Coordinated induction of drug transporters and phase I and II metabolism in human liver slices

Although regulation of phase I drug metabolism in human liver is relatively well studied, the regulation of phase II enzymes and of drug transporters is incompletely characterized. Therefore, we used human liver slices to investigate the PXR, CAR and AhR-mediated induction of drug transporters and phase I and II metabolic enzymes. Precision-cut human liver slices were incubated for 5 or 24 h with prototypical inducers: phenobarbital (PB) (50 μM) for CAR, β-naphthoflavone (BNF) (25 μM) for AhR, and rifampicin (RIF) (10 μM) for PXR, and gene expression of the phase I enzymes CYP1A1, 1A2, 3A4, 3A5, 2B6, 2A6, the phase II enzymes UGT1A1 and 1A6, and the transporters MRP2, MDR1, BSEP, NTCP and OATP8 was measured. BNF induced CYP1A1, UGT1A1 and UGT1A6 and MRP2, NTCP and MDR1. RIF induced CYP3A4, 3A5, 2B6, 2A6, UGT1A1, UGT1A6 and BSEP, MRP2 and MDR1 and slightly downregulated OATP8. PB induced CYP3A4, 3A5, 2B6 and 2A6, UGT1A1 and all transporters.

Large interindividual differences were found with respect to the level of induction.

Enzyme activity of CYP3A4, measured by testosterone metabolism, was increased after 24 h by RIF. 7-Ethoxycoumarin O-deethylation activity, mediated predominantly by CYP 1A1/1A2 but also by other CYPs, was increased after 24 h with PB.

We have shown that regulation of all phases of the (in)activation of a drug via the CAR, AhR and the PXR pathways can be studied in human liver slices. The concomitant induction of metabolic enzymes and transporters shows that also in the human liver transporters and metabolic enzymes are regulated coordinately.     

Effect of aflatoxin B1 on nuclear receptors PXR, CAR, and AhR and their target cytochromes P450 mRNA expression in primary cultures of human hepatocytes

Aflatoxin B1 (AFB1), one of the most common mycotoxins found in human foods and animal feed, is principally hepatotoxic and hepatocarcinogenic. The aim of the present study was to explore the effect of AFB1 on messenger RNA (mRNA) expression of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) and some of their target cytochromes using primary cultures of human hepatocytes. Our results showed that AFB1, at noncytotoxic increasing concentrations, caused a significant upregulation of cytochrome P 2B6 (CYP2B6), CYP3A5, and to a lesser extent CYP3A4 and CYP2C9. Pregnane X receptor and CAR mRNA expression increased in the 3 treated livers. Aflatoxin B1 was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that AFB1 could activate PXR, CAR, and AhR; however, further investigations are needed to confirm nuclear receptor activation by AFB1.     

中草药对孕烷X受体和组成型雄烷受体等核受体通路影响的研究进展

孕烷X受体（pregnane X receptor,PXR）是核受体亚家族的成员之一,参与大量的外源性和内源性化学物质的生物转化,能被多种中草药激活,调节下游靶基因的表达,在药物代谢酶和转运体的调节中起重要作用。组成型雄烷受体（constitutive androstane receptor,CAR）和PXR一样能与外源性配体结合调节CYP2B6、CYP3A4、CYP2C19、UGT1A1的表达,共同参与CYP药物代谢酶的调节,成为药物作用的靶标。     

Impact of obese levels on hepatic expression of drug-metabolizing enzymes in adult and offspring mice

OBJECTIVE To investigate the effect of obese levels on the expression of drug-metabolizing enzymes(DMEs) in the liver of adult and offspring mice.METODS C57 BL/6 mice(4-5 weeks) were maintained on either a high-fat diet(HFD, 60 kcal% from fat) or low-fat diet(LFD, 10 kcal% from fat). After 4, 8, or 18 weeks of diet treatment, some mice were sacrificed and liver tissues were collected to study the impact of obese levels on the expression of DMEs in adult mice. The remaining mice were mated and offspring of these mice were sacrificed at different ages(day 5-60 after birth) to assess the consequence of parental obese levels on the ontogenic expression of DMEs in the liver of offspring mice. The m RNA expression of nine DMEs(CYP1 A1, 1 A2, 2 B10, 2 C29,2 E1, 3 A11, 3 A16, UGT1 A1, and SULT1 A1) was determined by RT-q PCR. RESULTS Compared with the LFD groups, the m RNA expression of CYP1 A1 was induced in males fed with a HFD for 18 weeks(18-HD) and in females fed with a HFD for 4 or 8 weeks(4-HD and8-HD, respectively). For CYP1 A2 and 2 E1, the m RNA expression was only elevated in the male 4-HD group and in the female 8-HD group. However, consumption with a HFD only induced the expression of CYP2 B10 in males but not in females. Higher m RNA expression of UGT1 A1 was seen in the male 8-HD and 18-HD groups as well as in the female 8-HD group. Concomitantly, the expression of nuclear receptors, including constitutive androstane receptor(CAR), pregnane X-receptor(PXR),hepatocyte nuclear factor(HNF4α), and peroxisome proliferator-activated receptor α(PPARα), aryl hydrocarbon receptor(Ah R) was found to be increased by a HFD in adult mice. For the offspring, family based HFD consumption tended to induce the expression of most DMEs(eg,CYP1 A1, 1 A2, 2 E1, and UGT1 A1) in the offspring derived from 8-HD groups(O-8-HD mice). Altered ontogeny of CYP1 A2 and 2 E1 by HFD was also observed, which changed from an adolescent-enriched pattern to an adultenriched pattern. Whereas, decreased expression of CYP2 C29, 3 A11, and SULT1 A1 was seen in the offspring derived from 4-HD mice(O-4-HD mice). The expression of CYP1 A1, 1 A2, 2 E1, and UGT1 A1 was also lower in liver of the O-4-HD mice compared with the related LFD mice.Consistent with the altered expression of DMEs in offspring mice, the hepatic expression of CAR, PXR, HNF4α,PPARα, and Ah R was increased in O-8-HD mice whereas decreased in O-4-HD mice compared with the related LFD group. CONCLUSION Obese levels affect the hepatic expression of DMEs in adult mice and the ontogenic expression of DMEs in offspring mice which nuclear receptors may be an underlying mechanism.     

Zearalenone activates pregnane X receptor, constitutive androstane receptor and aryl hydrocarbon receptor and corresponding phase I target genes mRNA in primary cultures of human hepatocytes

The mycotoxin zearalenone (ZEN) is found worldwide as a contaminant in cereals and grains. ZEN subchronic and chronic toxicities are dominated by reproductive disorders in different mammalian species which have made ZEN established mammalian endocrine disrupter. Over the last 30 years of ZEN biotransformation study, the toxin was thought to undergo reductive metabolism only, with the generation in several species of α- and β-isomers of zearalenol. However, recent investigations have noticed that the mycoestrogen is prone to oxidative metabolism leading to hydroxylation of ZEN though the involvement of different cytochromes P450 (CYPs) isoforms. The aim of the present study was to further explore the effect of ZEN on regulation of some CYPs using primary cultures of human hepatocytes. For this aim, using real time RT-PCR, we monitored in a first time, the effect of ZEN on mRNA levels of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR), nuclear receptors known to be involved in the regulation of some CYPs. In a second time, we looked for ZEN effect on expression of PXR, CAR and AhR corresponding phase I target genes (CYP3A4, CYP3A5, CYP2B6, CYP2C9, CYP1A1 and CYP1A2). Finally, we realised the luciferase assay in HepG2 treated with the toxin and transiently transfected with p-CYP3A4-Luc in the presence of a hPXR vector or transfected with p-CYPA1-Luc.Our results clearly showed that ZEN activated human PXR, CAR and AhR mRNA levels in addition to some of their phase I target genes mainly CYP3A4, CYP2B6 and CYP1A1 and at lesser extent CYP3A5 and CYP2C9 at ZEN concentrations as low as 0.1 μM.     

Pyrene-induced CYP1A2 and SULT1A1 may be regulated by CAR and not by AhR

Aryl hydrocarbon receptor (AhR) plays important roles in the regulation and induction of xenobiotic-metabolizing enzymes including the cytochromes P450 1 family (CYP1) and UDP-glucuronosyltransferases 1A (UGT1As) by polycyclic aromatic hydrocarbons as well as chlorinated aromatic hydrocarbons. To determine whether pyrene-induced xenobiotic-metabolizing enzymes are regulated by AhR, male AhR (+/+) and (-/-) mice were used. Both genotyped mice were exposed to 0, 205, 300 or 410 mg/(kgday pyrene), once daily, for four consecutive days by gavage. Exposure to pyrene did not influence hepatic CYP1A1-mRNA in mice of both genotypes, whereas it induced hepatic CYP1A2 protein and mRNA expression and associated 7-ethoxyresorufin O-deethylase and pyrene 1-hydroxylation activities in both AhR (+/+) and (-/-) mice. Similar effects were also found with sulfotransferase 1A1 expression and the associated 1-hydroxypyrene sulfation activity. In contrast, pyrene exposure increased expression of the UGT1A1 and 1A6, and glucuronidation activities associated with 1-hydroxypyrene and 1-naphthol in the liver only in AhR (-/-) mice, although pyrene treatment dose-dependently decreased the latter activity. Pyrene exposure did not increase AhR-mRNA expression in AhR (+/+) mice. In contrast, pyrene-induced expression of the hepatic constitutive androstane receptor (CAR) and one of its target genes, CYP2B10, in both AhR (+/+) and (-/-) mice. These results strongly suggest that pyrene-induced CYP1A2 and SULT1A1 are regulated by CAR, not by AhR. However, the mechanisms of UGT1A1 and 1A6 induction by pyrene were not elucidated in this study.     

Regulation of Pregnane X Receptor (PXR) Function and UGT1A1 Gene Expression by Posttranslational Modification of PXR Protein

Human UDP-glucuronosyltransferase (UGT) 1A1 is a critical enzyme responsible for detoxification and metabolism of endogenous and exogenous lipophilic compounds such as bilirubin. The present study shows how cyclin-dependent kinase (CDK) inhibitor roscovitine stimulated the expression of UGT1A1 in HepG2 cells. Pregnane X receptor (PXR)-mediated transactivation of UGT1A1 reporter gene was more prominently enhanced by roscovitine, compared with the basal-, constitutive androstane receptor (CAR)-, and aryl hydrocarbon receptor-mediated activities. We determined the regulatory mechanism of UGT1A1 expression through PXR's stimulation by roscovitine. Although phosphomimetic mutations at Thr290 and Thr408 retained the PXR protein in cytoplasm and attenuated the induction of UGT1A1 expression by both roscovitine and rifampicin, a mutation at Ser350 specifically reduced the activity of PXR induced by roscovitine. Immunoprecipitation analysis revealed that the T290D but not T408D mutant protein remained in cytoplasm by forming a complex with heat shock protein 90 and cytoplasmic CAR retention protein, whereas treatment with proteasome inhibitor MG-132 accumulated the T408D mutant protein in cytoplasm. Transfection with anti-CDK2 small interfering RNA (siRNA) but not anti-CDK1 or CDK5 siRNA led to enhanced expression of UGT1A1. S350D yellow fluorescent protein-PXR fusion protein could translocate from cytoplasm to nucleus similar to the wild-type protein but was detected as an acetylated protein, whose binding with retinoid X receptor (RXR) and histone deacetylase was impaired. Cotransfection with coactivator steroid receptor coactivator (SRC) 2 but not SRC-1 partly recovered its PXR activity. These results indicate that roscovitine stimulated the expression of UGT1A1 by inhibiting CDK2, which phosphorylated PXR at Ser350 to suppress binding with RXR and coactivator and maintain the acetylation of PXR protein.