UGT1A Enzymes Research Articles

Polymorphisms in genes encoding interleukin-10 and drug metabolizing enzymes GSTP1, GSTT1, GSTA1 and UGT1A1 influence risk and outcome in Hodgkin lymphoma

We genotyped 224 patients with Hodgkin lymphoma (HL) and 1056 healthy controls and related the risk for HL and outcome of chemotherapy treatment to polymorphisms in genes encoding interleukins and metabolizing enzymes by capillary electrophoresis. Patients with the UGT1A1 TA tandem repeat TA6/6 genotype had a poorer overall survival (OS) (relative risk [RR] 3.63, p = 0.004), and patients above 40 years with the GSTA1 AA genotype had poorer event-free survival (EFS) (RR 4.38, p = 0.003) after chemotherapy. In patients above 40 years, the IL-10 rs1800890 T-allele was associated with lower risk for HL (TT genotype vs. AA, odds ratio [OR] 0.38 [95% confidence interval 0.21–0.69], p = 0.001; AT/TT combined genotypes vs. AA, OR 0.45 [0.27–0.74], p = 0.001). The GSTP1 rs1695 A-allele reduced the risk for HL (GG vs. AG, OR 0.64 [0.42–0.99], p = 0.04; GG vs. AG/AA combined genotypes, OR 0.70 [0.47–1.04], p = 0.07), and the GSTT1 deleted genotype increased the risk for HL (OR 3.17 [1.97–5.09], p < 0.001) regardless of age.

Geographic difference in advanced gastric cancer prevalence and chemotherapy treatment results: could it really be an independent prognostic factor?

Although quite a few reports of retrospective studies have been published, definitive evidence for the source of the disparities between East and West associated with gastric cancer chemotherapy, safety, and efficacy has not been shown to date. In order to shed light on these long-lasting discussions, Hsu et al. [1] have accumulated published information from 25 randomized clinical trials and investigated geographic differences in the safety and efficacy of systemic chemotherapy for advanced gastric carcinoma. According to their meta-analysis and meta-regression analysis, patients in Asian trials have an 8.2% lower incidence of grade 3–4 neutropenia and a 2.1% lower incidence of grade 3–4 diarrhea than patients in non-Asian trials. It is true that most gastric cancer oncologists and investigators have been realizing that there might be several factors favoring Asian patients. In colon cancer clinical trials using a fluorinated pyrimidine as the key drug, Haller et al. [2] reported more grade 3–4 adverse events and discontinuation of the treatment in United States patients compared with nonUnited States patients. Focusing on East Asia and the United States, grade 3–4 gastrointestinal toxicity was significantly higher (hazard ratio 3.62, 95% confidence interval 2.11–6.20, p 0.01) in the United States. This disparity in the tolerability profiles of fluorinated pyrimidines could be explained as arising from: (1) genetic polymorphisms of genes involved in fluorinated pyrimidine metabolism, (2) differences in dietary folate intake, and (3) cultural differences in patients’ behavior in using an oral self-administered drug. Moreover, Ajani et al., according to the result of their phase I study of the oral fluorinated pyrimidine S-1, plus cisplatin, have postulated that the efficacy of the CYP26 enzyme is higher in whites than in Asians [3]. They presume that this difference causes the conversion of ftorafur to active fluorouracil (FU) to occur at a faster rate, achieving a higher AUC of FU in whites than in Asians. From these results, the S-1 dose was reduced from 80 mg/m in a Japanese study to 50 mg/m in a global trial. Also, in a genomic analysis of the UGT1A1 enzyme, which is responsible for the glucuronization and detoxification of SN-38, the active metabolite of irinotecan, the UGT1A1*28 polymorphism was detected to differ between Caucasians and Asians [4]. A Japanese study has also demonstrated that the UGT1A1*6 polymorphism, rather than the UGT1A1*28 polymorphism, is a potential predictor of toxicity caused by irinotecan in Japanese patients [5]. All these complicated findings and assumptions have now alerted clinicians on the need to establish optimal regimens of various chemotherapeutic agents for diverse cancer populations. Another finding from Hsu et al. [5] is that the frequent use of second-line chemotherapy was commonly observed in Asian trials, and this was assumed to lead to a better overall survival in Asian patients, whereas progression-free survival (PFS) is better in Western trials. This difference could be explained simply by the fact that twoto threedrug combination regimens are common as first-line chemotherapy in Western trials. In such cases, first-line chemotherapy might be able to show a better response and better PFS than the monoor two-drug combination therapies routinely used in Asia. After the failure of first-line chemotherapy that has exhausted all promising This editorial refers to the article doi:10.1007/s10120-011-0106-5.

Glucuronidation of trans-3′-hydroxycotinine by UGT2B17 and UGT2B10

Trans-3'-Hydroxycotinine (3HC) and its glucuronide are major nicotine metabolites excreted in the urine of smokers and other tobacco users. Although several members of the UDP-glucuronosyltransferase (UGT) family of enzymes were previously shown to be active in catalyzing the formation of 3HC and its glucuronide, a comprehensive screening of all known human UGT1A and 2B enzymes for glucuronidation activity against 3HC was not previously performed. In the present study, human liver microsomes (HLM), eight UGT1A and six UGT2B enzymes were screened for activity against 3HC. UGT2B17 exhibited the highest O-glucuronidation activity, exhibiting a four-fold lower (P<0.005) KM (8.3 mmol/l) compared with that observed for UGTs 1A9 (35 mmol/l) or 2B7 (31 mmol/l) and a KM smaller compared with that observed for human liver microsomes (HLM; 26 mmol/l). The KM for 3HC-O-Gluc formation was 3.1-fold lower (P<0.0005) in HLM from male participants exhibiting the wild-type genotype UGT2B17 (*1/*1) compared with that in HLM from participants homozygous for the UGT2B17 deletion genotype [UGT2B17 (*2/*2)]. Both UGTs 2B10 and 1A4 exhibited 3HC-N-Gluc formation activity, with UGT2B10 exhibiting a four-fold lower (P<0.05) KM (13 mmol/l) compared with that observed for UGT1A4 (57 mmol/l) and, which was similar to the KM observed in HLM (14 mmol/l). There was 91 (P<0.0001) and 39% (P<0.001) decreases in the 3HC-N-Gluc formation activities in HLM from participants with the UGT2B10 (*2/*2) and UGT2B10 (*1/*2) genotypes, respectively, compared with that of HLM from participants with the wild-type UGT2B10 (*1/*1) genotype. These results suggest that UGT2B17 and UGT2B10 play key roles in the glucuronidation of 3HC in the human liver and that functional polymorphisms in UGT2B17 and UGT2B10 are associated with significantly reduced glucuronidation activities against 3HC.

Evaluation of the role of UGT1A1 genotype testing in colorectal cancer patients administered irinotecan and the occurence of grade 3 and 4 neutropenia.

412 Background: Irinotecan is metabolized primarily in the liver by the carboxylesterase enzyme to the SN-38 active metabolite and is then inactivated through conjugation by the UGT1A1 enzyme, a polymorphic enzyme. Individuals genotyped with the homozygous allele have an increased risk for grade 3 and 4 neutropenia. Methods: A retrospective cohort analysis was conducted in the Kaiser Permanente California regions. The study period was November 1, 2005 to July 1, 2010 and included patients that were 18 years of age or older, newly initiated on irinotecan for colorectal cancer and had no previous irinotecan therapy within six months of the initiation dose. Patients were excluded if they were enrolled in clinical trials, on granulocyte-colony stimulating factor prophylaxis, and genotype tested for UGT1A1 and subsequently not treated with irinotecan. Patients tested with the UGT1A1 assay were grouped according to their genotype results: wild-type, heterozygous, or homozygous*28. The incidence of grade 3 and 4 neutropenia were compared among patients tested for UGT1A1 variant alleles by their genotype results. Results: A total of 305 (28%) patients were tested with the UGT1A1 assay with a mean age of 62 (+/- 12) years, and 52% of the population female. There were 161 (53%) wild-type, 123 (40%) heterozygous, and 21 (7%) homozygous patients. The median irinotecan dose was 150 mg/m2 (124-180 mg/m2) and median number of irinotecan cycles were 6 (3-12). The wild-type, heterozygous, homozygous*28 population had a 21% (33/161), 24% (29/123), and 48% (10/21) rate of grade 3 and 4 neutropenia. When the homozygous*28 group was compared to the heterozygous and wild-type genotype the adjusted Cox Proportional Hazard was 3.05 (95% CI, 1.55-5.99), p = 0.001. The Kaplan-Meier Log Rank Test yielded a p-value of 0.002. Conclusions: The adjusted risk for homozygous genotyped patients was three times higher compared to the wild-type and heterozygous group for grade 3 and 4 neutropenia. Additional investigational studies examining the benefits of UGT1A1 genotyping as a prognostic test and further effect of dosage adjustments in UGT1A1*28 homozygous initiated on irinotecan therapy are needed.

Predictors of Irinotecan Toxicity and Efficacy in Treatment of Metastatic Colorectal Cancer

The colorectal cancer ranks high among the malignant tumours in incidence and mortality and irinotecan is standardly used in palliative treatment of metastatic disease in every therapeutic line. Unfortunately, the treatment with irinotecan is often associated with severe toxicities, especially neutropenia and diarrhea. The majority of the toxic manifestation is caused by the insufficient deactivation (glucuronidation) of irinotecan active metabolite SN-38 by UGT1A enzyme. The elevated SN-38 plasma concentration is responsible for the hematological and gastrointestinal toxicity that can become life-threatening. The patients carrying the mutation of the gene encoding UGT1A enzyme lack the ability of bilirubin glucuronidation, and suffer from the inherited un-conjugated hyperbilirubinemia (Gilbert syndrome, Crigler-Najjar type 1 and 2 syndrome). The mutations in other enzyme systems also play role in the etiopathogenesis of the irinotecan toxicity: CYP3A (cytochrome P-450), ABC family of transmembrane transporters (adenosine-triphosphate binding cassette). The goal of the contemporary research is to determine the predictive factors that will enable the individual adjustment of the individual drug dosage while minimising the adverse effects and maintaining the treatment benefit.

Abstract A93: Safety, efficacy and pharmacokinetics of two schedules of navitoclax (ABT-263) in combination with irinotecan: Preliminary results of an open-label phase 1 study.

Abstract Introduction: Navitoclax (Nav), a novel BH3 mimetic that binds with high affinity and inhibits antiapoptotic Bcl-2 family proteins, enhances chemotherapy activity by lowering the apoptotic threshold. Preclinically, navitoclax enhanced irinotecan (Ir) efficacy in colon, gastric, pancreatic, and small cell lung cancer tumor models. Methods: This phase 1 study in cancer patients (pts), assessed safety, pharmacokinetics (PK), and tumor activity of Nav in combination with Ir on 2 different schedules. Eligible pts had an ECOG performance score of ≤2 and a malignancy refractory to standard therapy. Pts were assigned to 1 of 2 dosing schedules. A continual reassessment method for dose escalation was used. Pts were assigned to 21-day (D)-cycle dosing schedules, either 3-week (Q3W) (Nav D1–3 [18 off]; Ir D1) or weekly (W) (Nav D1–3 and D8–10 [11 days off]; Ir D1 and D8). Oral Nav was dosed once daily starting at 150 mg in both schedules; Ir was dosed intravenously over 90 min at 180, 250 or 350 mg/m2 (Q3W), and over 45 min at 75 or 100 mg/m2 (W). PK was assessed for Nav and Ir; safety was assessed per NCI-CTCAE v3.0; disease response/progression was assessed per standard of care for each tumor type (RECIST, PSA, CEA, CA-125, etc). Results: 31 pts (15 women, 16 men) were enrolled; median age (range) 58 years (31–73); median (range) prior therapies was 4 (1–17). The most common adverse events (Q3W and W) that were Nav-related, irrespective of relationship to Ir, were diarrhea 77% (86% and 71%), nausea 45% (50% and 41%), vomiting 39% (29% and 47%), fatigue 32% (21% and 41%), and febrile neutropenia 26% (36% and 18%). Dose-limiting toxicities (DLT) (Q3W and W) were reported by 11 pts (6 and 5), and included febrile neutropenia 7 pts (5 and 2), diarrhea 7 pts (4 and 3), fatigue 2 pts (0 and 2), and colitis 1 pt (1 and 0). The recommended phase 2 dose (RPTD) in the W schedule was Nav 150 mg and Ir 75 mg/m2. The lowest level explored on the Q3W schedule (Nav 150 mg and IR 180 mg/m2) was determined to be not tolerable and therefore the highest administrated dose exceeded a maximum tolerated dose (MTD). Tumor response is available for 17 pts (Q3W and W); 2 had a partial response (1 Merkel cell carcinoma and 1 colon cancer; neither pt had received prior Ir), 6 had stable disease (2 and 4), 9 had progressive disease (PD) (5 and 4). Objective response rate was 6.5% [95% CI: 0.8, 21.4]. 19 pts discontinued treatment due to PD, 7 due to AEs, 3 withdrew consent, and 1 due to DLT (1 pt is still on study). Median [95% CI] time on study (Q3W and W) was 25 days [10, 32] (25 days [3, 32], 26 days [3, 53]). Preliminary results indicate no apparent PK interaction between Nav and Ir. The average SN-38 exposure appeared to be higher in pts with DLTs. After febrile neutropenia was observed in the first dose level explored, the study was amended to include only those pts with functional UGT1A1 enzyme activity. The frequency of UGT1A1 genotypes (%) was *1/*1 (32); *1/*28 (52); *28/*28 (13); *28/*37 (3). Conclusions: The RPTD in the W schedule was Nav 150 mg and Ir 75 mg/m2. The Q3W schedule had a high rate of toxicities and an MTD was not identified. Despite limiting enrollment to those with functional UGT1A1 enzyme activity, the SN-38 exposures remained highly variable and may have contributed to the high rate of toxicities observed. The 2 tumor responses were notable in this highly chemotherapy-refractory pt population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A93.

In vitro interaction of a novel neutrophil growth factor with human liver microsomal cytochromes P450 and the contribution of UDP-glucuronosyltransferases to its metabolism

Neutrophil growth factors (NGFs) stimulate neutrophil growth and survival. The first synthetic cytokinin-derived NGF was recently discovered and is a prospective drug owing to its potential use in anti-inflammatory therapy. The metabolism of some cytokinin-derived drugs (e.g. R-roscovitine, olomoucine II) has already been studied and it has been shown that they may give rise to drug-drug interactions.In this in vitro study, the interactions of the novel neutrophil growth factor NGF1568 with two of the main classes of human drug-metabolizing enzymes, cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), were tested. Of the CYPs evaluated, NGF1568 was found to inhibit only CYP2C9, by an uncompetitive mechanism and with a Ki value of 349 μM.Formation of a glucuronide of NGF1568 was detected by LC/MS/MS analysis after it was incubated with human liver microsomes and UDP-glucuronic acid. The human recombinant UGT1A9 enzyme (major liver expression) and UGT1A7, UGT1A8, UGT1A10 enzymes (expressed in gastrointestinal tract instead of liver) were found to be responsible for NGF1568 glucuronidation.These results show that interaction of NGF1568 with CYPs is not as important as it is in the case of the cytokinin CDK inhibitors R-roscovitine and olomoucine II, but the conjugation enzymes (UGTs) play a major role in its metabolism. Thus, possible interference of NGF1568 with metabolism of other coadministered drugs at least on level of liver, kidney or intestinal UGTs should be thoroughly considered.

Is Gilbert Syndrome a new risk factor for breast cancer?

Patients with Gilbert Syndrome have an impaired function of the enzyme UGT1A1, responsible for the degradation of 4-OH-estrogens. These elements are produced by the degradation of estrogens and are well-known carcinogens. In theory, patients with Gilbert Syndrome accumulate 4-OH-estrogens and, therefore, might have a higher risk for breast cancer, especially when exposed to higher levels of estrogens. If this theory is true, a new risk group for breast cancer would be described, producing new insights in breast carcinogenesis.

Dissecting the Genetic Heterogeneity of Gallbladder Stone Formation

Gallstone disease affects almost 20% of individuals in Westernized countries. As its incidence in the developing countries is rising considerably, currently, it is the second most common gastroenterological condition worldwide. Gallstone formation is driven by an interaction between genetic and environmental risk factors. Previous studies have demonstrated that the genetic background accounts for ~25% of the total disease risk. Linkage and case-control studies of candidate genes and recent genome-wide studies have identified multiple lithogenic genes, in particular the hepatocanalicular cholesterol transporter ABCG5/G8 and the bilirubin conjugating enzyme UGT1A1, as major genetic determinants of gallstones in humans. In this review, we summarize the recent findings related to the genetics of cholelithiasis, update the "inventory" of human lithogenic genes, and relate the genetic studies to the pathobiologic background of the disease. In closing, future applications of genetic testing for gallstone carriers and asymptomatic family members are addressed.

Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined.The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification.Hepatocytes and microsomes formed silybin A-7-O-β-d-glucuronides, B-7-O-β-d-glucuronides, A-20-O-β-d-glucuronides and B-20-O-β-d-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation.The predominant formation of 7-O-β-d-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

Severe insomnia related to high concentrations of raltegravir

Severe insomnia related to high concentrations of raltegravir

Deteriorating effect of fluvastatin on the cholestatic liver injury induced by bile duct ligation in rats

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.

Metabolic Pathways of the Camptothecin Analog AR-67

7-tert-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67; also known as DB-67) is a novel lipophilic camptothecin analog in early-phase anticancer clinical trials. In support of these studies, we evaluated the metabolism of AR-67 in vitro and identified potential metabolites in patient samples. The lactone form of AR-67 was found to be preferentially metabolized over AR-67 carboxylate in human microsomes. Subsequently, the lactone form was tested as a substrate in a panel of CYP450 and UDP-glucuronosyltransferase (UGT) enzymes known to metabolize the majority of clinically approved molecules. AR-67 was metabolized by CYP3A5, CYP3A4, CYP1A1, and CYP1A2, in order of activity. Extrahepatic UGT1A8 and UGT1A7 possessed at least 6-fold higher metabolizing activity than UGT1A1 and other UGT enzymes tested. CYP1A1 and UGT1A7 displayed Michaelis-Menten kinetics, whereas CYP3A4, CYP3A5, and UGT1A8 displayed kinetics consistent with substrate inhibition. Chromatographic analysis of representative patient plasma and urine samples demonstrated the presence of AR-67 glucuronides and oxidized products in the urine but only in very minimal amounts. We conclude that limited in vivo metabolism of AR-67 by UGT1A1 may partly explain the absence of AR-67 glucuronides in plasma and hypothesize that UGT1A8- and CYP3A-mediated biotransformation within the gastrointestinal epithelium may provide protective mechanisms against AR-67 gastrointestinal toxicity.

1029 Polymorphic Variants of Ugt1A1 in Neonatal Jaundice in Southern Brazil

Background and aims: Neonatal jaundice seems a benign condition but babies must be monitored in order to identify those who are at higher risk of encephalopathy. Abnormalities in hepatic conjugation of bilirubin were proposed in those cases - polymorphisms of the hepatic enzyme UGT1A1. Herein, we estimated the frequency of alleles and of genotypes of the promoter region of UGT1A1 gene in newborns and evaluated its association with severe hyperbilirubinemia.Methods: Case-control study, including all neonates who were in phototherapy at NICU/HCPA from March to December 2007, born > 35w of gestational age and weight > 2000g. Controls were neonates not jaundiced. Informed consent form applied. PCR performed and analyzed in GeneMapper® program.Results (490 NBs included): There were 7 of the 10 possible UGT1A1 genotypes identified; those related to Gilbert's syndrome were in 16%. Prevalence of polymorphic genotypes in icteric patients was 13.5% and in normal patients was 18.2%, p=0.08. In 86.6% of white neonates - lowrisk genotypes, 13.4%, Gilbert's genotypes, while among blacks, 34% showed the latter genotypes, and 76%, low-risk (p=0.001).Conclusions: In the present sample the presence of polymorphic variants of UGT1A1 could not be associated to severe hyperbilirubinemia. Variants showed allelic prevalence and frequencies similar to those observed in other populations, with a highlight to the genotypes of higher risk in blacks and mulattos. Possibly due to the high miscegenation found in our state, other factors and genic interactions should be sought in order to explain severe neonatal hyperbilirubinemia, including the study of other polymorphisms.

Inhibitory effects of commonly used herbal extracts on UGT1A1 enzyme activity

Commonly used herbal supplements were screened for their potential to inhibit UGT1A1 activity using human liver microsomes. Extracts screened included ginseng, echinacea, black cohosh, milk thistle, garlic, valerian, saw palmetto, and green tea epigallocatechin gallate (EGCG). Estradiol-3-O-glucuronide (E-3-G) formation was used as the index of UGT1A1 activity.All herbal extracts except garlic showed inhibition of UGT1A1 activity at one or more of the three concentrations tested. A volume per dose index (VDI) was calculated to estimate the volume in which the daily dose should be diluted to obtain an IC50-equivalent concentration. EGCG, echinacea, saw palmetto, and milk thistle had VDI values >2.0 L per dose unit, suggesting a higher potential for interaction.Inhibition curves were constructed for EGCG, echinacea, saw palmetto, and milk thistle. IC50 values were (mean ± SE) 7.8 ± 0.9, 211.7 ± 43.5, 55.2 ± 9.2, and 30.4 ± 6.9 µg/ml for EGCG, echinacea, saw palmetto, and milk thistle extracts, respectively.Based on our findings, inhibition of UGT1A1 by milk thistle and EGCG and to a lesser extent by echinacea and saw palmetto is plausible, particularly in the intestine where higher extract concentrations are anticipated. Further clinical studies are warranted.

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 (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.

Differentiation of HepG2 and Huh7 cells using dimethyl sulfoxide

The secondary hepatocytes HepG2 and Huh7 are phenotypically poorly differentiated compared with hepatocytes in situ, limiting their use in drug development. This study assessed the efficacy of dimethyl sulfoxide (DMSO) to promote mature hepatocyte‐like differentiation. mRNA of two hepatocyte maturity markers (albumin (ALB) and transferrin (TF)), one of immaturity (α‐fetoprotein (AFP)), and key metabolic enzymes CYP3A4 and UGT1A1, was measured by qPCR. Responses to DMSO were normalised to confluent untreated cells (CUC). 1% DMSO increased ALB and TF mRNA in both cell lines with maximal expression at 15 to 20 days, followed by a return to control levels (eg. HepG2: ALB: confluent untreated cells (CUC): 1.0 ± 0.1; 15d: 6.6 ± 2.3, p&lt;0.05. TF: CUC: 1.0 ± 0.2; 20d: 13.8 ± 0.6, p&lt;0.05). DMSO did not reduce AFP mRNA in either cell line. CYP3A4 mRNA was low in both cell lines and not affected by DMSO. 2% DMSO increased UGT1A1 mRNA in both cell lines (HepG2: CUC: 1.0 ± 0.2; 5d: 2187.0 ± 153.0, p&lt;0.05. Huh7: CUC: 1.0 ± 0.2; 28d: 4.0 ± 0.3, p&lt;0.05). The data indicate these cell lines may effectively be differentiated to provide suitable models for early stage drug testing. Funded by BBSRC and Sanofi‐Aventis.

The clinical application of UGT1A1pharmacogenetic testing: Gene-environment interactions

Over the past decade, the number of pharmacogenetic tests has increased considerably, allowing for the development of our knowledge of their clinical application. The uridine diphosphate glucuronosyltransferase 1A1 gene (UGT1A1) assay is an example of a pharmacogenetic test. Numerous variants have been found in UGT1A1, the main conjugating enzyme of bilirubin and drugs such as the anticancer drug irinotecan. Recently, the US Food and Drug Administration (FDA) recommended testing for the presence of UGT1A1*28, an allele correlated with decreased transcriptional activity, to predict patients at risk of irinotecan toxicity. The administration of other drugs -- such as inhibitors of the UGT1A1 enzyme -- can clinically mimic the *28 phenotype, whereas inducers of UGT1A1 can increase the glucuronidation rate of the enzyme. The *28 polymorphism is not present in all ethnicities at a similar frequency, which suggests that it is important to study different populations to determine the clinical relevance of testing for UGT1A1*28 and to identify other clinically relevant UGT1A1 variants. Environmental factors such as lifestyle can also affect UGT1A1 activity. This review is a critical analysis of studies on drugs that can be affected by the presence of UGT1A1*28, the distribution of this polymorphism around the globe, distinct variants that may be clinically significant in African and Asian populations and how lifestyle can affect treatment outcomes that depend on UGT1A1 activity.

Modulation of the Human Glucuronosyltransferase UGT1A Pathway by Splice Isoform Polypeptides Is Mediated through Protein-Protein Interactions

This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.

Interactions between human UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A enzymes

Glucuronidation catalyzed by UDP-glucuronosyltransferase (UGT) enzymes is an important pathway in the metabolism of drugs as well as environmental chemicals. In this study, protein–protein interactions between human UGT2B7 and UGT1As and their effects on the enzymatic activities were investigated using double expression systems in HEK293 cells (UGT2B7/UGT1A1, UGT2B7/UGT1A4, UGT2B7/UGT1A6, and UGT2B7/UGT1A9). Native-PAGE analysis clearly revealed that UGT2B7 forms homo-oligomers. Furthermore, hetero-oligomers of UGT2B7 with UGT1As were observed by native-PAGE analysis. Immunoprecipitation assay revealed associations of UGT2B7 with UGT1A1, UGT1A4, UGT1A6, and UGT1A9. The thermal stability of UGT2B7 was significantly increased by the coexpressed UGT1A1, UGT1A4, UGT1A6, and UGT1A9, indicating an interaction between UGT2B7 and the UGT1As. To examine the effects of the protein–protein interactions on the enzymatic activities, kinetic analyses were performed. Coexpression of the UGT1As significantly decreased Km and increased Vmax of zidovudine O-glucuronidation by UGT2B7. Coexpression of UGT2B7 also affected the kinetics of estradiol 3-O-glucuronidation by UGT1A1, imipramine N-glucuronidation by UGT1A4, serotonin O-glucuronidation by UGT1A6, and propofol O-glucuronidation by UGT1A9. In conclusion, it was clearly demonstrated that human UGT2B7 interacts with UGT1A enzymes, affecting their kinetics. That such interactions might occur in human liver microsomes underscores the complexities in glucuronidations in human liver. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:442–454, 2010