Expression Of Hepatic Drug Metabolizing Enzymes Research Articles

Constitutive Androstane Receptor Agonist, TCPOBOP: Maternal Exposure Impairs the Growth and Development of Female Offspring in Mice.

Environmental chemicals, which are known to impact offspring health, have become a public concern. Constitutive activated receptor (CAR) is activated by various environmental chemicals and participates in xenobiotic metabolism. Here, we described the effects of maternal exposure to the CAR-specific ligand 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP, TC) on offspring health outcomes. Maternal TC exposure exhibited a stronger inhibition of body weight in 3-week-old and 8-week-old first-generation (F1) offspring female mice compared to controls. Further, maternal TC exposure obtained a strong increase in hepatic drug-metabolizing enzyme expression in 3-week-old female mice that persisted into 8-week-old adulthood. Interestingly, we observed distorted intestinal morphological features in 8-week-old F1 female mice in the TC-exposed group. Moreover, maternal TC exposure triggered a loss of intestinal barrier integrity by reducing the expression of intestinal tight junction proteins. Accordingly, maternal exposure to TC down-regulated serum triglyceride levels as well as decreased the expression of intestinal lipid uptake and transport marker genes. Mechanistically, maternal TC exposure activated the intestinal inflammatory response and disrupted the antioxidant system in the offspring female mice, thereby impeding the intestinal absorption of nutrients and seriously threatening offspring health. Altogether, these findings highlight that the effects of maternal TC exposure on offspring toxicity could not be ignored.

Shikonin upregulates the expression of drug-metabolizing enzymes and drug transporters in primary rat hepatocytes

Ethnopharmacological relevanceShikonin, a naphthoquinone pigment abundant in the root of the Chinese herb Lithospermum erythrorhizon, has been widely used to treat inflammatory diseases for thousands of years. Whether shikonin changes drug metabolism remains unclear. Aim of the studyWe investigated whether shikonin modulates the expression of hepatic drug-metabolizing enzymes and transporters as well as the possible mechanisms of this action. Materials and methodsPrimary hepatocytes isolated from Sprague-Dawley rats were treated with 0–2 μM shikonin and the protein and mRNA levels of drug-metabolizing enzymes and transporters as well as the activation of aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) were determined. ResultsShikonin dose-dependently increased the protein and RNA expression of phase I enzymes, i.e., cytochrome P450 (CYP) 1A1/2, CYP3A2, CYP2D1, and CYP2C6; phase II enzymes, i.e., glutathione S-transferase (GST), NADP(H) quinone oxidoreductase 1 (NQO1), and UDP glucuronosyltransferase 1A1; and phase III drug transporters, i.e., P-glycoprotein, multidrug resistance-associated protein 2/3, organic anion transporting polypeptide (OATP) 1B1, and OATP2B1. Immunoblot analysis and EMSA revealed that shikonin increased AhR and Nrf2 nuclear contents and DNA binding activity. AhR and Nrf2 knockdown by siRNA attenuated the ability of shikonin to induce drug-metabolizing enzyme expression. In addition, shikonin increased p38, JNK, and ERK1/2 phosphorylation, and inhibitors of the respective kinases inhibited shikonin-induced Nrf2 nuclear translocation. ConclusionsShikonin effectively upregulates the transcription of CYP isozymes, phase II detoxification enzymes, and phase III membrane transporters and this function is at least partially through activation of AhR and Nrf2. Moreover, Nrf2 activation is dependent on mitogen-activated protein kinases.

Effect of experimental kidney disease on the functional expression of hepatic reductases.

Chronic kidney disease (CKD) affects the nonrenal clearance of drugs by modulating the functional expression of hepatic drug-metabolizing enzymes and transporters. The impact of CKD on oxidative and conjugative metabolism has been extensively studied. However, its effect on hepatic drug reduction, an important phase I drug-metabolism pathway, has not been investigated. We aimed to assess the effect of experimental CKD on hepatic reduction using warfarin as a pharmacological probe substrate. Cytosolic and microsomal cellular fractions were isolated from liver tissue harvested from five-sixths-nephrectomized and control rats (n = 10 per group). The enzyme kinetics for warfarin reduction were evaluated in both fractions, and formation of warfarin alcohols was used as an indicator of hepatic reductase activity. Selective inhibitors were employed to identify reductases involved in warfarin reduction. Gene and protein expression of reductases were determined using quantitative real-time polymerase chain reaction and Western blotting, respectively. Formation of RS/SR-warfarin alcohol was decreased by 39% (P < 0.001) and 43% (P < 0.01) in cytosol and microsomes, respectively, in CKD rats versus controls. However, RR/SS-warfarin alcohol formation was unchanged in the cytosol, and a trend toward its decreased production was observed in microsomes. Gene and protein expression of cytosolic carbonyl reductase 1 and aldo-keto reductase 1C3/18, and microsomal 11β-hydroxysteroid dehydrogenase type 1 were significantly reduced by >30% (P < 0.05) in CKD rats compared with controls. Collectively, these results suggest that the functional expression of hepatic reductases is selectively decreased in kidney disease. Our findings may explain one mechanism for altered nonrenal clearance, exposure, and response of drugs in CKD patients.

Role of Constitutive Androstane Receptor in Toll-Like Receptor-Mediated Regulation of Gene Expression of Hepatic Drug-Metabolizing Enzymes and Transporters

Impairment of drug disposition in the liver during inflammation has been attributed to downregulation of gene expression of drug-metabolizing enzymes (DMEs) and drug transporters. Inflammatory responses in the liver are primarily mediated by Toll-like receptors (TLRs). We have recently shown that activation of TLR2 or TLR4 by lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, leads to the downregulation of gene expression of DMEs/transporters. However, the molecular mechanism underlying this downregulation is not fully understood. The xenobiotic nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the expression of DMEs/transporter genes. Downregulation of DMEs/transporters by LTA or LPS was associated with reduced expression of PXR and CAR genes. To determine the role of CAR, we injected CAR(+/+) and CAR(-/-) mice with LTA or LPS, which significantly downregulated (~40%-60%) RNA levels of the DMEs, cytochrome P450 (Cyp)3a11, Cyp2a4, Cyp2b10, uridine diphosphate glucuronosyltransferase 1a1, amine N-sulfotransferase, and the transporter, multidrug resistance-associated protein 2, in CAR(+/+) mice. Suppression of most of these genes was attenuated in LTA-treated CAR(-/-) mice. In contrast, LPS-mediated downregulation of these genes was not attenuated in CAR(-/-) mice. Induction of these genes by mouse CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene was sustained in LTA- but not in LPS-treated mice. Similar observations were obtained in humanized CAR mice. We have replicated these results in primary hepatocytes as well. Thus, LPS can downregulate DME/transporter genes in the absence of CAR, whereas the effect of LTA on these genes is attenuated in the absence of CAR, indicating the potential involvement of CAR in LTA-mediated downregulation of DME/transporter genes.

Differential Role of Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein in Toll-Like Receptor 2-Mediated Regulation of Gene Expression of Hepatic Cytokines and Drug-Metabolizing Enzymes

Pharmacological activities of drugs are impaired during inflammation because of reduced expression of hepatic drug-metabolizing enzyme genes (DMEs) and their regulatory nuclear receptors (NRs): pregnane X receptor (PXR), constitutive androstane receptor (CAR), and retinoid X receptor (RXRα). We have shown that a component of Gram-positive bacteria, lipoteichoic acid (LTA) induces proinflammatory cytokines and reduces gene expression of hepatic DMEs and NRs. LTA is a Toll-like receptor 2 (TLR2) ligand, which initiates signaling by recruitment of Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) to the cytoplasmic TIR domain of TLR2. To determine the role of TIRAP in TLR2-mediated regulation of DME genes, TLR2(+/+), TLR2(-/-), TIRAP(+/+), and TIRAP(-/-) mice were given LTA injections. RNA levels of the DMEs (Cyp3a11, Cyp2b10, and sulfoaminotransferase), xenobiotic NRs (PXR and CAR), and nuclear protein levels of the central NR RXRα were reduced ∼ 50 to 60% in LTA-treated TLR2(+/+) but not in TLR2(-/-) mice. Induction of hepatic cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-6), c-Jun NH(2)-terminal kinase, and nuclear factor-κΒ was blocked in TLR2(-/-) mice. As expected, expression of hepatic DMEs and NRs was reduced by LTA in TIRAP(+/+) but not in TIRAP(-/-) mice. Of interest, cytokine RNA levels were induced in the livers of both the TIRAP(+/+) and TIRAP(-/-) mice, whereas LTA-mediated induction of serum cytokines was attenuated in TIRAP(-/-) mice. LTA-mediated down-regulation of DME genes was attenuated in hepatocytes from TLR2(-/-) or TIRAP(-/-) mice and in small interfering RNA-treated hepatocytes. Thus, the effect of TLR2 on DME genes in hepatocytes was mediated by TIRAP, whereas TIRAP was not involved in mediating the effects of TLR2 on cytokine expression in the liver.

Mechanisms responsible for the altered pharmacokinetics of Bosentan: analysis utilizing rats with bile duct ligation‐induced liver dysfunction

The purpose of this study was to evaluate the mechanisms responsible for the pharmacokinetic variability of bosentan utilizing rats with liver dysfunction induced by 7-day bile duct ligation (BDL). Bosentan was administered intravenously at a constant infusion rate (I) of 24, 40 or 60 microg/min/kg. The blood bosentan concentration (BBC) following infusion was measured by HPLC, and apparent clearance (CL) of the drug was estimated as I/BBC. The CL values in normal rats were 30.5 and 19.3 ml/min/kg at infusion rates of 24 and 60 microg/min/kg, respectively, suggesting non-linear pharmacokinetics of bosentan. The BBC in BDL rats was much higher than that in normal rats, and the CL values in BDL rats were 3.80 and 3.08 ml/min/kg at infusion rates of 24 and 60 microg/min/kg, respectively. The CL value of bosentan at an infusion rate of 40 microg/min/kg in normal rats was decreased significantly by the coadministration of taurocholic acid or bilirubin. In addition, the hepatic mRNA expression of CYP2C6, CYP3A2, Oatp1a1, Oatp1a4 and Oatp1b2 in BDL rats decreased to 77.6%, 34.0%, 65.4%, 84.8% and 44.2% of that in normal rats, respectively. These results suggested that bile acids and/or bilirubin accumulated in BDL rat plasma inhibited the hepatic uptake of bosentan, and that the decreased bosentan clearance in BDL rats was caused at least partly by the impaired expression of hepatic drug-metabolizing enzymes and uptake transporters. Moreover, because the pharmacokinetics of bosentan was non-linear at the tested doses, the increased BBC in BDL rats might further induce the saturation of hepatic uptake and/or metabolism of bosentan.

Methadone Induces the Expression of Hepatic Drug-Metabolizing Enzymes through the Activation of Pregnane X Receptor and Constitutive Androstane Receptor

Methadone (MD) is the most established substance abuse pharmacotherapy of choice for the management of heroin dependence. To date, drug-drug interactions involving MD have been characterized asymmetrically among existing reports, which describe how other drugs affect the metabolic or pharmacokinetic profiles of MD; however, limited information is available regarding the potential for MD to influence similar fates of coadministered drugs. Moreover, little to no mechanistic evidence has been explored. Here, we show that MD induces hepatic drug-metabolizing enzymes (DMEs) through the activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Real-time polymerase chain reaction analysis of human hepatocyte cultures revealed that MD induces the mRNA expression of CYP2B6, CYP3A4, UGT1A1, and multidrug resistance 1 in a concentration-related manner, with the maximal induction of CYP2B6 challenging that of the induction by rifampicin. Furthermore, MD-mediated induction of CYP2B6 and CYP3A4 proteins was observed in Western blot analysis. In cell-based reporter assays, MD significantly increased human (h) PXR-mediated CYP2B6 reporter activities but exhibited minimal effect on hCAR activation as a result of the constitutive activity of hCAR in HepG2 cells. Further studies revealed that treatment with MD resulted in significant nuclear accumulation of adenovirus/enhanced yellow fluorescent protein tagged-hCAR in human hepatocytes, which has been regarded as the initial step of CAR activation. Additional analysis of the two enantiomers of MD, R-(-)-MD (active) and S-(+)-MD (inactive), indicates the lack of stereoselectivity pertaining to MD-mediated DME induction. Overall, our results show that MD induces the hepatic expression of multiple DMEs by activating PXR- and CAR-mediated pathways.

Bioactive Terpenoids and Flavonoids from Ginkgo Biloba Extract Induce the Expression of Hepatic Drug-Metabolizing Enzymes Through Pregnane X Receptor, Constitutive Androstane Receptor, and Aryl hydrocarbon Receptor-Mediated Pathways

The objective of the current study is to investigate the hypothesis that bioactive terpenoids and flavonoids of Ginkgo biloba extract (GBE) induce human hepatic drug metabolizing enzymes (DMEs) and transporters through the selective activation of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). Human primary hepatocyte (HPH), and HepG2 cells are used as in vitro models for enzyme induction and nuclear receptor activation studies. A combination of real-time RT-PCR, transient transfection, and cell-based reporter assays were employed. In human primary hepatocytes, real-time PCR analysis showed induction of CYP2B6, CYP3A4, UGT1A1, MDR1, and MRP2 by EGb 761, ginkgolide A (GA) and ginkgolide B (GB), but not by bilobalide (BB) or the flavonoids (quercetin, kaempferol and tamarixetin) of GBE. Cell-based reporter assays in HepG2 revealed that GA and GB are potent activators of PXR; quercetin and kaempferol activate PXR, CAR, and AhR, whereas BB exerts no effects on these xenobiotic receptors. Notably, the flavonoids induced the expression of UGT1A1 and CYP1A2 in HepG2 cells but not in HPH. Our results indicate that terpenoids and flavonoids of GBE exhibit differential induction of DMEs through the selective activation of PXR, CAR, and AhR.

Ginkgo biloba Extracts Induce the Expression of Hepatic Drug-metabolizing Enzymes through Activation of PXR and AhR

Ginkgo biloba extract (GBE) is extensively used as an herbal remedy for enhancing mental focus and delaying onset of age-related loss of cognitive function. Potentials of related drug-herbal interaction have been highlighted by recent reporters, which indicated GBE causes enhancement of several P450 expression in rodent animals. However, data pertaining to GBE induction of human metabolism is limited, and the underlying mechanisms are not known. In the present study, we aimed to evaluate the metabolism induction profile of EGB761, ginkgolide A (GA), ginkgolide B (GB), and bilobalide (BB) using human primary hepatocyte cultures. Real-time PCR assay was employed to assess mRNA levels of CYP3A4, CYP2B6, CYP1A2, UGT1A1, and MDR1. To assess the potential for GBE activation of transcription factors, cell-based reporter assays for PXR, CAR, and AhR were conducted in HepG2 cells. Results demonstrated that GA and GB strongly induced CYP3A4 and CYP2B6 in human hepatocytes, and activated PXR but not CAR or AhR; EGB761 and BB only resulted in marginal changes in CYP2B6 expression, without activation of either PXR or CAR. In contrast, EGB761 but not GA or GB activated AhR in reporter assays, indicating EGB761 may contain components other than GA or GB, which mediated the activation of AhR. Current results revealed that GBE induces hepatic expression of CYP3A4 and CYP2B6 through the activation of PXR, and GA/GB is the major component responsible for this induction. EGB761 activation of AhR may through an unknown component other than GA. Human CAR is not involved in GBE mediated CYP inductions.

DNA damage and altered gene expression of enzymes for metabolism and DNA repair by tamoxifen and toremifene in the female rat liver

Effects of hepatocarcinogenic TAM and non-hepatocarcinogenic TOR on the formation of hepatic DNA adducts and on the gene expression of hepatic drug-metabolizing enzymes and DNA repair enzymes/proteins were comparatively examined in female Sprague-Dawley rats treated with TAM (20 or 40 mg/kg/day, i.g.) or TOR (40 mg/kg/day, i.g.) for 1, 2 or 8 weeks. Hepatic TAM-DNA adducts were formed even after 1 week of treatment with TAM at either dose, and the adduct levels increased in a dose- and treatment period-dependent manner, whereas no DNA adducts were detected in any of the TOR-treated rats. Conversely, TAM and TOR showed almost the same capacity for increasing the gene expression of drug-metabolizing enzymes responsible for metabolic activation and detoxification, at least up to the 2-week treatment mark. Accordingly, differences in DNA adduct formation between TAM- and TOR-treated rats would not be primarily dependent on the capacity for inducing hepatic drug-metabolizing enzymes. In addition, a drastic increase in the gene expression of cytochrome P4503A2 (CYP3A2), an activation enzyme of TAM, by the 8-week treatment with TAM might have contributed to the increased formation of DNA adducts. Gene expressions of DNA repair enzymes/proteins responsible for a nucleotide excision repair system were not significantly changed in any of the rats treated with either drug. The present findings suggest that the difference between TAM and TOR in hepatocarcinogenic potency is dependent on the capacity to form DNA adducts rather than modulating the expression of drug-metabolizing enzymes and DNA repair enzymes/proteins.

Traditional Chinese Medicines Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch) Activate Pregnane X Receptor and Increase Warfarin Clearance in Rats

The traditional Chinese medicines (TCMs) are essential components of alternative medicines. Many TCMs are known to alter the expression of hepatic drug-metabolizing enzymes and transporters. The molecular mechanism by which TCMs and/or their constituents regulate enzyme and transporter expression, however, has remained largely unknown. In this report, we show that two TCMs, Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch), and their selected constituents activate the xenobiotic orphan nuclear receptor pregnane X receptor (PXR). Treatment with TCM extracts and the Schisandrol and Schisandrin constituents of Wu Wei Zi induced the expression of drug-metabolizing enzymes and transporters in reporter gene assays and in primary hepatocyte cultures. The affected enzymes and transporters include CYP3A and 2C isozymes and the multidrug resistance-associated protein 2. In transient transfection and reporter gene assays, the Schisandrin constituents of Wu Wei Zi had an estimated EC50 of 2 and 1.25 microM on hPXR and mPXR, respectively. Interestingly, mutations that were intended to alter the pore of the ligand-binding cavity of PXR had species-specific effects on the activities of the individual Schisandrols and Schisandrins. In rats, the administration of Wu Wei Zi and Gan Cao increased the metabolism of the coadministered warfarin, reinforcing concerns involving the safe use of herbal medicines and other nutraceuticals to avoid PXR-mediated drug-drug interactions. Meanwhile, the activation of PXR and induction of detoxifying enzymes provide a molecular mechanism for the hepatoprotective effects of certain TCMs.

Ebselen protects against the reduction in levels of drug-metabolizing enzymes in livers of rats with deoxycholic acid-induced liver injury.

Ebselen is a seleno-organic compound that inhibits oxidative stress by lipid peroxidation through a glutathione peroxidase-like activity. We studied the effect of ebselen on the expression of hepatic drug-metabolizing enzymes in rats with deoxycholic acid-induced liver injury. Hydrophobic bile acids, such as deoxycholic acid, are known to cause cholestatic liver injury, and it was reported that expression of hepatic cytochrome P-450 (CYP) was reduced by deoxycholic acid administration in rats. Hydrophobic bile acids induce lipid peroxidation in the liver, and this may be one mechanism of the development of liver injury. In the present study, we investigated the effect of ebselen (30 mg/kg/day for 10 days) on rats ingesting deoxycholic acid (1% of diet for 10 days). Deoxycholic acid decreased levels of CYP1A1, 2B1, 2E1 and 3A2 to 34, 58, 62 and 37% of control values, respectively, and increased serum alkaline phosphatase (ALP) and alanine aminotransferase (ALT) activities to 1.8 and 8.6 times the levels of controls, respectively. Administration of ebselen with deoxycholic acid prevented the decreases in levels of CYP1A1 and 3A2 (86 and 65% of control, respectively) and the increases in serum ALP and ALT activities (1.4 and 1.9 times of control, respectively) caused by deoxycholic acid. These results indicate that ebselen may have a protective effect against hydrophobic bile acid-induced liver injury.