Tocotrienols activate the steroid and xenobiotic receptor, SXR, and selectively regulate expression of its target genes.

Vitamin E is an essential nutrient with antioxidant activity. Vitamin E is comprised of eight members, alpha-, beta-, gamma-, and delta-tocopherols and alpha-, beta-, gamma-, and delta-tocotrienols. All forms of vitamin E are initially metabolized by omega-oxidation, which is catalyzed by cytochrome P450 enzymes. The steroid and xenobiotic receptor (SXR) is a nuclear receptor that regulates drug clearance in the liver and intestine via induction of genes involved in drug and xenobiotic metabolism. We show here that all four tocotrienols specifically bind to and activate SXR, whereas tocopherols neither bind nor activate. Surprisingly, tocotrienols show tissue-specific induction of SXR target genes, particularly CYP3A4. Tocotrienols up-regulate expression of CYP3A4 but not UDP-glucuronosyltransferase 1A1 (UGT1A1) or multidrug resistance protein-1 (MDR1) in primary hepatocytes. In contrast, tocotrienols induce MDR1 and UGT1A1 but not CYP3A4 expression in intestinal LS180 cells. We found that nuclear receptor corepressor (NCoR) is expressed at relatively high levels in intestinal LS180 cells compared with primary hepatocytes. The unliganded SXR interacts with NCoR, and this interaction is only partially disrupted by tocotrienols. Expression of a dominant-negative NCoR enhanced the ability of tocotrienols to induce CYP3A4 in LS180 cells, suggesting that NCoR plays an important role in tissue-specific gene regulation by SXR. Our findings provide a molecular mechanism explaining how vitamin supplements affect the absorption and effectiveness of drugs. Knowledge of drug-nutrient interactions may help reduce the incidence of decreased drug efficacy.     

Characterization of human cytochrome P450 induction by pesticides

Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR.     

The pregnane X receptor binds to response elements in a genomic context-dependent manner, and PXR activator rifampicin selectively alters the binding among target genes.

The pregnane X receptor (PXR) is a key regulator of genes encoding several major types of cytochrome P450 enzymes and transporters (e.g., multidrug resistance-1, MDR1); therefore, PXR contributes significantly to drug-drug interactions. PXR binds to response elements and confers transactivation. Several target genes such as CYP3A4 and 3A7 contain two PXR elements (distant and proximal) that are separated by more than 7000 nucleotides in the genome. Disruption of the distant element causes a 73% decrease of the reporter activity, whereas inactivation of the proximal element decreases by only 53%. This study was undertaken to test the hypothesis that PXR differentially binds to the elements with the distant enhancer being bound to a higher extent. To test this hypothesis, a stable transfected line (hPXR-HRE) was prepared to constitutively express human PXR and harbor a chromatinized CYP3A4-ER6 reporter. This line responded to rifampicin and dexamethasone similarly as hepatocytes based on the relative potency and activation kinetics. Contrary to the hypothesis, chromatin immunoprecipitation experiments showed that the genomic fragment harboring the proximal element was preferably precipitated over the fragment containing the distant element in the CYP3A4 gene, but the opposite was true with the CYP3A7 gene. In addition, the promoters from the MDR1 and CYP2B6 genes were abundantly present in the PXR immunocomplexes from the vehicle-treated cells. However, such abundant interactions were markedly diminished when cells were treated with PXR activator rifampicin. These findings suggest that PXR binding is dependent on the genomic context and PXR activators modulate such bindings.     

孕烷X受体(PXR)对药物代谢途径的调控

目的 从孕烷X受体（PXR）对药物代谢途径的调控入手,在代谢性药物相互作用、PXR在CYP3A4调控中的作用及其调控机制等方面作分析和阐述。方法 结合近年来国内外相关文献进行评述。结果 PXR是CYP3A4的主要转录调控因子,药物通过PXR介导的信号通路调节CYP3A4的表达是影响药物体内代谢变化的重要途径。结论 就临床药物而言,由PXR介导的CYP3A4酶蛋白表达的改变可造成合用药物药效的减弱甚至丧失,因此必须引起广大临床药师的足够重视。     

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.     

3,4-methylenedioxymethamphetamine (MDMA) interacts with therapeutic drugs on CYP3A by inhibition of pregnane X receptor (PXR) activation and catalytic enzyme inhibition

Metabolism of MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) by the major hepatic drug-metabolizing enzyme cytochrome P450 3A (CYP3A), plays an important role in MDMA-induced liver toxicity. In the present study, we investigated interactions between MDMA and several therapeutic and recreational drugs on CYP3A and its regulator pregnane X receptor (PXR), using a human PXR-mediated CYP3A4-reporter gene assay, rat primary hepatocytes and microsomes. MDMA significantly inhibited hPXR-mediated CYP3A4-reporter gene expression induced by the human PXR activator rifampicin (IC₅₀ 1.26 ± 0.36 mM) or the therapeutic drugs paroxetine, fluoxetine, clozapine, diazepam and risperidone. All these drugs concentration-dependently inhibited CYP3A activity in rat liver microsomes, but in combination with MDMA this inhibition became more efficient for clozapine and risperidone. In rat primary hepatocytes that were pretreated with or without the rodent PXR activator pregnenolone 16alpha-carbonitrile (PCN), MDMA inhibited CYP3A catalytic activity with IC₅₀ values of 0.06 ± 0.12 and 0.09 ± 0.13 mM MDMA, respectively. This decrease appeared to be due to decreased activation of PXR and subsequent decreased CYP3A gene expression, and catalytic inhibition of CYP3A activity. These data suggest that in situations of repeated MDMA use in combination with other (therapeutic) drugs, adverse drug-drug interactions through interactions with PXR and/or CYP3A cannot be excluded.     

Endocrine disruptors induce cytochrome P450 by affecting transcriptional regulation via pregnane X receptor

Pregnane X receptor (PXR) is a nuclear receptor that regulates the expression of genes for cytochrome P450 3A (CYP3A), multidrug resistance 1 (MDR1), and organic anion-transporting peptide 2 (OATP2). These genes control the metabolism (CYP3A subfamily) and aspects of the pharmacokinetics (MDR1 and OATP2) of both endogenous and xenobiotic compounds. Since PXR is important in understanding the actions of endocrine disruptors (EDs), we determined the ability of suspected EDs to interact with PXR. In our study, 7 of 54 xenobiotics compounds interacted with PXR, including methoxychlor and benzophenone. All of the chemicals activated PXR in vitro and induced CYP3A mRNA in the male rat liver. In addition, CYP2C11 was also induced by some PXR agonists and converted methoxychlor into xenoestrogen. These findings suggest that some EDs affect sex hormone receptor indirectly by induction of metabolic enzyme via PXR, to produce rapidly higher concentrations of effective metabolites, leading to disturbance of the endocrine system.     

Effect of prototypical inducers on ligand activated nuclear receptor regulated drug disposition genes in rodent hepatic and intestinal cells

Aim:

The aim of this study was to investigate the impact on expression of mRNA and protein by paradigm inducers/activators of nuclear receptors and their target genes in rat hepatic and intestinal cells. Furthermore, assess marked inter laboratory conflicting reports regarding species and tissue differences in expression to gain further insight and rationalise previously observed species differences between rodent and human based systems.

Methods:

Quantitative real time-polymerase chain reaction (QRT-PCR) and immunoblots were used to assess messenger RNA (mRNA) and protein expression for CYP2B2, CYP3A1, CYP3A2, CYP3A9, ABCB1a, ABCB1b, ABCC1, ABCC2, pregnane X receptor (PXR), farnesoid X receptor (FXR) and constituitive androstane receptor (CAR) in rat hepatoma cell line H411E, intestinal cells, Iec-6, and rat primary hepatocytes, in response to exposure for 18 h with prototypical inducers.

Results:

Dexamethasone (DEX) and pregnenolone 16α carbonitrile (PCN) significantly induced PXR, CYP3A9, ABCB1a and ABCB1b. However, when co-incubated, DEX appeared to restrict PCN-dependent induction. Chenodeoxycholic acid (CDCA) was the only ligand to induce FXR in all three cell types. Despite previously reported species differences between PCN and rifampicin (RIF), both compounds exhibited a similar profile of induction.

Conclusion:

Data presented herein may explain some of the discrepancies previously reported with respect to species differences from different laboratories and have important implications for study design.     

Clofibrate and perfluorodecanoate both upregulate the expression of the pregnane X receptor but oppositely affect its ligand-dependent induction on cytochrome P450 3A23

The pregnane X receptor (PXR) interacts with a vast array of structurally dissimilar chemicals and confers induction of several major types of drug metabolizing enzymes such as cytochrome P450s (CYP). We previously reported that the expression of PXR was markedly increased in rats treated with clofibrate and perfluorodecanoic acid (PFDA). The present study was undertaken to test the hypothesis that induced expression of PXR increases PXR ligand-dependent induction on CYP3A23. Rat hepatocytes were treated with clofibrate or PFDA individually, or along with PXR ligand pregnenolone 16alpha-carbonitrile (PCN), and the levels of PXR and CYP3A23 were determined by Western blots. Both clofibrate and PFDA markedly increased the expression of PXR with PFDA being more potent, and the induction was abolished by actinomycin D, an inhibitor for mRNA synthesis. As expected, PCN alone markedly induced the expression of CYP3A23. Interestingly, co-treatment with clofibrate enhanced the induction, whereas co-treatment with PFDA suppressed it. Clofibrate and PFDA represent multi-classes of chemicals called peroxisome proliferators including many therapeutic agents and industrial pollutants. The opposing effects of clofibrate and PFDA on the PCN-induced expression of CYP3A23 suggest that peroxisome proliferators likely increase the expression of PXR but differentially alter its ligand-dependent induction. The interaction between PXR inducer and ligand provides a novel mechanism on how functionally and structurally distinct chemicals cooperatively regulate the expression of xenobiotic-metabolizing enzymes and transporters.     

Rifampicin, a keystone inducer of drug metabolism: from Herbert Remmer's pioneering ideas to modern concepts

In 1972, Herbert Remmer's group at the University of Tübingen had developed a micro method to assess cytochrome P450 contents and activities of drug metabolising enzymes in needle biopsies from human liver. Upon application of this method to patients receiving different kinds of drug therapy, Herbert Remmer was the first to describe that total human hepatic cytochrome P450 was markedly elevated by the new anti-tuberculosis drug rifampicin. Similar observations were made for the antimycotic clotrimazol. In 1975, Herbert Remmer's group described the unique species difference that induction of cytochrome P450 by rifampicin did not occur in the rat. After the first clinical reports of impaired effectiveness of oral contraception in persons treated with rifampicin, studies at Herbert Remmer's Institute showed a 4-fold increase, after repetitive rifampicin administration to humans, in the ability of hepatic microsomes to ortho-hydroxylate the contraceptive estrogen ethinylestradiol, compared to microsomes from untreated normal subjects. Subsequent pharmacokinetic investigations were compatible with this induction of the estrogen-2-hydroxylase by rifampicin and provided a rational explanation for the classical drug interaction between rifampicin and oral contraceptives. These early studies, in the 1970s in Tübingen, were followed by further developments. It was realized that the cytochrome P450 isoenzyme 3A4 (CYP3A4) is the major CYP isozyme in the human liver metabolizing a variety of xenobiotics and endobiotics, being also responsible for the 2-hydroxylation of ethinylestradiol. The inducibility of CYP3A4 by barbiturates and rifampicin explains the effects of inducers to enhance the clearance of ethynylestradiol and thereby to reduce the effectiveness of oral contraceptives, rifampicin being one of the most potent inducers of human CYP3A4 gene expression. Since 1998, novel "orphan" members of the nuclear hormone receptor superfamily were cloned from mouse, rat, rabbit, and human origin. These so-called pregnane X receptors (PXR), across species, are activated by inducers of CYP3A4 expression. It now appears that PXR is a key mediator of complex induction processes of xenobiotic processing enzymes, which are triggered by rifampicin and other inducers. Studies of the structure and substrate affinities of PXR have provided the rational explanation of the unique species difference of rifampicin induction between humans and rats that was first described by Herbert Remmer.