducers (6/6) showed positive responses in the PXR assay. One out of five clinical non-inducers, pioglitazone, also showed a positive response. An additional series of 18 commonly used drugs with no reports of clinical induction was also evaluated as putative negative controls. Sixteen of these were negative (89%), whereas two of these, flutamide and haloperidol showed 16-fold (RIF ratio 0.79) and 10-fold (RIF ratio 0.48) maximal induction, respectively in the reporter-gene system. Flutamide and haloperidol were further demonstrated to cause CYP3A4 induction in human cryopreserved hepatocytes based on testosterone 6beta-hydroxylation activity. The induction potential index calculated based on the maximum RIF ratio, EC50, and in vivo maximum plasma concentration was used to predict the likelihood of CYP3A4 induction in humans. When the induction potential index is greater than 0.08, the compound is likely to cause induction in humans. A high-throughput screening strategy was developed based on the validation results at 1microM and 10microM for the same set of drugs. A RIF ratio of 0.4 was set as more practical screening cut-off to minimize the possibility of generating false positives. Thus, a tiered approach was implemented to use the human PXR reporter-gene assay from early lead optimization to late lead characterization in drug discovery.     

Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene

CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene.In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRα heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4.Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRα heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells.We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.     

Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR^−/− mice, but not in hypothyroid PXR^−/− mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR^−/− hypothyroid mice, and this induction was abolished in CAR^−/− mice and in and CAR^−/− PXR^−/− double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR^−/− mice and lowest in WT and PXR^−/− mice. Hypothyroid WT or PXR^−/− mice survived chronic CBZ treatment, but all hypothyroid CAR^−/− and CAR^−/− PXR^−/− mice died, with CAR^−/−PXR^−/− mice surviving longer than CAR^−/− mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge.     

The CYP2B2 5' flank contains a complex glucocorticoid response unit

Rat CYP2B1 and CYP2B2 and mouse CYP2B10 are dramatically induced by phenobarbital (PB) in liver. PB responsiveness requires the constitutive androstane receptor (CAR). However, dexamethasone treatment can also induce CYP2B genes in both rat and mouse liver. Three regions have been shown to be involved in conferring dexamethasone responsiveness on CYP2B2 reporter constructs. They are the PB response unit, a functional glucocorticoid response element at −1.3 kb in the 5′ flank and a weak element in the basal promoter. We report here the identification, by deletion analysis of the CYP2B2 5′ flank, of new glucocorticoid response elements or accessory factor sites. Moreover, we show that CAR acts as an accessory factor in the dexamethasone response in vivo of CYP2B10 protein in mice, by increasing both the basal and induced levels. We propose a model to explain the dexamethasone responsiveness of the CYP2B2 gene in which induction is mediated by a complex glucocorticoid response unit.     

Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C

Pregnane X receptor (PXR, NR1I2) regulates the inducible expression of the 3A sub-family of cytochrome P450 genes (CYP3A). CYP3A enzymes are responsible for the oxidative metabolism of a wide array of endobiotic and xenobiotic compounds. Hepatic CYP3A gene expression is rapidly down-regulated during inflammation and sepsis. There are twelve protein kinase C (PKC) isoforms, classified into three subfamilies according to the structure of the N-terminal regulatory domain and their sensitivity to calcium and diacylglycerol. It is now well accepted that cytokine stimulation of hepatocytes increases intracellular PKC activity during inflammation and sepsis. We show here that protein kinase C alpha (PKCα) and phorbol ester-dependent PKC signaling dramatically repressed PXR activity in both, cell-based reporter gene assays and in hepatocytes. Moreover, treatment with the protein phosphatase PP1/PP2A inhibitor okadaic acid (OA) totally abolished PXR activity in reporter gene assays and in cultured hepatocytes. In mammalian two-hybrid assays, treatment with phorbol 12-myristate 13-acetate (PMA) increased the strength of interaction between PXR and the nuclear receptor co-repressor protein (NCoR). Treatment with PMA also abolished the ligand-dependent interaction between PXR and the steroid receptor co-activator 1 protein (SRC1). Our findings suggest that activation of the protein kinase C signaling pathway represses PXR activity through alterations in PXR-protein co-factor complexes, possibly through direct alterations in the phosphorylation status of one or all of these proteins. In addition, our data potentially provide important insights into the molecular mechanism of the repression of hepatic CYP3A gene expression that occurs during the inflammatory response.     

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies.