Regulation of mRNA expression of xenobiotic transporters by the pregnane x receptor in mouse liver, kidney, and intestine.

Multiple transporter systems are involved in the disposition of xenobiotics and endogenous compounds. The pregnane X receptor (PXR) is a major chemical sensor known to activate the expression of CYP3A/Cyp3a in humans and rodents. The purpose of this study is to systematically determine whether the major xenobiotic transporters in liver, kidney, duodenum, jejunum, and ileum are induced by pregnenolone-16alpha-carbonitrile (PCN), and whether this increase is mediated by the nuclear receptor PXR. In liver, PCN induced the expression of Oatp1a4 and Mrp3 mRNA in wild-type (WT) mouse liver, but not in PXR-null mice. In kidney, PCN did not alter the expression of any drug transporter. In duodenum, PCN increased Abca1 and Mdr1a mRNA expression in WT mice, but not in PXR-null mice. In jejunum and ileum, PCN increased Mdr1a and Mrp2 mRNA, but decreased Cnt2 mRNA in WT mice, but none of these transporters was altered when PCN was administered to PXR-null mice. Therefore, PCN regulates the expression of some transporters, namely, Oatp1a4 and Mrp3 in liver, as well as Abca1, Cnt2, Mdr1a, and Mrp2 in small intestine via a PXR-mediated mechanism.     

Gender dictates the nuclear receptor-mediated regulation of CYP3A44.

The CYP3As are broad-spectrum drug-metabolizing enzymes that are collectively responsible for more than 50% of xenobiotic metabolism. Unlike other CYP3As, murine CYP3A44 is expressed predominantly in the female liver, with much lower levels in male livers and no detectable expression in brain or kidney in either gender. In this study, we examined the role of nuclear hormone receptors in the regulation of Cyp3a44 gene expression. Interestingly, we observed differential effects of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) -mediated activation of Cyp3a44 gene expression, which was gender-specific. For example, activation of PXR by pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone (DEX) induced CYP3A44 mRNA levels in a PXR-dependent fashion in male mice, whereas no induction was detected in female mice. In contrast, PCN and DEX down-regulated CYP3A44 expression in female PXR null animals. Similar to PXR, CAR activation also showed a male-specific induction with no effect on CYP3A44 levels in females. When PXR knockout mice were challenged with the CAR activator phenobarbital, a significant up-regulation of male CYP3A44 levels was observed, whereas levels in females remained unchanged. We conclude that gender has a critical impact on PXR- and CAR-mediated effects of CYP3A44 expression.     

Effect of lipopolysaccharide on the xenobiotic-induced expression and activity of hepatic cytochrome P450 in mice

Infection-associated inflammation can alter the expression levels and functions of cytochrome P450s (CYPs). Cyp gene expression is regulated by the activation of several nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). These receptors can be activated by xenobiotics, including medicines. Here, to study the xenobiotic-induced fluctuations in CYP during inflammation, we examined the effect of lipopolysaccharide (LPS) treatment on the level of mRNAs encoding hepatic CYPs induced by xenobiotic-activated nuclear receptors, in mice. Both the mRNA induction of Cyp genes and the metabolic activities of CYP proteins were examined. LPS treatment caused a significant decrease in the induced expression of the mRNAs for Cyp3a11, 2c29, 2c55, and 1a2, but not for Cyp2b10. To assess the CYP enzymatic activities, CYP3A-mediated testosterone 6β-hydroxylation and the intrinsic clearance (CL(int)) of nifedipine in liver microsomes were measured in mice treated with the xenobiotic pregnenolone-16alpha-carbonitrile (PCN) with or without LPS administration. Both assays revealed that the CYP3A activity, which was induced by PCN, declined significantly after LPS treatment, and this decline correlated with the Cyp3a11 mRNA level. In addition, we found that the mRNAs for interleukin (IL)-1β and tumor necrosis factor (TNF) α were increased after treatment with LPS plus xenobiotics. Our findings demonstrated that LPS treatment reduces the PXR- and AhR-mediated, and possibly CAR-mediated Cyp gene expression and further suggest that these decreases are dependent on inflammatory cytokines in the liver.     

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats

1. The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats).

2. The treatment of WT rats for 7?d with 500?ppm NaPB in the diet and 100?mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats.

3. Male C57BL/6?J (WT) and CAR KO/PXR KO mice were given 1000?ppm NaPB in the diet for 7?d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes.

4. These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

     

Tissue distribution, ontogeny, and regulation of aldehyde dehydrogenase (Aldh) enzymes mRNA by prototypical microsomal enzyme inducers in mice.

Aldehyde dehydrogenases (Aldhs) are a group of nicotinamide adenine dinucleotide phosphate-dependent enzymes that catalyze the oxidation of a wide spectrum of aldehydes to carboxylic acids. Tissue distribution and developmental changes in the expression of the messenger RNA (mRNA) of 15 Aldh enzymes were quantified in male and female mice tissues using the branched DNA signal amplification assay. Furthermore, the regulation of the mRNA expression of Aldhs by 15 typical microsomal enzyme inducers (MEIs) was studied. Aldh1a1 mRNA expression was highest in ovary; 1a2 in testis; 1a3 in placenta; 1a7 in lung; 1b1 in small intestine; 2 in liver; 3a1 in stomach; 3a2 and 3b1 expression was ubiquitous; 4a1, 6a1, 7a1, and 8a1 in liver and kidney; 9a1 in liver, kidney, and small intestine; and 18a1 in ovary and small intestine. mRNAs of different Aldh enzymes were detected at lower levels in fetuses than adult mice and gradually increased after birth to reach adult levels between 15 and 45 days of age, when the gender difference began to appear. Aromatic hydrocarbon receptor (AhR) ligands induced the liver mRNA expression of Aldh1a7, 1b1, and 3a1, constitutive androstane receptor (CAR) activators induced Aldh1a1 and 1a7, whereas pregnane X receptor (PXR) ligands and NF-E2 related factor 2 (Nrf2) activators induced Aldh1a1, 1a7, and 1b1. Peroxisome proliferator activator receptor alpha (PPAR alpha) ligands induced the mRNA expression in liver of almost all Aldhs. The Aldh organ-specific distribution may be important in elucidating their role in metabolism, elimination, and organ-specific toxicity of xenobiotics. Finally, in contrast to other phase-I metabolic enzymes such as CYP450 enzymes, Aldh mRNA expression seems to be generally insensitive to typical microsomal inducers except PPAR alpha ligands.     

Oleanolic acid alters bile acid metabolism and produces cholestatic liver injury in mice

Oleanolic acid (OA) is a triterpenoids that exists widely in plants. OA is effective in protecting against hepatotoxicants. Whereas a low dose of OA is hepatoprotective, higher doses and longer-term use of OA produce liver injury. This study characterized OA-induced liver injury in mice. Adult C57BL/6 mice were given OA at doses of 0, 22.5, 45, 90, and 135 mg/kg, s.c., daily for 5 days, and liver injury was observed at doses of 90 mg/kg and above, as evidenced by increases in serum activities of alanine aminotransferase and alkaline phosphatase, increases in serum total bilirubin, as well as by liver histopathology. OA-induced cholestatic liver injury was further evidenced by marked increases of both unconjugated and conjugated bile acids (BAs) in serum. Gene and protein expression analysis suggested that livers of OA-treated mice had adaptive responses to prevent BA accumulation by suppressing BA biosynthetic enzyme genes (Cyp7a1, 8b1, 27a1, and 7b1); lowering BA uptake transporters (Ntcp and Oatp1b2); and increasing a BA efflux transporter (Ostβ). OA increased the expression of Nrf2 and its target gene, Nqo1, but decreased the expression of AhR, CAR and PPARα along with their target genes, Cyp1a2, Cyp2b10 and Cyp4a10. OA had minimal effects on PXR and Cyp3a11. Taken together, the present study characterized OA-induced liver injury, which is associated with altered BA homeostasis, and alerts its toxicity potential.     

Effect of nuclear receptor downregulation on hepatic expression of cytochrome P450 and transporters in chronic hepatitis C in association with fibrosis development

Analysis of mRNAs from liver biopsy samples of patients with chronic hepatitis C revealed that the levels of nuclear receptor expression were correlated with those of drug-metabolizing enzymes and transporters in relation to the development of fibrosis. Overall, the median mRNA level was largely dependent on fibrosis stage (F), and that for stage 3 patients (F3) was about 50% less than that for F1 patients. Levels of expression of AhR, together with CAR and PXR, were lowest in livers of F3 patients. Multivariate linear regression analysis revealed that AhR expression appeared to be involved in the regulation of CYP1A2, 2E1, 2D6, UGT1A, MDR1/3, MRP2/3, NTCP and OCT1 in the livers of patients with chronic hepatitis C. These results suggest that downregulation of AhR during the progression of liver fibrosis is associated with decreased expression levels of these phase I and II enzymes and drug transporters during inflammation-related signal transduction between AhR and other nuclear receptors.     

Regulation of drug transporter gene expression by nuclear receptors.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are key regulators of xenobiotic-inducible cytochrome P450 gene expression. Whereas much is known about their role in regulating drug metabolism, little is known regarding their role in regulating drug transport in vivo. Wild-type mice and mice lacking PXR (PXR-KO) were used to examine the inducible expression of two drug transporter genes, Oatp2 (Slc21a5) and Mrp3 (Abcc3), in liver following treatment with selective PXR and CAR activators. Selective activation of PXR or CAR induced Oatp2 and Mrp3 expression in wild-type mice but not in PXR-KO mice. Basal expression levels of Oatp2 and Mrp3 gene were significantly higher in PXR-KO mice when compared with wild-type mice. Additionally, phenobarbital (PB)-inducible Oatp2 and Mrp3 gene expression was significantly increased in the PXR-KO mice when compared with wild-type PB-treated mice. We also examined the effect of PXR ablation on PB-inducible hepatic CYP3A activity in vivo. Microsomes isolated from PB-treated PXR-KO mice exhibited a significantly elevated rate of testosterone 6 beta-hydroxylation when compared with microsomes isolated from wild-type PB-treated mice. PB treatment produced significantly increased levels of hepatomegaly in PXR-KO mice when compared with wild-type PB-treated mice. Taken together, these results suggest that nonliganded PXR plays a net negative role in coregulating shared PXR/CAR-target gene expression in vivo and extend the hypothesis that PXR and CAR coregulate not only drug metabolism but also drug transport.     

Coordinate regulation of xenobiotic and bile acid homeostasis by pregnane X receptor.

Identification and characterization of the pregnane X receptor (PXR) as a key regulator of cytochrome P450 3A (CYP3A) gene expression has led to an increased understanding of the molecular basis of many drug-drug interactions. Mice lacking PXR (PXR-KO) were used in the present study to delineate the role of PXR in regulating hepatomegaly and regulating the activity of CYP3A, organic anion transporting polypeptide-2 (Oatp2), and Cyp7a1 (cholesterol 7alpha-hydroxylase) gene products in vivo. Pregnenolone-16alpha-carbonitrile (PCN) produced hepatomegaly in the wild-type mice but not in the PXR-KO mice. PCN increased both the number of proliferating cell nuclear antigen immuno-positive nuclei and apparent cell size in the wild-type mice but not in the PXR-KO mice. To determine the role PXR plays in regulating CYP3A activity, 6beta-hydroxylation of testosterone and the duration of the loss of righting reflex following administration of the muscle-relaxant zoxazolamine were measured. PCN increased the level of testosterone 6beta-hydroxylation and decreased the duration of the loss of righting-reflex time following zoxazolamine administration in wild-type mice, but did not effect either of these parameters in PXR-KO mice. PCN increased the hepatic uptake of [(3)H]digoxin, an Oatp2 substrate, in wild-type mice but not in the PXR-KO mice. Similarly, PCN decreased bile acid excretion in wild-type mice but not in the PXR-KO mice. Taken together, these data demonstrate a pivotal role for PXR in the regulation of drug-induced hepatomegaly and in the metabolism (CYP3A), transport (Oatp2), biosynthesis (Cyp7a1), and excretion of xenobiotics and bile acids in vivo.