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.     

Induction of cytochrome P450 3A by paclitaxel in mice: pivotal role of the nuclear xenobiotic receptor, pregnane X receptor.

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.     

Disruption of thyroid hormone homeostasis in Ugt1a-deficient Gunn rats by microsomal enzyme inducers is not due to enhanced thyroxine glucuronidation

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T₄), thus reducing serum T₄, and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T₄ glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T₄ glucuronidation, decreased serum T₄, and increased TSH after MEI treatment. Male Wistar and Ugt1a-deficient Wistar (Gunn) rats were fed a control diet or diet containing pregnenolone-16α-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum T₄, triiodothyronine (T₃), and TSH concentrations, hepatic T₄/T₃ glucuronidation, and thyroid histology and follicular cell proliferation were investigated. PCN, 3-MC, and PCB treatments decreased serum T₄, whereas serum T₃ was maintained in both Gunn and Wistar rats (except for PCB treatment). TSH was increased in Wistar and Gunn rats after PCN (130 and 277%) or PCB treatment (72 and 60%). T₄ glucuronidation in Wistar rats was increased after PCN (298%), 3-MC (85%), and PCB (450%), but was extremely low in Gunn rats, and unchanged after MEI. T₃ glucuronidation was increased after PCN (121%) or PCB (58%) in Wistar rats, but only PCN increased T₃ glucuronidation in Gunn rats (43%). PCN treatment induced thyroid morphological changes and increased follicular cell proliferation in both strains. These data demonstrate that T₄ glucuronidation cannot be increased in Ugt1a-deficient Gunn rats. Thus, the decrease in serum T₄, increase in TSH, and increase in thyroid cell proliferation after MEI are not dependent on increased T₄ glucuronidation, and cannot be attributed to Ugt1a enzymes.     

Characterization of UDP-glucuronosyltransferases (UGTS) involved in the metabolism of troglitazone in rats and humans

UDP-glucuronosyltransferases (UGTs) involved in troglitazone glucuronidation in rats and humans have been characterized to support the previous toxicity study on troglitazone in Gunn rats and to examine whether the UGT polymorphism or inhibition of bilirubin metabolism is related to the clinically reported rare cases of liver failure. The experiments using Gunn rats revealed that UGT1 enzymes are not involved in troglitazone glucuronidation and that the responsible enzyme in rats was suggested to be UGT2B2, an androsterone UGT, by inhibition studies. In humans, contribution of UGT1A1 was estimated to be about 30% of the total troglitazone glucuronidation by UGTs, using human liver microsomes and recombinant UGTs. Other UGT1 and UGT2 enzymes seem to be responsible for the rest of the troglitazone glucuronidation in humans. The multiplicity of UGTs involved in troglitazone glucuronidation in humans may allow even patients lacking bilirubin UGT (UGT1A1) activity to produce troglitazone glucuronide. These observations suggest that the polymorphism of UGT is not the reason behind the liver failure induced by the troglitazone treatment, and troglitazone does not inhibit bilirubin glucuronidation in clinical treatment. In addition, the increased bilirubin level in the blood of patients who have troglitazone-induced liver failure is a consequence of liver injury and not due to inhibition of bilirubin glucuronidation by troglitazone.     

Hepatoprotective role of PXR activation and MRP3 in cholic acid-induced cholestasis

BACKGROUND AND PURPOSE: Activation of the pregnane X receptor (PXR) has been shown to protect against cholestatic hepatotoxicity. As PXR alters the expression of numerous hepatic bile acid transporters, we sought to delineate their potential role in hepatoprotection. EXPERIMENTAL APPROACH: Wild-type (PXR+/+) and PXR-null (PXR-/-) mice were fed a 1% cholic acid (CA) diet with or without the PXR activator, PCN. Liver function was assessed along with the corresponding changes in hepatic gene expression. KEY RESULTS: CA administration caused significant hepatotoxicity in PXR+/+ mice and was associated with induction of several FXR and PXR regulated genes, which encode for bile acid transport and metabolizing proteins. Compared to CA alone, co-administration of PCN to CA-fed PXR+/+ mice significantly decreased hepatotoxicity and was associated with induction of MRP3 mRNA as well as CYP3A11 mRNA and functional activity. Unexpectedly, PXR-/- mice, which expressed significantly higher basal and CA-induced levels of MRP2, MRP3, OSTalpha, OSTbeta, OATP2 and CYP3A11, were dramatically less sensitive to CA hepatotoxicity than PXR+/+ mice. CONCLUSIONS: Protection of PXR+/+ mice against CA-induced hepatotoxicity by PCN is associated with the induction of MRP3 and CYP3A11 expression. Resistance against CA-induced hepatotoxicity in PXR-/- mice may result from higher basal and induced expression of bile acid transporters, particularly MRP3. These findings emphasize the importance of transport by MRP3 and metabolism as major protective pathways against cholestatic liver injury.     

Inhibition of human UGT2B7 gene expression in transgenic mice by the constitutive androstane receptor

The xenobiotic receptors, constitutive androstane receptor (CAR), and pregnane X receptor (PXR) regulate and alter the metabolism of xenobiotic substrates. Among the 19 functional UDP-glucuronosyltransferases (UGTs) in humans, UGT2B7 is involved in the metabolism of many structurally diverse xenobiotics and plays an important role in the clearance and detoxification of many therapeutic drugs. To examine whether this gene is regulated by CAR and PXR in vivo, transgenic mice expressing the entire UGT2B7 gene (TgUGT2B7) were created. Gene expression profiles revealed that UGT2B7 is differentially expressed in liver, kidney, adipocytes, brain, and estrogen-sensitive tissues, such as ovary and uterus. Liver UGT2B7 expression levels were decreased when TgUGT2B7 mice were treated with the CAR ligand 1,4-b-s-[2-(3,5,-dichloropyridyloxy)] (TCPOBOP) but not the PXR ligand pregnenolone 16α-carbonitrile. Although TCPOBOP decreased the levels of UGT2B7 mRNA in TgUGT2B7 mice, it had no affect on Tg(UGT2B7)Car(-/-) mice, adding support for a CAR-dependent mechanism contributing toward UGT2B7 gene suppression. Expression of promoter constructs in HepG2 cells showed the CAR-dependent inhibition was linked to hepatocyte nuclear factor-4α (HNF4α)-mediated transactivation of the UGT2B7 promoter. The inhibitory effect of CAR on UGT2B7 gene expression was validated in chromatin immunoprecipitation assays in which TCPOBOP treatment blocked HNF4α binding to the UGT2B7 promoter. These results suggest that HNF4α plays an important role in the constitutive expression of hepatic UGT2B7, and CAR acts as a negative regulator by interfering with HNF4α binding activity.     

CYP3A induction by liver x receptor ligands in primary cultured rat and mouse hepatocytes is mediated by the pregnane X receptor.

The effects of oxysterol and drug ligands of the liver X receptor (LXR) on cytochrome P450 expression were evaluated in primary cultured rodent hepatocytes. Treatment of rat hepatocyte cultures with either 25-hydroxycholesterol or 24(S),25-epoxycholesterol (10(-5) to 5 x 10(-5) M) produced concentration-dependent elevations in CYP3A mRNA and immunoreactive protein levels but did not increase the amounts of CYP1A1, CYP2B, or CYP4A gene products. The effects of 24(S),25-epoxycholesterol on CYP3A content were much greater than were those of 25-hydroxycholesterol, consistent with the relative abilities of these sterols to bind and activate LXR. To understand the mechanistic basis of these observations, experiments were performed using primary cultured hepatocytes prepared from LXRalpha/beta- or pregnane X receptor (PXR)-null mice. CYP3A mRNA levels were increased after treatment with 24(S),25-epoxycholesterol in both wild-type and LXR-null mouse hepatocytes. In contrast, neither 24(S),25-epoxycholesterol nor either of two additional potent LXR ligands, 22(R)-hydroxycholesterol and N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)ethyl-]phenyl]-benzenesulfonamide (T0901317), altered CYP3A mRNA levels in hepatocytes prepared from PXR-null mice, although these agents induced CYP3A mRNA content in wild-type cultures. As evidence that the LXR ligands also activated PXR in rat hepatocytes, cotransfection of primary cultures with a dominant negative PXR abolished reporter gene induction after treatment with any of the test agents. These results indicate that selected LXR ligands are capable of activating PXR, probably as a defensive measure to prevent the accumulation of these potentially toxic endogenous molecules.     

Tissue- and gender-specific mRNA expression of UDP-glucuronosyltransferases (UGTs) in mice.

UDP-glucuronosyltransferases (UGTs) catalyze phase II biotransformation reactions in which lipophilic substrates are conjugated with glucuronic acid to increase water solubility and enhance excretion. Currently, little information regarding tissue- or gender-specific expression of mouse UGTs is available. Mice are increasingly popular models in biomedical research, and therefore, thorough characterization of murine drug metabolism is desired. The purpose of the present study was to determine both tissue- and gender-specific UGT gene expression profiles in mice. RNA from 14 tissues was isolated from male and female C57BL/6 mice and UGT expression was determined by the branched DNA signal amplification assay. UGTs highly expressed in mouse liver include Ugt1a1, Ugt1a5, Ugt1a6, Ugt1a9, Ugt2a3, Ugt2b1, Ugt2b5/37/38, Ugt2b34, Ugt2b35, and Ugt2b36. Several isoforms were expressed in the gastrointestinal (GI) tract, including Ugt1a6, Ugt1a7c, Ugt2a3, Ugt2b34, and Ugt2b35. In kidney, Ugt1a2, Ugt1a7c, Ugt2b5/37/38, Ugt2b35, and Ugt3a1/2 were expressed. UGT expression was also observed in other tissues: lung (Ugt1a6), brain (Ugt2b35), testis and ovary (Ugt1a6 and Ugt2b35), and nasal epithelia (Ugt2a1/2). Male-predominant expression was observed for Ugt2b1 in liver, Ugt2b5/37/38 in kidney, and Ugt1a6 in lung. Female-predominant expression was observed for Ugt1a1 and Ugt1a5 in liver, Ugt1a2 in kidney, Ugt2b35 in brain, and Ugt2a1/2 in nasal epithelia. UDP-glucose pyrophosphorylase was highly expressed in liver, kidney, and GI tract, whereas UDP-glucose dehydrogenase was highly expressed in the GI tract. In conclusion, marked differences in tissue- and gender-specific expression patterns of UGTs exist in mice, potentially influencing drug metabolism and pharmacokinetics.     

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.     

Glucuronidation enzymes,genes and psychiatry

The phase I cytochrome P450 (CYP) isoenzymes have received substantial attention in the pharmacogenetic literature. Researchers are beginning to examine the role of the phase II UDP-glucuronosyltransferase (UGT) enzymes, which produce products that are more water-soluble, less toxic and more readily excreted than the parent compounds. Several reasons may have contributed to neglect of UGTs (compared to CYPs) including: (1) the overlapping activity of UGTs and lack of selective probes; (2) the complexity of the glucuronidation cycle; and (3) the difficulty in developing analytic methods to measure glucuronides. Current CYP knowledge is used as a model to predict advances in UGT knowledge. At least 24 different UGT human genes have been identified and are classified in two families (UGT1 and UGT2) based on sequence homology. The UGT1A subfamily (genes located on chromosome 2) glucuronidates bilirubin, thyroid hormones, and some medications. UGT1A4 metabolizes tricyclic antidepressants and some antipsychotics. The UGT2B subfamily (genes located on chromosome 6) glucuronidates sexual steroids and bile acids. Oxazepam and lorazepam are mainly metabolized by glucuronidation. Anti-epileptics with mood-stabilizing properties are frequently metabolized by UGTs. Opioid and nicotine addiction may also be influenced by glucuronidation. Glucuronidation of serotonin may be important during fetal development. UGTs appear to be in small concentrations in brain tissue (and higher concentrations at brain capillaries). However, UGTs may be localized in certain brain areas to provide a neuroprotective function. This review illustrates the importance of glucuronidation and the implications for psychiatry.     

A high throughput assay for the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin by recombinant human UDP-glucuronosyltransferases and liver microsomes

Abstract

1.?UDP-glucuronosyltransferases (UGTs) are versatile and important conjugation enzymes in the metabolism of drugs and other xenobiotics.

2.?We have developed a convenient quantitative multi-well plate assay to measure the glucuronidation rate of 7-hydroxy-4-trifluoromethylcoumarin (HFC) for several UGTs.

3.?We have used this method to screen 11 recombinant human UGTs for HFC glucuronidation activity and studied the reaction kinetics with the most active enzymes. We have also examined the HFC glucuronidation activity of liver microsomes from human, pig, rabbit and rat.

4.?At a substrate concentration of 20?µM, the most active HFC glucuronidation catalysts were UGT1A10 followed by UGT1A6 >UGT1A7 >UGT2A1, whereas at 300?µM UGT1A6 was about 10 times better catalyst than the other recombinant UGTs. The activities of UGTs 1A3, 1A8, 1A9, 2B4 and 2B7 were low, whereas UGT1A1 and UGT2B17 exhibited no HFC glucuronidation activity. UGT1A6 exhibited a significantly higher V_max and Km values toward both HFC and UDP-glucuronic acid than the other UGTs.

5.?Human, pig and rabbit, but not rat liver microsomes, catalyzed HFC glucuronidation at high rates.

6.?This new method is particularly suitable for fast activity screenings of UGTs 1A6, 1A7, 1A10 and 2A1 and HFC glucuronidation activity determination from various samples.     

Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans.     

Effect of Cigarette Smoke on UDP-Glucuronosyltransferase Activity and Cytochrome P450 Content in Liver,Lung and Kidney Microsomes in Mice

Abstract: The effect of cigarette smoke on the expression of several cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGT) was studied in mice. The animals were exposed to cigarette smoke for 4 to 30 days. Enzymatic activities supported by CYP1A1, 1A2, 2B, 2E1 and the glucuronidation activity toward phenols were measured in lung, liver and kidney microsomes. Cigarette smoke induced several CYPs, especially in lung. CYP2E1 was more induced than CYP1A1 in this organ. The expression of CYP2E1 was also increased in kidney (5.6 times after 30 days). The glucuronidation in kidney was non-sensitive to the treatment whatever substrate used. In contrast, this activity was enhanced in liver and particularly in lung, in which the glucuronidation of 1-naphthol and 2-hydroxybiphenyl was increased by 122 and 180%, respectively. Interestingly, the times of induction differed according to the substrate used, thus suggesting the presence of different UGTs active toward phenols that were differentially affected by cigarette smoke. The LIGT activities toward phenols were low in lung, when compared with those measured in liver or kidney. In conclusion, cigarette smoke greatly affected both glucuronidation activity and the hydroxylation reactions supported by CYPs in mouse liver and lung.     

Kinetics of acetaminophen glucuronidation by UDP-glucuronosyltransferases 1A1, 1A6, 1A9 and 2B15. Potential implications in acetaminophen-induced hepatotoxicity

The importance of uridine 5'-diphosphate-glucuronosyltranferases (UGT) 2B15 and other UGT enzymes (1A1, 1A6, and 1A9) in glucuronidating acetaminophen (APAP) is demonstrated. The kinetics and contributions of various UGTs in glucuronidating APAP are presented using clinically and toxicologically relevant concentrations of the substrate. UGT 1A9 and UGT 2B15 contribute significantly toward glucuronidating APAP when incubations were conducted in either phosphate or Tris-HCl buffers at 0.1 and 1.0 mM substrate concentrations. At 10 mM APAP, UGT 1A9 is a significant enzyme responsible for metabolizing APAP in either one of the buffers. UGT 1A1 is the next most important enzyme in glucuronidating APAP at this high substrate concentration. The contribution of UGT 1A6 at 10 mM APAP concentration became obscured by similar relative activities exhibited by UGTs 1A7, 1A8, and 2B7. These observations may reflect the differences in kinetic parameters for APAP glucuronidation by the individual UGTs. UGT 1A1 demonstrated Hill kinetics while UGT 1A9 displayed Michaelis-Menten kinetics. Substrate inhibition kinetics is observed with UGT 1A6, UGT 2B15, and human liver microsomes. The substrate inhibition is confirmed by employing stable isotope-labeled APAP as the substrate, while APAP glucuronide is used to test for inhibition of d4-APAP glucuronide. The in vitro hepatotoxicity caused by APAP in combination with phenobarbital or phenytoin is demonstrated in this study. The inhibition of APAP glucuronidation by phenobarbital leads to an increase in APAP-mediated toxicity in human hepatocytes. The toxicity to hepatocytes was further increased by coadministering APAP with phenytoin and phenobarbital. This synergistic increase in toxicity is postulated to be due to inhibition of UGTs (1A6, 1A9, and 2B15) responsible for detoxifying APAP through the glucuronidation pathway.