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The transgender adult population is growing globally, but clinical pharmacology has lagged behind other areas of transgender medicine. Medical care for transgender adults may include long-term testosterone or estrogen treatment to align secondary sex characteristics with gender identity. Clinicians often use drug-drug interaction data from the general adult population to predict medication disposition or safety among transgender adults. However, this approach does not address the complex pharmacodynamic effects of hormone therapy in transgender adults. In this review, we critically examine sex-related and gender-related differences in clinical pharmacology and apply these data to discuss current gaps in transgender medicine., (© 2021 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Metribuzin is a herbicide that inhibits photosynthesis and has been used for over 40 years. Its main target organ is the liver and to some extent the kidney in rats, dogs, and rabbits. Metribuzin shows a specific thyroxine (T4) profile in rat studies with T4 increases at low doses and T4 decreases at higher doses. Only the T4 decreases occur together with histopathological changes in the thyroid and weight changes of liver and thyroid. A set of experiments was conducted to investigate metribuzin's endocrine disruptor potential according to European guidance and regulations. The results indicate that a liver enzyme modulation, i.e. of the uridine 5'-diphospho-glucuronosyltransferase (UDPGT, UGT), is most likely responsible for both increased and decreased plasma thyroxine level and for thyroid histopathological observations. Animals with high T4 levels show low UGT activity, while animals with low T4 levels show high UGT activity. A causal relationship was inferred, since other potentially human-relevant mode of action (MOA) pathways were excluded in dedicated studies, i.e. inhibition of deiodinases (DIO), inhibition of thyroid peroxidase (TPO) or of the sodium importer system (NIS). This liver metabolism-associated MOA is considered not relevant for human hazard assessment, due to species differences in thyroid homeostasis between humans and rats and, more importantly, based on experimental data showing that metribuzin affects UGT activity in rat but not in human hepatocytes. Further, we discuss whether or not increased T4 levels in the rat, in the absence of histopathological changes, should be considered as adverse and therefore used as an appropriate hazard model for humans. Based on a weight of evidence approach, metribuzin should not be classified as an endocrine disruptor with regard to the thyroid modality., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Polygoni Multiflori Radix, the dried root of Polygonum multiflorum Thunb., and its processed products have been used as restoratives for centuries in China. However, the reports of Polygoni Multiflori Radix-induced liver injury (PMR-ILI) have received wide attention in recent years, and the components and mechanism of PMR-ILI are not completely clear yet. Our previous studies found that the PMR-ILI was related to the down-regulation of some drug metabolism enzymes (DME)., Aim of the Study: To explore the effect of the inhibition of CYP3A4 or UGT1A1 on PMR-ILI, screen the relevant hepatotoxic components and unveil its mechanism., Methods: RT-qPCR was used to detect the effects of water extract of Polygoni Multiflori Radix (PMR) and its main components on the mRNA expression of CYP3A4 and UGT1A1 in human hepatic parenchyma cell line L02. High-performance liquid chromatography (HPLC) was employed to detect the content of major components in the PMR. And then, the stable CYP3A4 or UGT1A1 knockdown cells were generated using short hairpin RNAs (shRNA) in L02 and HepaRG cells. Hepatotoxic components were identified by cell viability assay when PMR and its four representative components, 2,3,5,4'-tetrahydroxy stilbene glycoside (TSG), emodin (EM), emodin-8-O-β-D-glucoside (EG), and gallic acid (GA), acted on CYP3A4 or UGT1A1 knockdown cell lines. The PMR-ILI mechanism of oxidative stress injury and apoptosis in L02 and HepaRG cells were detected by flow cytometry. Finally, the network toxicology prediction analysis was employed to excavate the targets of its possible toxic components and the influence on the metabolic pathway., Results: PMR and EM significantly inhibited the mRNA expression of CYP3A4 and UGT1A1 in L02 cells, while TSG and GA activated the mRNA expression of CYP3A4 and UGT1A1, and EG activated CYP3A4 expression while inhibited UGT1A1 expression. The contents of TSG, EG, EM and GA were 34.93 mg/g, 1.39 mg/g, 0.43 mg/g and 0.44 mg/g, respectively. The CYP3A4 or UGT1A1 knockdown cells were successfully constructed in both L02 and HepaRG cells. Low expression of CYP3A4 or UGT1A1 increased PMR cytotoxicity remarkably. Same as PMR, the toxicity of EM and GA increased in shCYP3A4 and shUGT1A1 cells, which suggested EM and GA may be the main components of hepatotoxicity in PMR. Besides, EM not only inhibited the expression of metabolic enzymes but also reduced the cytotoxicity threshold. EM and GA affected the level of ROS, mitochondrial membrane potential, Ca 2+ concentration, and dose-dependent induced hepatocyte apoptosis in L02 and HepaRG cells. The network toxicology analysis showed that PMR-ILI was related to drug metabolism-cytochrome P450, glutathione metabolism, and steroid hormone biosynthesis., Conclusion: The inhibition of mRNA expression of CYP3A4 or UGT1A1 enhanced hepatotoxicity of PMR. EM and GA, especially EM, may be the main hepatotoxic components in PMR. The mechanism of PMR, EM and GA induced hepatotoxicity was proved to be related to elevated levels of ROS, mitochondrial membrane potential, Ca 2+ concentration, and induction of apoptosis in liver cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: Nilotinib is a selective inhibitor of the BCR-ABL tyrosine kinase receptor and is used in the management of chronic myelogenous leukemia (CML). Nilotinib therapy at high doses is associated with elevated serum bilirubin levels. If the serum bilirubin level exceeds 3 times the upper limit of normal, the recommendation is to either adjust nilotinib dosage or temporarily discontinue the treatment. However, it is unclear whether hyperbilirubinemia indicates obvious liver histology damage., Patient Concerns: A 24-year-old man with confirmed CML was treated with nilotinib therapy and developed hyperbilirubinemia after the treatment. Although the first remission of the hyperbilirubinemia was achieved after dose adjustment, the hematological parameters deteriorated. Thus, we initiated an antineoplastic therapy (at the standard dose) until complete remission of the CML was achieved. The pathogenic mechanism of hyperbilirubinemia may be related to the inhibition of uridine diphosphate-glucuronosyltransferase (UGT1A1) activity. Liver histological analysis revealed no significant liver damage. In addition, the patient had no family history of hyperbilirubinemia and liver disease., Diagnosis: The patient was admitted to our hospital under the diagnosis of hyperbilirubinemia, and histopathology by liver biopsy showed no obvious damage. We also detected a UGT1A1 mutation [ex1 c.686C > A (p.Pro229Gln)] in the patient and his mother., Interventions: When the nilotinib dose was decreased to 300 mg daily, the total bilirubin (TBIL) level decreased to 30 to 50 μmol/L for 1 month. However, because the Bcr-Abl/Abl ratio did not correspond to the major molecular response (MMR; <0.1%), the nilotinib dose was readjusted to 400 mg daily. One week later, the TBIL and indirect bilirubin levels increased to 89 and 79 μmol/L, respectively. The levels of alanine transaminase and other liver functional indicators were normal., Outcomes: A Naranjo Adverse Drug Reaction (ADR) Probability Scale score of 13 indicates that hyperbilirubinemia is attributed to ADR caused by nilotinib rather than by drug-induced liver injury., Conclusion: Although reducing the nilotinib dose can alleviate the occurrence of hyperbilirubinemia, the effect of MMR is also reduced. Treatment of CML without dose adjustment or discontinuation of nilotinib therapy may be more advantageous.

In arsenic-endemic regions of the world, arsenic exposure correlates with diabetes mellitus. Multiple animal models of inorganic arsenic (iAs, as As 3+ ) exposure have revealed that iAs-induced glucose intolerance manifests as a result of pancreatic β-cell dysfunction. To define the mechanisms responsible for this β-cell defect, the MIN6-K8 mouse β-cell line was exposed to environmentally relevant doses of iAs. Exposure to 0.1-1 µM iAs for 3 days significantly decreased glucose-induced insulin secretion (GIIS). Serotonin and its precursor, 5-hydroxytryptophan (5-HTP), were both decreased. Supplementation with 5-HTP, which loads the system with bioavailable 5-HTP and serotonin, rescued GIIS, suggesting that recovery of this pathway was sufficient to restore function. Exposure to iAs was accompanied by an increase in mRNA expression of UDP-glucuronosyltransferase 1 family, polypeptide a6a (Ugt1a6a), a phase-II detoxification enzyme that facilitates the disposal of cyclic amines, including serotonin, via glucuronidation. Elevated Ugt1a6a and UGT1A6 expression levels were observed in mouse and human islets, respectively, following 3 days of iAs exposure. Consistent with this finding, the enzymatic rate of serotonin glucuronidation was increased in iAs-exposed cells. Knockdown by siRNA of Ugt1a6a during iAs exposure restored GIIS in MIN6-K8 cells. This effect was prevented by blockade of serotonin biosynthesis, suggesting that the observed iAs-induced increase in Ugt1a6a affects GIIS by targeting serotonin or serotonin-related metabolites. Although it is not yet clear exactly which element(s) of the serotonin pathway is/are most responsible for iAs-induced GIIS dysfunction, this study provides evidence that UGT1A6A, acting on the serotonin pathway, regulates GIIS under both normal and pathological conditions.

Aims: A population pharmacokinetic (PK) model was developed for cediranib to simulate cediranib exposure for different doses, including comedication with strong uridine glucuronosyl transferase/P-glycoprotein inducers such as rifampicin, in cancer patients., Methods: Plasma concentrations and covariates from 625 cancer patients after single or multiple oral cediranib administrations ranging from 0.5 to 90 mg in 19 Phase I and II studies were included in the analysis. Stepwise covariate modelling was used to develop the population PK model. The final model was used to simulate cediranib exposure in cancer patients to evaluate cediranib target coverage and the need for dose adjustment for covariates or coadministration with rifampicin., Results: A two-compartment model with sequential zero- and first-order absorption and first-order elimination adequately described the cediranib concentration-time courses. Body weight and age were identified as having statistically significant impact on cediranib PK, but only <21% impact on AUC and maximum concentrations. Simulated lower bounds of 90% prediction interval or median of unbound cediranib concentrations after cediranib 15 or 20 mg exceeded the IC 50 for vascular endothelial growth factor receptors-1, -2 and -3. Exposures of cediranib 20 or 30 mg with coadministration of rifampicin were comparable to those of 15 or 20 mg, respectively, without coadministration., Conclusions: No covariate was identified to require dose adjustment for cediranib. Cediranib exposure following 15 or 20 mg daily dose administration is adequate overall for inhibition of in vitro estimated vascular endothelial growth factor receptor-1, -2 and -3 activities. An increase in cediranib dose may be needed for cediranib coadministered with strong uridine glucuronosyl transferase/P-glycoprotein inducers such as rifampicin., (© 2017 The British Pharmacological Society.)

Inhibition of UDP-glucuronosyltransferases (UGTs) can result in many undesired side effects. Diethylstilbestrol (DES), a synthetic oestrogen famous for its multiple toxicities, was once widely administered to women in high dosages and now still gains application in clinics. This study investigated in vitro inhibitory effects of DES on catalytic activities of human UGTs, aiming at disclosing new potential toxic mechanisms on the basis of interactions between DES and metabolizing enzymes. DES (10 μM) could decrease activities of UGT1A1, 1A3, 1A9, 2B4 and 2B7 in catalysing 4-methylumbelliferone (4-Mu) glucuronidation. Further kinetic analyses showed that inhibition of these UGTs followed competitive (UGT1A1 and 1A9), mixed (UGT1A3 and 2B4) and non-competitive (UGT2B7) mechanisms, with K i values ranging from 0.91 to 4.1 μM. The inhibition potentials of UGT1A9 and 2B7 in human liver microsomes (HLM) were further tested by employing propofol and zidovudine as probe substrates, respectively. The inhibition of human liver microsomal UGT1A9 followed mixed mechanism, with the K i value of 3.5 μM and α of 4.1. On the other hand, DES displayed non-competitive inhibition against UGT2B7 in HLM, with the K i value of 9.8 μM. The risks of in vivo inhibition of human UGTs were also predicted by calculation of plasma C/K i values. Results suggest that DES can trigger in vivo inhibition of UGT1A1, 1A3, 1A9, 2B4 and 2B7 after the intravenous administration in high doses., (© 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Introduction: We have previously observed that rat synovial membranes incubated in medium containing cartilage interstitial fluid (CIF) responded by changes in the expression of hyaluronan synthases (HAS1 and HAS2), collagen type I, versican, aggrecan, lubricin, matrix metalloproteinases 2 and 3 (MMP2 and MMP3), tissue inhibitors of metalloproteinases (TIMP1, 2 and 3), transforming growth factor β1 (TGFβ1), tumor necrosis factor (TNF), interleukin (IL) 1β and IL-6. The aim of the study was to evaluate the influence of particular cytokines found in CIF on the gene expression of extracellular matrix (ECM) proteins in synovial membrane cells., Material and Methods: Synovial membranes (SMs) were removed from the knee joints of inbred, male Lewis rats and incubated with insulin-like growth factor 1 (IGF1), TGFβ1, basic fibroblast growth factor (bFGF) and granulocyte- macrophage colony-stimulating factors (G-CSF and M-CSF), either individually or in the combinations TGFβ1/IGF1, TGFβ1/IGF1/bFGF or G-CSF/M-CSF. Next, total RNA was isolated and the studied genes' expression was evaluated by real-time PCR., Results: TGFβ1/IGF1 stimulated expression of HAS1, lubricin, collagen type I, aggrecan and TGFβ1, inhibited expression of MMP3 and TNF and had no effect on TIMP1 and IL-6 mRNA levels. TGFβ1/IGF1/bFGF stimulated the expression of HAS1, lubricin and TGFβ1 genes, inhibited the expression of TNF and had no effect of the expression of collagen 1, aggrecan, MMP3, TIMP1 and IL-6 genes. G-CSF/M-CSF stimulated the expression of aggrecan. TGFβ1, bFGF, and IGF1 applied individually exerted inhibitory effect on the expression of lubricin. TGFβ1 and bFGF inhibited expression of MMP3 and bFGF inhibited also the expression of aggrecan and TNF., Conclusions: The response of the studied genes represents a resultant activity of all major cell types building the synovial membrane, i.e. highly specialized synovial fibroblasts, macrophages, epithelial cells and adipocytes. The results of our study can improve understanding of synovial membrane responses to the intraarticular injections of platelet-rich plasma (PRP) used for the treatment of joint ailments, since PRP contains factors which are also present in CIF.

Pigmented rice bran has been suggested to be a valuable source of beneficial phytochemicals. We investigated genotoxic and anti-genotoxic effects of purple rice bran extract (PRBE) in rats using a liver micronucleus assay. Purple rice bran was extracted with methanol, obtaining large amounts of phenolic compounds, including anthocyanins and small amounts of gamma-oryzanol. The experimental protocols were divided into two sets. Male rats were divided into three groups. Group 1 was a negative control, while Groups 2 and 3 were fed with 100 and 500 mg/kg bw of PRBE, respectively, for 28 days. PRBE had no effect on micronucleus formation or xenobiotic metabolizing enzymes in rat liver. Experiments concerning the effect of PRBE on AFB1 showed that PRBE significantly lessened the amount of micronucleated hepatocytes in AFB1 treated rats. Furthermore, it modulated metabolic activation of AFB1 metabolism in the liver by suppressing activity and protein expression of CYP1A2, CYP3A and CYP 450 reductase, and enhancing phase II enzymes including GST and UGT. Overall, purple rice bran extract was not genotoxic in rats. It exhibited anti-genotoxicity by modulation some xenobiotic enzymes active in AFB1 metabolism.

Herb-drug interaction strongly limits the clinical application of herbs and drugs, and the inhibition of herbal components towards important drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reasons. The present study aims to investigate the inhibition potential of andrographolide derivatives towards one of the most important phase II DMEs UDP-glucuronosyltransferases (UGTs). Recombinant UGT isoforms (except UGT1A4)-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation were employed to firstly screen the andrographolide derivatives' inhibition potential. High specific inhibition of andrographolide derivatives towards UGT2B7 was observed. The inhibition type and parameters (Ki) were determined for the compounds exhibiting strong inhibition capability towards UGT2B7, and human liver microsome (HLMs)-catalyzed zidovudine (AZT) glucuronidation probe reaction was used to furtherly confirm the inhibition behavior. In combination of inhibition parameters (Ki) and in vivo concentration of andrographolide and dehydroandrographolide, the potential in vivo inhibition magnitude was predicted. Additionally, both the in vitro inhibition data and computational modeling results provide important information for the modification of andrographolide derivatives as selective inhibitors of UGT2B7. Taken together, data obtained from the present study indicated the potential herb-drug interaction between Andrographis paniculata and the drugs mainly undergoing UGT2B7-catalyzed metabolic elimination, and the andrographolide derivatives as potential candidates for the selective inhibitors of UGT2B7., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Colon cancer is the third most global oncologic problem faced by medical fraternity. Troxerutin, a flavonoid present in tea, coffee, cereal grains, and a variety of fruits and vegetables, exhibits various pharmacological and biological activities. This study was carried out to investigate the effect of troxerutin on xenobiotic metabolizing enzymes, colonic bacterial enzymes and the development of aberrant crypt foci (ACF) during 1,2-dimethylhydrazine (DMH) induced experimental rat colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control. Group 2 received troxerutin (50 mg/kg b.w., p.o. every day) for 16 weeks. Groups 3-6 received subcutaneous injections of DMH (20 mg/kg b.w.) once a week, for the first four weeks. In addition, groups 4-6 received different doses of troxerutin (12.5, 25, 50 mg/kg b.w., p.o. every day respectively) along with DMH injections. Our results reveal that DMH treated rats exhibited elevated activities of phase I enzymes such as cytochrome P450, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase and reduced activities of phase II enzymes such as glutathione-S-transferase (GST), DT-diaphorase (DTD) and uridine diphospho glucuronyl transferase (UDPGT) in the liver and colonic mucosa of control and experimental rats. Furthermore, the activities of fecal and colonic mucosal bacterial enzymes, such as β-glucronidase, β-glucosidase, β-galactosidase and mucinase were found to be significantly higher in DMH alone treated rats than those of the control rats. On supplementation with troxerutin to DMH treated rats, the alterations in the activities of the biotransforming enzymes, bacterial enzymes and the pathological changes were significantly reversed, the effect being more pronounced when troxerutin was supplemented at the dose of 25 mg/kg b.w. Thus troxerutin could be considered as a good chemopreventive agent against the formation of preneoplastic lesions in a rat model of colon carcinogenesis., (© 2013.)

Unlabelled: Bilirubin biotransformation occurs with the participation of the glucuronyl transferase (GTF) system of the liver and hepatocyte membranes. Disturbances in these systems may result in a rise of blood bilirubin levels and disbalance between direct and indirect bilirubin leading to jaundice. Gilbert's syndrome (GS) is a genetic disorder associated with the enhanced level of indirect bilirubin due to GTF insufficiency., Materials and Methods: The study included adolescents aged 13.4 ± 0.42 yr divided into 2 groups depending on anti-Helicobacter therapy (AHBT). We measured levels of direct and indirect bilirubins, their ratio, and direct bilirubin content as percentage of total bilirubin at admission to and discharge from the hospital. The daily incremental growth of both bilirubin fractions was calculated, Results: Detailed analysis revealed negative effect of AHBT on the GTF system attributable to its impaired stability in patients with abnormal genotype., Conclusion: Prescription of AHBT to children with Gilbert's syndrome requires the thorough choice of medications and monitoring of their potential effect on the GFT system.

Pollution is one of today's greatest problems, and the release of contaminants into the environment can cause adverse changes in vitally important biological pathways. In this study, we exposed three-spined stickleback Gasterosteus aculeatus to produced water (PW), i.e. wastewater from offshore petroleum production. PW contains substances such as alkylphenols (APs) and aromatic hydrocarbons (PAHs) known to induce toxicant stress and endocrine disruption in a variety of organisms. Following exposure to PW, a standardized confinement treatment was applied as a second stressor (PW-stress), testing how fish already under stress from the pollutant would respond to an additional stressor. The endpoint for analysis was a combination of blood levels of cortisol and glucose, in addition to transcribed levels of a set of genes related to toxicant stress, endocrine disruption and general stress. The findings of this study indicate that low doses of PW do not induce vitellogenin in immature female stickleback, but do cause an upregulation of cytochrome (CYP1A) and UDP-glucuronsyltransferase (UDP-GT), two biomarkers related to toxicant stress. However, when the second stressor was applied, both genes were downregulated, indicating that the confinement exposure had a suppressive effect on the expression of toxicant biomarkers (CYP1A and UDP-GT). Further, two of the stress related genes, heat shock protein 90 (HSP90) and stress-induced phosphoprotein (STIP), were upregulated in both PW- and PW-stress-treatment, but not in the water control confinement treatment, indicating that PW posed as a larger stress-factor than confinement for these genes. The confinement stressor caused an increased level of glucose in both control and PW-treated fish, indicating hyperglycemia, a commonly reported stress response in fish., (© 2013.)

Aims: Dapagliflozin is a selective sodium glucose cotransporter 2 (SGLT2) inhibitor that decreases serum glucose by reducing renal glucose reabsorption, thereby promoting urinary glucose excretion. Dapagliflozin is primarily metabolized via the uridine diphosphate-glucuronosyltransferase (UGT)1A9 pathway to its major inactive metabolite, dapagliflozin 3-O-glucuronide. The aim of this study was to evaluate the potential for drug-drug interaction between dapagliflozin and two potential UGT1A9 modulators., Methods: The results of two open-label, non-randomized, single-sequence studies are reported in which the effects of rifampin (a pleiotropic drug-metabolizing enzyme inducer; study 1) and mefenamic acid (a strong UGT1A9 inhibitor; study 2) were evaluated on the pharmacokinetics and pharmacodynamics (assessed by urinary glucose excretion [UGE]) of dapagliflozin in healthy subjects. In study 1, 14 subjects received single doses of dapagliflozin 10 mg alone and in the presence of rifampin 600 mg QD (6 days). In study 2, 16 subjects received single doses of dapagliflozin 10 mg alone and in the presence of mefenamic acid 250 mg q6h (5 days)., Results: Rifampin reduced total exposure (area under the concentration-time curve from time 0 to infinity [AUC0-inf]) to dapagliflozin by 22% and mefenamic acid increased dapagliflozin AUC0-inf by 51%. No clinically meaningful effect of rifampin or mefenamic acid on the pharmacokinetics of dapagliflozin or on dapagliflozin-mediated urinary glucose excretion was observed., Conclusion: Modest changes in dapagliflozin exposure were seen with rifampin and mefenamic acid with minor changes in UGE, none of which were considered clinically meaningful., (© 2012 Blackwell Publishing Ltd.)

This work tests the mode-of-action (MOA) hypothesis that maternal and developmental triclosan (TCS) exposure decreases circulating thyroxine (T4) concentrations via up-regulation of hepatic catabolism and elimination of T4. Time-pregnant Long-Evans rats received TCS po (0-300mg/kg/day) from gestational day (GD) 6 through postnatal day (PND) 21. Serum and liver were collected from dams (GD20, PND22) and offspring (GD20, PND4, PND14, PND21). Serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) concentrations were measured by radioimmunoassay. Ethoxy-O-deethylase (EROD), pentoxyresorufin-O-depentylase (PROD) and uridine diphosphate glucuronyltransferase (UGT) enzyme activities were measured in liver microsomes. Custom Taqman(®) qPCR arrays were employed to measure hepatic mRNA expression of select cytochrome P450s, UGTs, sulfotransferases, transporters, and thyroid hormone-responsive genes. TCS was quantified by LC/MS/MS in serum and liver. Serum T4 decreased approximately 30% in GD20 dams and fetuses, PND4 pups and PND22 dams (300mg/kg/day). Hepatic PROD activity increased 2-3 fold in PND4 pups and PND22 dams, and UGT activity was 1.5 fold higher in PND22 dams only (300mg/kg/day). Minor up-regulation of Cyp2b and Cyp3a expression in dams was consistent with hypothesized activation of the constitutive androstane and/or pregnane X receptor. T4 reductions of 30% for dams and GD20 and PND4 offspring with concomitant increases in PROD (PND4 neonates and PND22 dams) and UGT activity (PND22 dams) suggest that up-regulated hepatic catabolism may contribute to TCS-induced hypothyroxinemia during development. Serum and liver TCS concentrations demonstrated greater fetal than postnatal internal exposure, consistent with the lack of T4 changes in PND14 and PND21 offspring. These data support the MOA hypothesis that TCS exposure leads to hypothyroxinemia via increased hepatic catabolism; however, the minor effects on thyroid hormone metabolism may reflect the low efficacy of TCS as thyroid hormone disruptor or highlight the possibility that other MOAs may also contribute to the observed maternal and early neonatal hypothyroxinemia., (Published by Elsevier Ireland Ltd.)

Highly active antiretroviral therapy (HAART) has increased the survival of HIV-infected patients. However, adverse effects play a major role in adherence to HAART. Some protease inhibitors (mainly atazanavir and indinavir) act as inhibitors of uridine diphosphate-glucuronosyltransferase (UGT1A1), the enzyme responsible for hepatic conjugation of bilirubin. Variations in the promoter region of the UGT1A1 gene (UGT1A1*28, rs8175347) can influence bilirubin plasma levels, modulating the susceptibility to hyperbilirubinemia. Aiming to analyze the association between UGT1A1*28 allele and hyperbilirubinemia in individuals exposed to HAART, we evaluated 375 HIV-positive individuals on antiretroviral therapy. Individuals carrying the UGT1A1*28 allele had a higher risk of developing severe hyperbilirubinemia [prevalence ratio (PR)=2.43, 95% confidence interval (CI) 1.08-5.45, p=0.032] as well as atazanavir users (PR=7.72, 95% CI=3.14-18.98, p<0.001). This is the first description of such an association in Brazilian HIV patients, which shows that in African-American and Euroamerican HAART users, the UGT1A1*28 allele also predisposes to severe hyperbilirubinemia, especially in those exposed to atazanavir.

Purpose: The pharmacokinetic interaction of etravirine and raltegravir is reviewed, with discussion of implications for clinical practice., Summary: Etravirine (a second-generation nonnucleoside reverse transcriptase inhibitor) and raltegravir (an integrase strand- transfer inhibitor) are two agents approved by the Federal Food and Drug Administration for use in human immunodeficiency virus (HIV) treatment-resistant patients. Minimal data exist on the concurrent use of raltegravir with etravirine. This combination would offer treatment-experienced HIV patients a novel pharmacotherapy plan including two new fully active agents. Etravirine induces uridine diphosphate- glucuronosyltransferase 1A1 and reduces the raltegravir minimum concentration (C(min)) by 34% when administered concurrently in healthy volunteers. In a case series of four HIV treatment-resistant patients initiated on an antiretroviral regimen including standard doses of etravirine and raltegravir, poor virological control was demonstrated. Two of these four patients had a raltegravir C(min) below the 95% minimum inhibitory concentration. In a larger study (n = 103), sustained virological control (viral loads of <50 copies/mL) resulted when HIV treatment-resistant patients received standard doses of darunavir, ritonavir, etravirine, raltegravir, and nucleoside analogs with or without enfuvirtide. Debate exists regarding the best raltegravir pharmacokinetic parameter to evaluate (C(min) or the area under the concentration curve/50% effective concentration). Recent data in HIV treatment-naive patients support a negative association between a low raltegravir C(min) (≤43 ng/mL) and virological suppression., Conclusion: The need to adjust the dosage of raltegravir in HIV-infected patients who are also receiving etravirine is unclear. In such patients who have an extensive history of HIV disease treatment, prescribing raltegravir 1200 mg/day, rather than the standard 800 mg/day, may be prudent to prevent the development of treatment-resistant virus and to achieve an optimal virological response.

The potential of three isothiocyanates, namely R,S-sulforaphane, erucin and phenethyl isothiocyanate, of two naturally occurring glucosinolates, namely glucoerucin and glucoraphanin, and of the enantiomers of sulforaphane to modulate glucuronosyl transferase and epoxide hydrolase, two major carcinogen-metabolising enzyme systems, was investigated in precision-cut rat liver slices. Following exposure of the slices to the isothiocyanates (0-25 μM), erucin and phenethyl isothiocyanate, but not R,S-sulforaphane, elevated glucuronosyl transferase and epoxide hydrolase activities and expression, determined immunologically. Of the two enantiomers of sulforaphane, the R-enantiomer enhanced, whereas the S-enantiomer impaired, glucuronosyl transferase activity and only the former increased protein expression; furthermore, R-sulforaphane was more effective than the S-enantiomer in up-regulating microsomal epoxide hydrolase. When precision-cut rat liver slices were exposed to the same concentrations of glucoerucin and glucoraphanin, both glucosinolates caused a marked increase in the activity and expression of the microsomal epoxide hydrolase but had no effect on glucuronosyl transferase activity. It may be inferred that the ability of isothiocyanates to enhance hepatic microsomal epoxide hydrolase and glucuronosyl transferase activities is dependent on the nature of the side chain. Moreover, in the case of sulforaphane, the naturally occurring R-enantiomer increased both activities, whereas, in contrast, activities were impaired in the case of the S-enantiomer. Finally, intact glucosinolates are potent inducers of epoxide hydrolase and can thus contribute directly to the chemopreventive potential associated with cruciferous vegetable consumption.

Aims: The only FDA approved medication for colorectal cancer (CRC) prevention is celecoxib. Its adverse effects underline the need for safer drugs. Polyphenols like resveratrol are in clinical trials for this purpose. This study aimed at examining effects of resveratrol alone and in combination with curcumin or chrysin on UGT induction in Caco-2 cells. Phytochemical combinations were selected using drug combination analyses of various anti-proliferation ratios of resveratrol+curcumin and resveratrol+chrysin., Main Methods: Cell proliferation and UGT1A1 induction assays were carried out with individual polyphenols and combinations. Cell viability was determined with AlamarBlue assays. UGT1A1 mRNA was quantified via real time RT-PCR. UGT activity was determined with 4-methylumbelliferone (4MU) glucuronidation., Key Findings: Cell proliferation IC(50) estimates (± SE) for resveratrol, curcumin and chrysin were 20.8 ± 1.2, 20.1 ± 1.1 and 16.3 ± 1.3μM respectively. Combination of anti-proliferative effects showed additivity for resveratrol+chrysin and resveratrol+curcumin. Resveratrol at its IC(50) mediated a four-fold induction of UGT1A1 mRNA in a concentration independent manner. Chrysin at its IC(50) induced UGT1A1 expression seven-fold while Curcumin at its IC(90) mediated a two-fold induction. The 20 μM:40μ M resveratrol+curcumin and 20 μM :32 μM resveratrol+chrysin combinations mediated the greatest increases in mRNA expression (12 and 22 folds respectively). Significant increase in 4-MU glucuronidation was observed with combinations exhibiting maximal mRNA induction., Significance: Phytochemical combinations can offer greater chemoprevention than single agents. These chemicals might offer safer options than present synthetic therapeutics for CRC prevention., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Antiinflammatory effect of statins mediated by the reduction of cytokine IL-6 in hepatocytes have been reported. Contrary to beneficial effect, statins can increase susceptibility to mitochondrial dysfunction. Extrahepatic biliary obstruction is associated with oxidative stress, pro-inflammatory response and hepatocyte mitochondrial dysfunction. The aim of our study was to verify the effect of fluvastatin on cholestatic liver injury. Cholestasis was induced in Wistar rats by bile duct ligation. Fluvastatin (1 or 5 mg/kg) was administered after surgery and then daily for 7 days. The dose of 5 mg/kg led to the deterioration of hepatocellular injury. Despite lower production of IL-6, decrease in GSH content, rise of TGFß and inhibition of respiratory complex I in mitochondria were determined. The mRNA expressions of canalicular transporter Mdr1b and basolateral transporter Mrp3 increased in cholestatic liver. Fluvastatin administration then led to the attenuation of this change. Analogously, mRNA expression of conjugative enzyme Ugt1a1 was diminished by fluvastatin administration to cholestatic rats. We can conclude that decrease in the antioxidative status and mitochondrial dysfunction could at least in part participate on the deteriorating effect of fluvastatin. Whether these processes can be a consequence of the alteration in metabolism and transport of potentially toxic substances remains to verify.

Irinotecan hydrochloride (CPT-11) is a useful drug for cancer chemotherapy but sometimes induces severe diarrhea clinically. CPT-11 is mainly activated to SN-38 by carboxylesterase (CES) and then detoxified to SN-38 glucuronide (SN-38G) by UDP-glucuronosyltransferase (UGT) in the liver. SN-38G is excreted via bile and de-conjugated to SN-38 by β-glucuronidase (β-GLU) in the intestinal content. In order to clarify the alleviative effect of antibiotics on CPT-11-induced diarrhea, we examined whether penicillin G and streptomycin (SM) alleviate CPT-11-induced delayed-onset diarrhea using three diarrheal models, i.e., Wistar rats with repeated dosing of CPT-11 (60 mg/kg/day i.v. for 4 consecutive days) and Wistar and Gunn rats with a single dosing of CPT-11 (200 and 20 mg/kg i.v., respectively). Gunn rats have an inherited deficiency of UGT1A and cannot conjugate SN-38 to SN-38G. Therefore, onset of CPT-11-induced diarrhea in Gunn rats is not affected by β-GLU activity. SM alleviated diarrhea in all three diarrheal models. The alleviation of diarrhea by SM in Gunn rats indicated that the effect of SM occurred by a mechanism other than the inhibition of β-GLU activity. SM decreased CPT-11 and/or SN-38 concentrations in intestinal tissues and alleviated epithelial damage from the ileum to colon. SM did not inhibit β-GLU activity in the cecal content. SM also inhibited the intestinal absorption of CPT-11 and decreased CES activity and increased UGT activity in the intestinal epithelium. These findings indicated that SM decreased the exposure of CPT-11 and SN-38 to the intestinal epithelium by inhibiting the absorption of CPT-11 from the intestinal lumen and the change of CES and UGT activities in the intestinal epithelium and alleviated delayed-onset diarrhea.

Purpose: To study the possible pharmacokinetic and pharmacodynamic interactions between irinotecan and methimazole., Methods: A patient treated for colorectal cancer with single agent irinotecan received methimazole co-medication for Graves' disease. Irinotecan pharmacokinetics and side effects were followed during a total of four courses (two courses with and two courses without methimazole)., Results: Plasma concentrations of the active irinotecan metabolite SN-38 and its inactive metabolite SN-38-Glucuronide were both higher (a mean increase of 14 and 67%, respectively) with methimazole co-medication, compared to irinotecan monotherapy. As a result, the mean SN-38 glucuronidation rate increased with 47% during concurrent treatment. Other possible confounding factors did not change over time. Specific adverse events due to methimazole co-treatment were not seen., Conclusions: Additional in vitro experiments suggest that these results can be explained by induction of UGT1A1 by methimazole, leading to higher SN-38G concentrations. The prescribed combination of these drugs may lead to highly toxic intestinal SN-38 levels. We therefore advise physicians to be very careful in combining methimazole with regular irinotecan doses, especially in patients who are prone to irinotecan toxicity.

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T(4)), thus reducing serum T(4), and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T(4) glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T(4) glucuronidation, decreased serum T(4), 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 T(4), triiodothyronine (T(3)), and TSH concentrations, hepatic T(4)/T(3) glucuronidation, and thyroid histology and follicular cell proliferation were investigated. PCN, 3-MC, and PCB treatments decreased serum T(4), whereas serum T(3) 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%). T(4) 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. T(3) glucuronidation was increased after PCN (121%) or PCB (58%) in Wistar rats, but only PCN increased T(3) glucuronidation in Gunn rats (43%). PCN treatment induced thyroid morphological changes and increased follicular cell proliferation in both strains. These data demonstrate that T(4) glucuronidation cannot be increased in Ugt1a-deficient Gunn rats. Thus, the decrease in serum T(4), increase in TSH, and increase in thyroid cell proliferation after MEI are not dependent on increased T(4) glucuronidation, and cannot be attributed to Ugt1a enzymes., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Imidazoles and triazoles represent major classes of antifungal azole derivatives. With respect to UDP-glucuronosyltransferase (UGT) enzymes, the drug metabolism focus has mainly concentrated on their inhibitory effects with little known about azoles as substrates for UGTs. N-Glucuronide metabolites of the imidazole antifungals, tioconazole and croconazole, have been reported, but there are currently no reports of N-glucuronidation of triazole antifungal agents. In this study, evidence for glucuronidation of azole-containing compounds was studied in human liver microsomes (HLM). When a glucuronide metabolite was identified, azoles were incubated in 12 recombinant UGT (rUGT) enzymes, and enzyme kinetics were determined for the UGT with the most intense glucuronide peak. Six imidazole antifungals, three triazoles, and the benzodiazepine alprazolam (triazole) were evaluated in this study. All compounds investigated were identified as substrates of UGT. UGT1A4 was the main enzyme involved in the metabolism of all compounds except for fluconazole, which was mainly metabolized by UGT2B7, probably mediating its O-glucuronide metabolism. UGT1A3 was also found to be involved in the metabolism of all imidazoles but not triazoles. In both HLM and rUGT K(m) values were lower for imidazoles (14.8-144 microM) than for triazoles (158-3037 microM), with the exception of itraconazole (8.4 microM). All of the imidazoles studied inhibited their own metabolism at high substrate concentrations. In terms of UGT1A4 metabolism, itraconazole showed kinetic features characteristic of imidazole rather than triazole antifungals. This behavior is attributed to the physicochemical properties of itraconazole that are similar to those of imidazoles in terms of clogP.

Glucuronidation is one of the major pathways of metabolism of endo- and xenobiotics. UDP-Glucuronosyltransferase (UGT)-catalyzed glucuronidation accounts for up to 35% of phase II reactions. The expression and function of UGT is modulated by gene regulation, post-translational modifications and protein-protein association. Many studies have focused on drug-drug interactions involving UGT, and there are a number of reports describing the inhibition of UGT by xenobiotics. However, studies about the role of endogenous compounds as an inhibitor or activator of UGT are limited, and it is important to understand any change in the function and regulation of UGT by endogenous compounds. Recent studies in our laboratory have shown that fatty acyl-CoAs are endogenous activators of UGT, although fatty acyl-CoAs had been considered as inhibitors of UGT. Further, we have also suggested that adenine and related compounds are endogenous allosteric inhibitors of UGT. In this review, we summarize the endogenous modulators of UGT and discuss their relevance to UGT function.

The present manuscript describes the development of a cell-based reporter transcriptional activation assay for evaluating induction of UGT1A1. A reporter construct (pGL-UGT1A1-Luc) encompassing the proximal promoter (nucleotide -254 to +38) and distal enhancer (-3483 to -3194) regions of the human UGT1A1 gene was generated by PCR cloning, and co-transfected with a previously generated PXR construct (pSG5-PXR) into HepG2 cells. The system was then validated using known ligands of PXR, rifampicin (RIF), clotrimazole (CLOT) sulfinpyrazone (SPZ) and phenobarbital (PB), which produced dose dependent induction of UGT1A1 luciferase activity by 4.4, 5.3, 4.7 and 3.7 fold, respectively, relative to the vehicle control, 0.1 % dimethylsulfoxide (DMSO). Aryl hydrocarbon receptor (AhR) ligands a-naphthoflavone (a-Naph), b-naphthoflavone (b-Naph) and 3-methylchloranthene (3-MC) increased UGT1A1 luciferase activity in a concentration dependent manner resulting in 17.2, 11.3 and 6.1 fold, respectively, at their highest concentrations, suggesting that endogenous AhR is also involved in the regulation of the UGT1A1 reporter construct in HepG2 cells. For comparison with transcriptional regulation of endogenous UGT1A1, 10 mM RIF, 50 mM SPZ, 10 mM CLOT, 4 mM 3-MC, 10 mM b-Naph and 25 mM a-Naph also induced UGT1A1 mRNA in human primary hepatocytes by 2.5, 2.8, 3.2, 3.7, 3.9 and 4.3 fold, respectively. In summary, by co-transfecting the UGT1A1 reporter and PXR constructs into HepG2 cells, we have developed a cellular model for evaluating induction of UGT1A1. Data from the reporter gene assay correlated with that generated in human primary hepatocytes. Based on these data, we suggest that this reporter gene assay can be used as a screening tool in the early stages of drug discovery, to evaluate potential induction of UGT1A1 by new chemical entities and to aid in lead selection and optimization.

Objective: The aim of this study was to explore the relationship between UGT1A7 gene polymorphisms and susceptibility to chromosomal damage among polycyclic aromatic hydrocarbons (PAH)-exposed workers., Methods: Blood and urine samples of 140 PAH-exposed workers and 66 non-PAH-exposed workers were collected. The polymorphisms of UGT1A7, cytokinesis-block micronucleus (CBMN) and 1-hydroxypyrene were analyzed., Results: UGT1A7 codon 208 Arg carriers exhibited significantly higher CBMN frequencies than did the Trp/Trp (P < 0.05). Stratification analysis revealed that the significant association between CBMN frequencies and the polymorphisms of UGT1A7 Asn129Lys, Arg131Lys, and Trp208Arg was most pronounced. Moreover, higher CBMN frequencies were found among subjects with *3 allele than those with *1 allele (P = 0.008) in younger PAH-exposed workers., Conclusion: Polymorphisms of UGT1A7 gene may alter the severity of PAH-induced chromosomal damage among the exposed workers.

The presence of drug metabolizing enzymes in extrahepatic tissues such as the choroid plexus (CP) suggests that the CP, like the blood-brain barrier, affords a metabolic protection to the brain against xenobiotics. The CP, which is the principal site of formation of the cerebrospinal fluid (CSF), controls the exchange of many endogenous compounds and exogenous molecules between brain tissue and CSF. We present the changes in mRNA expression and enzymatic activities of UDP-glucuronosyltransferase, UGT1A6 isoform and NADPH-cytochrome P450 reductase, after in vitro treatment with xenobiotic molecules known to act in the liver as inducers or inhibitors of these drug metabolizing enzymes. Five study groups of male Sprague-Dawley rats were treated separately with 3-methylcholantrene (3-MC), phenobarbital (PB), dexamethasone (DEX), cyclosporine (CsA) or paraquat (PQ). Choroidal 1-naphthol glucuronidation activities were significantly induced by 3-MC and PQ administration (354 +/- 85 and 257 +/- 49 vs. 115 +/- 24 nmol/h per mg protein, in control group), whereas the other molecules were without effect. Accordingly, UGT1A6 mRNA expression, measured by RT-PCR, was 2.3-fold higher after 3-MC treatment and 2.1-fold higher after PQ administration. By contrast, reductase activities and mRNA expression remained unchanged in the isolated choroids plexus in these experimental conditions. We present for the first time evidences that the choroids plexus express transcripts for both UGT1A6 and NADPH-cytochrome P450 reductase, and their mRNA expression can be differently regulated by exogenous factors. These results emphasize that xenobiotics could modulate the biotransformation of exogenous and/or endogenous compounds in the choroids plexus, and underline the role of UGTs in the maintenance of brain homeostasis.

Silymarin, which is extracted from the milk thistle (Silybum marianum), has been used for centuries for treating hepatic disorders and its hepatoprotective effects have been known for hundreds of years. Silymarin is a mixture of polyphenoic flavonoids, which include silibinin (silybin A and silybin B), isosilyin A and B, silychristin A and B, silydianin and other phenol compounds. The pharmacokinetics of silibinin shows fast absorption and elimination. Silymarin undergoes phase I and phase II metabolism, especially phase II conjugation reactions, it undergoes multiple conjugation reactions, and is primarily excreted into bile and urine. Silymarin has a good safety profile, but little is known regarding its potential for drug interaction. Silymarin has limited effect on the pharmacokinetics of several drugs in vivo; despite silymarin decreasing the activity of cytochrome P-450 (CYPs) enzymes, UDP-glucuronosyltransferase (UGT) enzyme, and reducing P-glycoprotein (P-gp) transport. Health-care practitioners should caution patients against co-administration of silymarin and pharmaceutical drugs.

Atazanavir (ATV) is known to inhibit UGT1A1-mediated glucuronidation. Here we report the effect of ATV and ATV/ritonavir (RTV) on another UGT1A isoenzyme, UGT1A4. Twenty-one healthy volunteers received a single dose of 100 mg of oral lamotrigine on days 1, 13, and 27; on each occasion blood was sampled before the dose was administered and through 120 h after ingestion of the drug. On days 8-17 the subjects received oral ATV 400 mg q.d. On days 18-30 the subjects received oral ATV 300 mg plus oral RTV 100 mg q.d. Seventeen subjects were evaluable for pharmacokinetic analysis. Geometric mean ratios (+90% confidence intervals (CIs)) of lamotrigine area under the plasma concentration-time curve (AUC)(0-inf) and peak plasma concentration (C(max)) for ATV + lamotrigine and for lamotrigine alone were 0.88 (0.86-0.91) and 0.99 (0.95-1.02), respectively; the corresponding ratios for ATV/RTV and for lamotrigine were 0.68 (0.65-0.70) and 0.94 (0.90-0.97), respectively. The mean ratio of lamotrigine-2N-glucuronide to lamotrigine AUC(0-inf) increased from 0.45 for lamotrigine to 0.71 for ATV/RTV + lamotrigine. ATV alone does not significantly influence glucuronidation of lamotrigine. In contrast, ATV/RTV results in moderately decreased exposure to lamotrigine.

Atazanavir use is associated with increases in serum bilirubin. Ribavirin, used to treat hepatitis-C infection, cause hemolysis and may worsen hyperbilirubinemia. We studied HIV/hepatitis-C virus-coinfected patients who initiated hepatitis-C therapy. Hyperbilirubinemia grade 3-4 increased from 9% to 45% after the start of hepatitis-C treatment in patients who used atazanavir concomitantly. Atazanavir use and hemoglobin (Hb) drops were predictors of increases in bilirubin. A substantial proportion of patients under atazanavir-therapy experienced significant hyperbilirubinemia and jaundice following initiation of hepatitis-C therapy.

Glucuronidation studies using microsomes and recombinant uridine diphosphoglucuronosyltransferases (UGTs) can be complicated by the presence of endogenous beta-glucuronidases, leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used beta-glucuronidase inhibitor, although it is not clear whether this reagent should be added routinely to glucuronidation incubations. Here we have determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and recombinant UGTs (rUGTs). Despite the use of buffered incubation solutions, it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM did not enhance any of the glucuronidation activities evaluated that could be considered consistent with inhibition of beta-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for glucuronidation of 5-hydroxytryptamine and estradiol by pHLMs, with a 35% decrease at 20 mM saccharolactone concentration. Endogenous beta-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes and insect-cell expressed rUGTs, but not for kidney, intestinal or human embryonic kidney HEK293 microsomes. However, the extent of hydrolysis was relatively small, representing only 9-19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations. If saccharolactone is used, concentrations should be titrated to achieve activity enhancement without inhibition.

Sulindac is a nonsteroidal antiinflammatory drug that has been demonstrated to be a potent chemopreventive agent against colorectal cancer in both human and animal models. In vivo, sulindac may be reversibly reduced to the active antiinflammatory compound, sulindac sulfide, or irreversibly oxidized to sulindac sulfone. Sulindac has also been shown to inhibit polycyclic aromatic hydrocarbon (PAH)-induced cancer, but the molecular mechanisms of its antitumor effect remain unclear. In this study, we investigated the effects of sulindac and its metabolites on the expression of enzymes that metabolize and detoxify PAHs in 2 human colon cancer cell lines, LS180 and Caco-2. Sulindac and sulindac sulfide induced a sustained, concentration-dependent increase in CYP enzyme activity as well as an increase in the mRNA levels of CYP1A1, CYP1A2 and CYP1B1. Sulindac and sulindac sulfide induced the transcription of the CYP1A1 gene, as measured by the level of heterogeneous nuclear CYP1A1 RNA and verified by the use of actinomycin D as a transcription inhibitor. Chromatin immunoprecipitation assays demonstrated that sulindac and sulindac sulfide also increased the nuclear level of activated aryl hydrocarbon receptor, the transcription factor which mediates CYP expression. Additionally, sulindac and both metabolites increased the activity and mRNA expression of the carcinogen detoxification enzyme NAD(P)H:quinone oxidoreductase, as well as the expression of UDP-glucuronosyltransferase mRNA. These results show an overall upregulation of carcinogen metabolizing enzymes in colon cancer cells treated with sulindac, sulindac sulfide and sulindac sulfone that may contribute to the established chemoprotective effects of these compounds., ((c) 2007 Wiley-Liss, Inc.)

Synthetic food dyes are xenobiotics, and, after ingestion, portions of these dyes may be absorbed and metabolized by phase I and II drug-metabolizing enzymes, and excreted by transporters of phase III enzymes. In the previous report, it was shown that inhibition of UDP-glucuronosyltrasnferase 1A6 occurred following ingestion of phloxine, erythrosine, and rose bengal present in 12 permitted synthetic food dyes. In this report, the influence of dyes was examined on CYP3A4, a major phase I drug-metabolizing enzyme, and P-glycoprotein, a major transporter by synthetic food dyes. Human cytochrome P-450 (CYP) 3A4 and P-glycoprotein were inhibited by xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the level of inhibition of UGT1A6 produced by three haloganated xanthene food dyes in the previous report, except acid red, which inhibited only CYP3A4. Data suggest that inhibition by dyes is not enzyme specific but may be in a membrane-specific or protein-specific manner, such as conformational changes in protein. In the previous study, it was suggested that inhibition by dyes depended upon light irradiation due to generation of (1)O2 from these dyes. In this study, the influence of superoxide dismutase and catalase on inhibition by dyes was examined. Superoxide dismutase but not catalase was effective in preventing the inhibition of UGT1A6 by the dyes. Data suggest that superoxide anions, originating from dyes via light irradiation, may attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins in skin and may lead to skin damage.

Background: Studies have reported that prenatal exposure to polychlorinated biphenyls (PCBs) may alter neurodevelopment in both humans and animals. Furthermore, prenatal exposure to some PCB congeners and commercial mixtures has been shown to decrease free and total thyroxine (T(4)) blood levels in animals. Because thyroid hormones (TH) are essential for normal neurologic development, it has been suggested that the deleterious neurodevelopmental effect of PCBs may occur through TH disruption. PCBs may in turn affect TH levels by inducing the microsomal enzyme uridinediphosphate glucuronosyltransferase (UDP-GT), which is involved in TH elimination., Objectives: Our goals were to group PCB congeners based on their potential to induce microsomal enzymes in animals, and to examine the relationship between neonatal TSH levels and prenatal exposure to PCB congeners grouped according to their structure and potential mechanisms of action., Methods: We measured the concentration of 34 PCB congeners in serum samples collected from 285 pregnant women and the thyroid-stimulating hormone (TSH) levels in their children's blood collected shortly after birth., Results: We found no association between the sum of PCB congeners, the toxic equivalents, or structure-based groupings (mono- or di-ortho substituted congeners), and TSH blood concentration. However, we found a positive association between the sum of congeners suspected to be UDP-GT inducers (more specifically cytochrome P450 2B inducers) in animals and neonatal TSH levels. In individual congener analyses, PCBs 99, 138, 153, 180, 183, 187, 194, and 199 were positively associated with neonatal TSH levels after adjustment for covariates. PCBs 194 and 199 remained significant after adjustment for multiple hypothesis testing., Conclusions: Our results support grouping PCB congeners based on their potential mechanism of action of enzyme induction when investigating associations with TH. Findings also suggest that PCBs affect TH homeostasis even at the low background level of exposure found in the CHAMA-COS (Center for the Health Assessment of Mothers and Children of Salinas) population.

We previously showed that the intake of sesamin, a major lignan in sesame seed, decreased lipid peroxidation and elevated tocopherol concentration in rat tissues. In this study, we examined the effect of dietary sesame seed and sesamin on the ascorbic acid concentration in rat tissues. Rats (4-wk-old) were fed either a vitamin E-free diet, or a diet containing 50 mg gamma-tocopherol/kg, one containing 2 g sesamin/kg, one containing 50 mg gamma-tocopherol/kg and 2 g sesamin/kg, or one containing 200 g sesame seed/kg for 28 d. The dietary sesamin and sesame seed elevated ascorbic acid concentrations in the liver and kidney, and increased urinary excretion in those Wistar rats. The dietary sesamin also elevated the hepatic mRNA levels of cytochrome P450 (CYP) 2B, and UDP-glucuronosyltransferase (UGT) 1A and 2B. In contrast, neither the sesamin nor the sesame seed affected the liver concentration of ascorbic acid in ODS rats with a hereditary defect in ascorbic acid synthesis, though the dietary sesame seed elevated the UGT1A and 2B mRNA levels in the liver. In addition, the sesame seed elevated the gamma-tocopherol concentration in the various ODS rat tissues and the ascorbic acid concentrations in the kidney, heart and lung, while reducing the thiobarbituric acid reactive substance concentration in the heart and kidney. These results suggest that dietary sesame seed and its lignan stimulate ascorbic acid synthesis as a result of the induction of UGT1A and the 2B-mediated metabolism of sesame lignan in rats. The data of ODS rat studies also suggest that dietary sesame seed enhances antioxidative activity in the tissues by elevating the levels of two antioxidative vitamins, vitamin C and E.

Objective: A number of nonsteroidal anti-inflammatory drugs (NSAIDs) are subject to glucuronidation in humans, and UDP-glucuronosyltransferase (UGT) 2B7 is involved in the glucuronidation of many NSAIDs. The objective of this study was to identify a NSAID with potent inhibitory potential against UGT2B7 using liquid chromatography with tandem mass spectrometry (LC-MS/MS)., Methods: A rapid screening method for detecting the inhibitory potential of various drugs against UGT2B7 was established using a LC-MS/MS system. The effects of nine NSAIDs (acetaminophen, diclofenac, diflunisal, indomethacin, ketoprofen, mefenamic acid, naproxen, niflumic acid, and salicylic acid) against UGT2B7-catalyzed 3'-azido-3'-deoxythymidine glucuronidation (AZTG) were investigated in human liver microsomes (HLM) and recombinant human UGT2B7., Results: Mefenamic acid inhibited AZTG most potently, with an IC(50) value of 0.3 microM, and its inhibition type was not competitive. The IC(50) values for diclofenac, diflunisal, indomethacin, ketoprofen, naproxen, and niflumic acid against AZTG were 6.8, 178, 51, 40, 23, and 83 microM, respectively, while those for acetaminophen and salicylic acid were >100 microM. The IC(50) values for NSAIDs against AZTG in recombinant human UGT2B7 were similar to those obtained in HLM., Conclusion: The method established in this study is useful for identifying drugs with inhibitory potential against human UGT2B7. Among the nine NSAIDs investigated, mefenamic acid had the strongest inhibitory effect on UGT2B7-catalyzed AZTG in HLM. Thus, caution might be exercised when mefenamic acid is coadministered with drugs possessing UGT2B7 as a main elimination pathway.

The advances in genetics and molecular biology have raised new areas in medicine, such as pharmacogenomics, which tries to predict drug responses and toxicities based on the individual genetic variability, describing the so called: pharmacogenomic syndromes. Oncology would find this development extremely useful because of the severe toxicity of chemotherapy. There are a lot of genetic loci under investigation for their potential in predicting drug toxicity, but only three of them have showed clinical usefulness up to now. In particular, quantification of the number of thymine-adenine (TA) dinucleotics in the promoter region of the UDP-glucuronosyl-transferase 1A1 enzime (TA indel) proved to be capable of predicting severe neutropenia in patients exposed to intermediate or high doses of irinotecan. Herein we report a case of a patient with small cell lung cancer who suffered severe hematological and gastrointestinal toxicity after being treated with relatively low doses (65 mg/m(2)) of irinotecan and whose leucocyte DNA analysis showed the presence of seven TA repetitions in both alleles. This case is an example of the clinical applicability and the utility of the test as a toxicity predictor. We also discuss the clinical decisions that may be taken with these patients.

To prevent rapid forming O-glucuronide of propofol (2,6-diisopropylphenol), an intravenous anesthetic, effects of propofol analogs were investigated. Propofol was predominately glucuronidated by human UGT1A9 and intestinal or liver microsomes. 2,5-Diisopropylphenol inhibited the propofol glucuronidation. A possibility of developing orally administrable agents or of reducing propofol dose by coadministration is suggested.

An integrated systems biology approach of measuring mRNA, protein and enzyme activity, was used to determine the molecular mechanisms responsible for reductions in thyroid hormone levels observed in rats given 1000 mg/kg/day of a nonsteroidal progesterone agonist (NSP). The effect of NSP on drug metabolizing enzyme (DME) expression was determined in livers from treated and vehicle control rats. In treated males, CYP1A1, CYP2B1, CYP2B2, CYP2C12, CYP3A1 and UGT1A mRNAs increased by 2.2, 31.0, 9.4, 13.0, 6.4 and 2.3 fold, while CYP2C11 and CYP3A2 levels decreased by 4.8 and 15.0 fold respectively. CYP1A, CYP2B and UGT1A enzyme activities increased by 2.9, 6.2 and 1.4 fold while CYP2C and CYP3A activities decreased by 2.2 and 1.8 fold respectively. CYP2B and CYP2C proteins increased by 2.1 and 1.3 fold but CYP2C11, the male-specific isozyme, and CYP3A protein decreased by 2.0 and 1.4 fold respectively. In treated females, CYP1A, CYP2B, CYP2C, CYP3A and UGT activities increased by 1.9, 12.0, 23.0, 13.0 and 2.2 fold respectively; with corresponding increases in mRNA ranging from 1.5 to 783 fold. CYP2B, CYP2C and CYP3A proteins increased by 3.6, 2.2 and 6.4 fold respectively, but CYP2C11 remained unchanged. These data suggest that NSP modulates the transcriptional regulation DME in rats and could account for the observed reductions in thyroid hormones, since UGT conjugation is the main pathway of thyroid hormone elimination in rats. These data also show gender and isozyme-specific regulation of some genes, thus demonstrating the value of an integrated approach in determining the contribution of individual genes in drug safety and metabolism observations.

To elucidate the roles of the transcription factor NF-E2-related factor (Nrf2) in hepatocarcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a mutagenic and carcinogenic heterocyclic amine, Nrf2-deficient mice were treated with 300 p.p.m. IQ in their diet for 1, 4 or 52 weeks. In the long-term experiment, the multiplicity and incidence of liver tumors in male and female IQ-treated Nrf2 deficient (-/-) mice were significantly higher than those in their counterpart wild-type (+/+) mice exposed to IQ. In the short-term experiment, although IQ exposure to Nrf2(+/+) mice of both sexes did not modify UDP-glucuronosyltransferase values, glutathione S-transferase values were significantly increased due to IQ treatment, in contrast to no alteration in male and female Nrf2(-/-) mice. Levels of oxidative stress markers such as 8-hydroxydeoxyguanosine and thiobarbituric acid reactive substances in the livers of all treated mice were not changed by IQ treatment. IQ-specific DNA adduct levels were elevated only in female Nrf2(-/-) mice, although the increase was not significant. IQ treatment caused an increase in proliferating cell nuclear antigen labeling indices only in male Nrf2(-/-) mice. The present data clearly show that Nrf2(-/-) mice of both sexes are susceptible to IQ hepatocarcinogenicity, which might result from IQ accumulation due to failure of metabolizing enzyme induction. In addition, inconsistent results concerning IQ-specific adducts and proliferating cell nuclear antigen labeling indices in male and female Nrf2(-/-) mice suggest the existence of different contributions of Nrf2 to IQ hepatocarcinogenesis between mice of the two sexes.

To elucidate the mechanism underlying suppression by curcumin of esophageal carcinogenesis induced by NMBA, we evaluated the CYP level and mutagenic activation of environmental carcinogens, by immunoblot analyses and Ames preincubation test, respectively, and bilirubin, 4-nitrophenol and testosterone UDPGT activities in F344 rats treated with curcumin and/or NMBA. No significant alterations in the hepatic levels of constitutive CYP proteins, mutagenic activation by liver S9 or hepatic UDPGT activities were produced by subcutaneous treatment with 0.5 mg/kg NMBA for 5 weeks and/or feeding of 0.05% and 0.2% curcumin for 6 weeks. In contrast, gavage of 0.2% curcumin decreased esophageal CYP2B1 and 2E1 by up to 60%, compared with vehicle control. Similarly, intragastric treatment with 270 mg/kg curcumin decreased esophageal and gastric CYP2B1 and CYP2E1, but not in lung, kidney or intestine. Conversely, large intestinal CYP2B1 was 2.8-fold higher in the treated rats than in control rats. Mutagenic activities of NOC, including NMBA, in the presence of esophagus and stomach S9 were markedly decreased in the treated rats, whereas those in the presence of large intestine S9 were 2.2-3.0-fold above control. These results show that modifying effects of curcumin on esophageal carcinogenesis can be attributed to a decrease in metabolic activation of NMBA by esophageal CYP2B1 during the initiation phase, without the contribution of metabolic activation and inactivation by liver. Further, the present findings suggest the potential of curcumin for modification of gastric and intestinal carcinogenesis initiated with NOC.

Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24-glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol-activated nuclear receptor liver X-receptor alpha (LXRalpha). In human hepatic cells and human UGT1A transgenic mice, LXRalpha activators induce UGT1A3 mRNA levels and the formation of CDCA-24glucuronide (24G) and LCA-24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site-directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXRalpha is found to interact with the SRC-1alpha and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC-1beta. In conclusion, these observations establish LXRalpha as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively.

Juvenile Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) were exposed for 3 weeks in a continuous water flow to 0.5 ppm of dispersed North Sea crude oil, 0.5 ppm of dispersed North Sea crude oil spiked with 0.1 ppm of a mixture of alkylphenols (offshore oil production), and 30 ppb of nonylphenol (NP). As potential markers of endocrine alteration, key enzymatic activities involved in both synthesis (17beta-hydroxysteroid dehydrogenases and P450 aromatase) and metabolism (liver UDP-glucuronosyltransferases (UGT) and sulfotransferases) of steroids were assessed together with circulating levels of testosterone and estradiol in plasma. NP-exposed turbot had lower ovarian P450 aromatase, lower levels of testosterone and estradiol in plasma, and lower glucuronidation rates of sex steroids than those from the control group. In contrast, higher liver UGT-testosterone, and a trend towards higher P450 aromatase was detected in oil-exposed specimens. Those exposed to the combination oil+alkylphenols had lower levels of estradiol in plasma than controls, and no significant effects on any the enzymatic activities tested was observed. All these alterations were more evident in turbot than in cod. In fact, apart from a higher glucuronidation rate of estradiol detected in the liver of NP-exposed cod, no significant differences were observed between control and exposed cod.

Objective: The aim of this study was to investigate the respective influence of genetic and nongenetic factors on morphine dose requirements and adverse effects after colorectal surgery., Methods: Seventy-four patients who planned to undergo colorectal surgery were included in this pilot study. The cumulative 24-hour postoperative dose of morphine and postoperative nausea or vomiting requiring the antiemetic ondansetron were the 2 clinical end points. The association of patient characteristics, A118G mu-opioid receptor (OPRM1) single-nucleotide polymorphism (SNP); T802C uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7) SNP; and 2 adenosine triphosphate-binding cassette, subfamily B, member 1 (ABCB1) (multidrug resistance 1 [MDR1]) exonic SNPs (G2677T/A and C3435T) with study end points was investigated., Results: Age, creatinine clearance, and the regular use of psychotropic agents were found to be significantly associated with postoperative morphine dose requirements by univariate analysis. Multivariate analysis identified that age (P = .01) and the use of psychotropic agents before surgery (P = .03) were positively associated with a higher rate of morphine consumption. A higher weight (P = .05) and the ABCB1 homozygous GG-CC diplotype (P = .03) were significantly associated with fewer morphine side effects by univariate analysis. The homozygous ABCB1 diplotype (GG-CC) conferred an odds ratio of 0.12 (95% confidence interval, 0.01-0.98) with regard to the use of ondansetron for postoperative nausea or vomiting. Multivariate analysis identified that the ABCB1 GG-CC diplotype was the only borderline-significant (P = .07) predictive factor of morphine side effects., Conclusion: Age and prior use of psychotropic agents are associated with postoperative morphine dose requirements. Whether ABCB1 polymorphisms might predict morphine side effects remains to be determined.

Rifampicin, an important drug in the treatment of tuberculosis, is used extensively despite its broad effects on drug-drug interactions, creating serious problems. The clinical importance of such interactions includes autoinduction leading to suboptimal or failed treatment. The concomitantly administered effects of rifampicin on other drugs can result in their altered metabolism or transportation that are metabolised by cytochromes P450 or transported by p-glycoprotein in the gastrointestinal tract and liver. This review paper summarises recent findings with emphases on the molecular mechanisms used to explain these broad drug-drug interactions. In general, rifampicin can act on a pattern: rifampicin activates the nuclear pregnane X receptor that in turn affects cytochromes P450, glucuronosyltransferases and p-glycoprotein activities. This pattern of action may explain many of the rifampicin inducing drug-drug interactions. However, effects through other mechanisms have also been reported and these make any explanation of such drug-drug interactions more complex.

A synergistic depletion of glutathione has been suggested to be one critical factor in the hepatic injury in mice induced by non-toxic doses of paracetamol (APAP) when co-administered with alpha-adrenergic agonists. Prazosin (an alpha-adrenergic antagonist) could confer hepatoprotection following a toxic APAP dose (530 mg/kg) by increasing glutathione levels and enhancing bioinactivation by glucuronidation and glutathione conjugation. The effect of prazosin pre-treatment on APAP-induced gluthathione depletion and bioinactivation in vivo was assessed. Prazosin (15 mg/kg) pre-treatment provided protection against APAP-induced hepatic injury as evidenced by a significant decrease in serum transaminase (ALT) levels after 5h (p<0.05). Interestingly, prazosin pre-treatment did not prevent the dramatic depletion of glutathione by high dose APAP and it had no effect on the quantity of the glutathione conjugate formed. However, prazosin pre-treatment caused a significant increase in recovery of the administered dose (530 mg/kg) as the glucuronide metabolite (p<0.05). UDP-glucuronosyltransferase (UGT) is involved in the bioinactivation of APAP by glucuronidation and we showed that prazosin had no effect on microsomal UGT kinetics. Thus, prazosin had no effect on either APAP-mediated glutathione depletion or the extent of APAP-glutathione conjugate formation and may be affecting other mechanisms to reduce oxidative stress caused by a toxic dose of APAP.

Hyaluronan (HA), is a high molecular mass extracellular matrix constituting connective tissue and plays a critical role in not only homeostasis but also inflammatory and wound-healing responses. In this study, we investigated the effect of fibroblast growth factor (FGF)-2 on the production of HA by human dental pulp cells (HDPC). An inhibition binding-protein assay showed that FGF-2 increased HA production by HDPC. In addition, expression of mRNA of hyaluronan synthase (HAS) 1 and HAS 2, both of which are related to the production of high molecular mass of HA, but not HAS 3, was enhanced in FGF-2-stimulated HDPC. These results provide new evidence for the involvement of FGF-2 in the regulation of HA production by HDPC possibly through HAS 1 and HAS 2.