Hepatic UDP-glucuronosyltransferase Research Articles

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): Role of UGT1A1,1A3 and 1A8

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.

The Interaction of UGT1A, HO1 and α-Thalassemia Variants with Bilirubin Levels and Gallstones in Sickle Cell Disease.

Chronic hyperbilirubinemia is common in patients with sickle cell disease (SCD), frequently resulting in the formation of gallstones. This hyperbilirubinemia (predominantly unconjugated) is attributed to hemolysis that exceeds the conjugating capacity of the hepatic UDP-glucuronosyltransferase (UGT1A) enzyme. Previous studies have shown that genetic variants ([TA]n repeats) in the promoter region of the UGT1A gene have a major influence on the levels of bilirubin and gallstones. Alpha-thalassemia, which is associated with reduced hemolysis, has also been shown to affect bilirubin levels. Another potential modulating factor is heme-oxygenase, a rate-limiting enzyme in the heme catabolic pathway that results in the production of bilirubin. While the severity of jaundice and cholelithiasis in patients with SCD is predisposed by the inheritance of certain variants of the UGT1A gene, inconsistencies have been observed. We propose that some of these inconsistencies may be explained by the modulating effects of genetic variants of HO1 and α-thalassemia. A total of 263 patients with SCD attending specialist clinics in two hospitals were studied: King's 116 SS, 5 Sβ0 and 59 SC; St Thomas' 83 SS. 81 ethnically matched subjects were recruited as controls (HbAA). Groups were age and sex matched. Cholelithiasis data, ascertained by liver ultrasound, was available for a subset of SCD patients (76 SS, 4 SC). Samples were genotyped for variants of UGT1A, HO1 and α-thalassemia. The different genetic allele distributions were statistically similar between groups. Data was analysed according to the sum of [TA] repeats on both alleles of UGT1A. A range of 10–15 [TA] repeats was observed. For HO1, the median sum of repeats on both alleles was 63, so samples were grouped as <63 or ≥ 63. α-thalassemia genotypes were as follows: SS, 127 αα/αα, 55 αα/α-, 11 α-/α-; SC, 37 αα/αα, 20 αα/α-; controls 51 αα/αα, 22 αα/α-, 4 α-/α-. Median bilirubin levels varied as expected between groups according to β-globin genotype and were as follows: King's SS 42 (15–269.5); St Thomas' SS 52.5 (15.5–696.5); King's SC 22 (10–81.8); AA Controls 10 (5–24), median (range) mmol/L. Regression analysis showed that serum bilirubin levels were strongly associated with UGT1A repeat length in all subjects (p<0.0001). Furthermore, the increase in serum bilirubin (21.3% mean for SS/Sβ0, 20.5% for SC) and cholelithiasis risk odds (86.5% mean for SS/Sβ0, 67.6% for SC) could be quantified per [TA] repeat. HO1 genotype did not affect serum bilirubin in SCD patients or the control cohort. The presence of α- thalassemia correlated (negatively) with serum bilirubin in SCD patients (p<0.0001) but not controls. This is the first time the relationship between UGT1A [TA] repeat length, serum bilirubin and cholelithiasis has been shown quantitatively. Additionally, co-existing α-thalassemia appears to moderate bilirubin levels but HO1 variants do not.

The Contribution of Intestinal UDP-Glucuronosyltransferases in Modulating 7-Ethyl-10-hydroxy-camptothecin (SN-38)-Induced Gastrointestinal Toxicity in Rats

Life-threatening diarrhea afflicts a considerable percentage of patients treated with irinotecan, an anticancer agent with effects elicited through its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). The primary detoxification pathway for SN-38 is glucuronidation. The purpose of this study was to evaluate the role that intestinal UDP-glucuronosyltransferases (UGTs) have from hepatic UGTs in modulating this diarrhea. To investigate this, Gunn rats devoid of UGT1A activity were injected with recombinant adenoviral vectors expressing UGT1A1, 1A6, and 1A7, resulting in reconstituted hepatic UGT expression comparable to a heterozygote. Hepatic microsome studies indicated that 4 to 7 days after adenoviral injection, transfected Gunn rats (j/jAV) had SN-38 glucuronide (SN-38G) formation rates three times higher than control heterozygote rats (j+AV). The adenovirus did not impart any glucuronidating capacity to the intestine in j/jAV rats, whereas j+AV rats possessed intestinal UGT function. After the administration of 20 mg/kg/day irinotecan i.p. to j/jAV rats 4 days after adenovirus injection, diarrhea ensued before the fourth irinotecan dose. j+AV rats were spared the diarrhea, and the toxicity was mild compared with the j/jAV rats, as measured by diarrhea scores, weight loss, and histological assessments of the cecum and colon. The pharmacokinetics of irinotecan, SN-38, and SN-38G indicate that the systemic exposure of SN-38 and SN-38G was higher and lower, respectively, in j/jAV rats. Despite this, the biliary excretion of irinotecan and metabolites was similar. Because intestinal UGTs are the main discriminating factor between j/jAV and j+AV rats, their presence seems to be critical for the gastrointestinal protection observed in j+AV rats.

Differences between the Antioxidation Effects of Butylated Hydroxyanisole and Butylated Hydroxytoluene on Acetaminophen - Induced Hepatic Metabolism in Rats

The effects of dietary administration of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on acetaminophen (APAP)-induced hepatic metabolism in rats were examined. The administration method involves providing the rats with 0.5% each of BHA and BHT separately added to their diets for 7 days. Based on the results of the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities for dietary administration, BHA and BHT in the diets fully prevented APAP-induced hepatotoxicity (500 mg / kg IP). By adding BHA and BHT to feedstuffs, hepatic UDP-glucuronosyltransferase (UDP-GTase) remained activated in the rats. The excreted amount of APAP-glucuronide increased and the residual APAP declined in the urine of the rats. After APAP administration in BHA or BHT pretreated rats, the excretion in plasma reached the largest amount for APAP-glucuronide at 2-4 h and APAP-sulfate at 6 h. It was clear that BHT excelled over BHA in playing the role of promoting hepatic metabolism. Data thus obtained showed the proposed different metabolisms in APAP-induced hepatotoxicity between BHA and BHT.

Gilbert's disease and atazanavir: From phenotype to UDP-glucuronosyltransferase haplotype

Gilbert's disease leads to intermittent non-hemolytic hyperbilirubinemia by a reduction of hepatic bilirubin glucuronidation associated with the presence of the UDP-glucuronosyltransferase (UGT) 1A1*28 polymorphism. It is considered benign because it does not result in hepatocellular damage. However, pharmacogenetic analyses have linked UGT1A1*28 to drug toxicity and cancer predisposition. The protease inhibitor atazanavir (ATV) is an inhibitor of hepatic UGT activity leading to hyperbilirubinemia in individual patients. Whether this is linked specifically to UGT1A1*28 or to more complex variants influencing glucuronidation is unclear. One hundred and six ATV-treated patients were characterized and genotyped for UGT1A1*28, the UGT1A3 (-66C) and UGT1A7 (-57G) promoter variants, and UGT1A7(129K/131K). ATV treatment increased median bilirubin levels from 10 to 41 micromol/L (P = .001) with hyperbilirubinemia exceeding 43 micromol/L in 37%. Hyperbilirubinemia over 43 micromol/L was significantly associated not only with UGT1A1*28 but also with UGT1A3-66C, UGT1A7-57G, and UGT1A7(129K/131K), although these variants do not naturally occur in linkage dysequilibrium in blood donors. Homozygous combinations of UGT1A1*28 with the other variants increased from 7.4% (normal bilirubin to 42 micromol/L) to 41% to 46.1% (43 to >85 micromol/L), and 100% (>85 micromol/L). All six patients with hyperbilirubinemia greater than 85 micromol/L were homozygous for all four variants identifying a haplotype inherited on a single allele. In conclusion, the genetic variant associated with Gilbert's disease is identified as part of a haplotype of four UGT1A variants spanning three genes at the UGT1A gene locus. This haplotype predisposes to hyperbilirubinemia in ATV treatment and may have an additional role as a pharmacogenomic risk factor for drug therapy.

EXPRESSION OF UDP-GLUCURONOSYLTRANSFERASE ISOFORM mRNAS DURING INFLAMMATION AND INFECTION IN MOUSE LIVER AND KIDNEY

Inflammation or infection down-regulates the activity and expression of cytochrome P450 (P450) enzymes involved in hepatic drug clearance, possibly altering drug effectiveness and leading to toxicity. The regulation of UDP-glucuronosyltransferases (UGTs) in inflammation and infection is less well characterized. To determine the response of hepatic and renal UGTs during inflammation and infection, mice were administered either saline or 1 mg/kg lipopolysaccharide (LPS) (16 h), or Citrobacter rodentium by oral gavage (6 days). Hepatic mRNA expression of UGT1A1, 1A9, and 2B5 was similarly down-regulated after LPS exposure and C. rodentium infection, whereas UGT1A2 and 1A6 mRNAs were unchanged. Effects of C. rodentium infection did not require a functional Toll-like receptor 4. Conversely, renal UGT isoforms were relatively unaffected, except for UGT2B5 induction after LPS treatment. Regulation of UGTs during the inflammatory response exhibits similarities to and differences from regulation of P450s, and may be cytokine-mediated.

Curcumin modulates drug metabolizing enzymes in the female Swiss Webster mouse

Curcumin, the yellow pigment found in turmeric, exhibits potent chemopreventative properties in both in vivo and in vitro cancer models. We hypothesized that this effect may occur via curcumin-mediated changes in enzymes involved in both carcinogen bioactivation and estrogen metabolism. Female Swiss Webster mice were treated with either curcumin (200 mg/kg or 400 mg/kg, p.o.) or vehicle control for 1 or 2 weeks. The results demonstrated that curcumin had no effect on the catalytic activities of ovarian aromatase, hepatic catechol- O-methyltransferase or hepatic UDP-glucuronosyltransferase. However, both doses of curcumin caused a 25% decrease in CYP1A catalytic activity, but not polypeptide levels, following 2 weeks of treatment. Additionally, following 2 weeks of curcumin at 400 mg/kg, there was a 20% decrease in the catalytic activity and a 28% decrease in polypeptide levels of CYP3A. While 2 weeks of curcumin treatment (400 mg/kg) caused a 20% increase in glutathione S-transferase activity, there was no parallel increase in hepatic stores of the co-factor glutathione. In conclusion small changes in CYP1A, CYP3A and GST following long term treatment (2 weeks) suggest that the combination of all three metabolic pathways may play a small role in curcumin's chemopreventative action.

Therapeutic Lentivirus-Mediated Neonatal in Vivo Gene Therapy in Hyperbilirubinemic Gunn Rats

Crigler–Najjar type 1 disease (CN-1) is a genetic disorder characterized by high levels of unconjugated bilirubin due to the absence of hepatic UDPglucuronosyltransferase (UGT1) activity. Here we show that in vivo neonatal hepatocyte transduction with a lentiviral vector expressing the defective enzyme resulted in long-term correction in Gunn rats, a model of CN-1. Lentiviral vectors harboring the human UGT1 cDNA (approved symbol UGT1A1) under the control of a liver-specific transthyretin promoter were produced. Two-day-old Gunn rats were injected with 50 μl of vector. Bilirubinemia was monitored at 6 weeks and monthly thereafter. At 6 weeks, bilirubinemia was completely normalized in treated animals, whereas it remained around 100 μM in control rats. The level of correction remained stable for up to 42 weeks. Large amounts of bilirubin conjugates were present in the bile of corrected animals. PCR and Western blots confirmed the presence and expression of UGT1 in liver. The estimated proportion of transduced hepatocytes was 40% and transduced cells were not detected in extrahepatic tissues except bone marrow in some animals. This work represents the first demonstration of a complete and permanent correction of hyperbilirubinemia in Gunn rats using lentiviral vectors.

METABOLISM OF N-HYDROXYGUANIDINES (N-HYDROXYDEBRISOQUINE) IN HUMAN AND PORCINE HEPATOCYTES: REDUCTION AND FORMATION OF GLUCURONIDES

The biotransformation of N-hydroxydebrisoquine, a model substrate for N-hydroxyguanidines, was studied in vitro with cultured and characterized porcine and human hepatocytes. The objective of the present work was to compare the N-oxidative and N-reductive metabolism of this compound using a monolayer culture system with previously described microsomal studies and to investigate the phase 2 metabolism, in particular, the glucuronidation of this class of compounds. At the same time, the suitability of pig hepatocytes as a model system for the human metabolism could be investigated. Two glucuronides of the parent compound N-hydroxydebrisoquine were analyzed. For the first time, one of these phase 2 metabolites could be identified as an O-glucuronide of an N-hydroxyguanidine by comparing it to a synthesized authentic compound. The involvement of certain human UDP-glucuronosyltransferases (UGTs) was evaluated by incubating the substrate with eight human hepatic recombinant UGT enzymes. Metabolites were determined by a newly developed LC-MS (liquid chromatography/mass spectrometry) analysis using electrospray ionization (ESI). The known microsomal reduction of the N-hydroxylated compound was also demonstrated with hepatocytes. The N-hydroxylation of the corresponding reduced compound (debrisoquine), which was previously described with microsomes, could not be detected in hepatocytes. There was no qualitative difference in the formation of the described derivatives by human and porcine hepatocytes. All phase 2 metabolites identified in hepatocyte culture were also formed by glucuronosyltransferases. In culture, the N-reduction of the N-hydroxylated substrate is the dominating reaction, indicating a predominance of N-reduction in vivo.

896. Complete and Permanent Correction of Hyperbilirubinemia in Newborn Gunn Rats by Lentiviral-Mediated Gene Transfer

Background: Crigler Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-Glucuronosyl transferase (UGT1A1, EC 2.4.1.17). The hallmark of CN1 is a very high level of circulating unconjugated bilirubin that may result in kernicterus. The only curative treatment of CN1 is orthotopic liver transplantation. The Gunn rat is the model for CN1 and many previous studies have attempted to correct hyperbilirubinemia in the Gunn rat using a large array of gene transfer vectors. However in most cases phenotypic correction was transient, mainly because of immune response directed against the transduced cells. Here we show that in vivo neonatal hepatocyte transduction with lentiviral vectors resulted in long-term complete phenotype correction in Gunn rats.

Metabolism of benzamidoxime (N-hydroxyamidine) in human hepatocytes and role of UDP-glucuronosyltransferases

N-Hydroxyamidines (amidoximes) can act as pro-drugs of amidines (e.g. ximelagatran, a novel direct thrombin inhibitor). This known pro-drug principle is based on the N-reduction of an oral bioavailable amidoxime to its active form. Previous study of the metabolism of the model substrate benzamidoxime by pig hepatocytes demonstrated the formation of benzamidoxime-O-glucuronide in addition to the well-established N-reduction. The objective of the present work was to investigate the glucuronidation of benzamidoxime by using cultivated cryopreserved human hepatocytes. Furthermore, the involvement of human UDP-glucuronosyltransferases (UGTs) was examined by incubating benzamidoxime in the presence of eight human hepatic recombinant UGT enzymes. Metabolites were analysed by liquid chromatography/mass spectrometry using electrospray ionization and compared with authentic synthetic compounds. For the first time, the O-glucuronidation of benzamidoxime was demonstrated in cultures of human hepatocytes. UGT1A9 is the most efficient enzyme conjugating benzamidoxime, whereas the conversion activities of UGT1A1 and UGT1A3 were 60-fold lower. Human hepatocytes form two non-mutagenic compounds: benzamidine, as the predominating metabolite, and benzamidoxime-O-glucuronide to a lesser extent. N-oxidation of benzamidine was not detected.

Successful gene therapy of the Gunn rat byin vivo neonatal hepatic gene transfer using murine oncoretroviral vectors

Crigler-Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-glucuronosyl transferase (UGT1), resulting in high levels of unconjugated bilirubin. CN1 is an attractive candidate disease for gene therapy. Here we show that in vivo neonatal hepatocyte transduction using recombinant oncoretroviral vectors results in long-term and complete phenotype correction in Gunn rats, a model for CN1. Two-day-old newborn Gunn rats were injected via the temporal vein with 200 microL UGT1 or control beta-galactosidase retroviral vectors. In UGT1-injected animals, bilirubinemia was normal at 6 weeks (3 micromol/L) and remained in the normal range (i.e., <10 micromol/L) for more than 34 weeks. In contrast, in beta-galactosidase-injected animals as well as in noninjected controls, bilirubinemia remained at a high level (i.e., >100 micromol/L) during the whole experimental follow-up. Large amounts of bilirubin monoglucuronides and diglucuronides were present in the bile of treated animals. Finally, polymerase chain reaction and reverse transcription polymerase chain reaction analysis as well as Western blot confirmed the presence and expression of UGT1 almost exclusively in the liver. The estimated proportion of transduced hepatocytes was in the range of 5% to 10%. In conclusion, complete and permanent correction of hyperbilirubinemia in newborn Gunn rats using retroviral vectors can be obtained, paving the way for future gene therapy for CN1.

Protective effects of Brussels sprouts, oligosaccharides and fermented milk towards 2-amino-3-methylimidazo[4,5- f]quinoline (IQ)-induced genotoxicity in the human flora associated F344 rat: role of xenobiotic metabolising enzymes and intestinal microflora

We investigated the chemoprotective effects of four common constituents of the human diet, i.e. a fermented milk, inulin, oligofructose and Brussels sprouts, towards 2-amino-3-methylimidazo[4,5- f]quinoline (IQ)-induced genotoxicity in male Fischer 344 rats harbouring a human intestinal microflora. We found that the four dietary components significantly reduced IQ-induced DNA damage in hepatocytes (reduction ranged from 74% with inulin to 39% with Brussels sprouts) and colonocytes (reduction ranged from 68% with inulin to 56% with Brussels sprouts). This chemoprotective effect correlated with the induction of hepatic UDP-glucuronosyl transferase following Brussels sprouts consumption, and with alterations of bacterial metabolism in the distal gut (acidification, increase of butyrate proportion, decrease of β-glucuronidase activity) following inulin consumption.

Effect of chronic renal failure on UDP-glucuronosyltransferase (UGT) enzymes in liver and kidney

In Chronic Renal Failure (CRF) patients, the hepatic clearance of many drugs is altered, which could be due to downregulation of hepatic enzymes. UGT1A6 is an important Phase II enzyme involved in the glucuronidation of planar phenols. The purpose of this study was to determine the catalytic activity and protein levels of rat hepatic and renal UGT1A6. CRF was induced in male Sprague-Dawley rats (n=4) by 5/6th Nephrectomy model in which two-thirds of left kidney and complete right kidney were excised and animals were sacrificed after 5 weeks; control animals (n=5) were sham operated and pair fed. UGT1A6 activity was determined by the glucuronidation of 4-methylumbelliferone (4MU) and Vmax and Km values were determined for hepatic UGT1A6. Protein content of UGT1A6 was examined by Western blot using a polyclonal antibody. There was no difference between CRF and CPF rats in hepatic and renal 4MU UGT activity (CRF liver: 32.08±4.82 nmole/min/mg, CPF liver: 30.26±5.54 nmole/min/mg; CRF kidney: 14.30±5.04 nmole/min/mg, CPF kidney: 13.63±5.55 nmole/min/mg) and UGT1A6 protein level. No difference was observed in the Vmax and Km values between the two groups. UGT1A6 protein levels were similar between the liver and the kidney. However the UGT1A6 activity was ~2-fold higher in the liver than the kidney, suggesting that 4MU may also be metabolized by other UGT isozymes in liver. CRF does not appear to influence the protein levels or catalytic activity of UGT1A6. Clinical Pharmacology & Therapeutics (2004) 75, P86–P86; doi: 10.1016/j.clpt.2003.11.327

Effect of food restriction on UDP-glucuronosyltransferase (UGT) enzymes in liver

Purpose The influence of diet and nutritional status on drug metabolism has been widely documented. Hepatic drug metabolism enzymes can be profoundly altered in patients suffering from malnutrition and starvation. In this research we have studied the effect of food restriction on UDP-glucuronosyltransferase (UGT) enzymes in rat liver. Methods Male Sprague-Dawley rats were maintained on a powdered commercial diet. Rats in control group (C, n=4) had ad libitum access to food and water. Animals in food restriction group (FRG, n=5) were fed with 60% of food intake of the control group. After 5 weeks, the rats were sacrificed and liver microsomes were prepared. Protein content was assessed by Western blot using polyclonal antibody for rat UGT1A1, human UGT1A6, and rat UGT1A7. Results The body weight of the FRG rats was reduced by 35% compared to the C rats. Hepatic and renal organ function was normal between the two groups. Western blot results revealed that antibody against human UGT1A6 cross-reacted with rat UGT1A6. The polyclonal antibodies were found to be specific for the UGTs. Food restriction did not have any influence on protein content of hepatic UGT1A1 and UGT1A7. However, the UGT1A6 protein levels were significantly lower (p<0.05) in the FRG rats (~3-fold reduction) compared to control animals. Conclusion Food restriction appears to selectively downregulate hepatic UGT enzymes in male rats. Clinical Pharmacology & Therapeutics (2004) 75, P85–P85; doi: 10.1016/j.clpt.2003.11.326

Correlation between the UDP-Glucuronosyltransferase ( UGT1A1 ) TATAA Box Polymorphism and Carcinogen Detoxification Phenotype

Of the hepatic UDP-glucuronosyltransferases (UGTs), only UGT1A1 and UGT1A9 exhibit activity against benzo(a)pyrene-trans-7R,8R-dihydrodiol [BPD(-)], precursor to the highly mutagenic anti-(+)-benzo(a)pyrene-7R,8S-dihydrodiol-9S,10R-epoxide. The UGT1A1*28 allelic variant contains an additional (TA) dinucleotide repeat in the "TATAA" box [(TA)(6)>(TA)(7)] of the UGT1A1 promoter that has been linked to decreased expression of the UGT1A1 gene and decreased bilirubin conjugation, leading to the relatively nondebilitating condition known as Gilbert's syndrome. To determine whether the UGT1A1 TATAA box polymorphism may play a role in the overall glucuronidation of BPD(-) in humans, we compared UGT1A1 TATAA box genotype with BPD(-) glucuronidating activity in normal liver microsomes. Significant decreases in UGT1A1 protein (P < 0.005) and bilirubin conjugation activity (P < 0.001) were observed in liver microsomes from subjects homozygous for the UGT1A1*28 allelic variant compared with subjects homozygous for the wild-type UGT1A1*1 allele. Significant decreases in BPD(-) glucuronidation activity (P < 0.02) were observed in subjects with the UGT1A1(*28/*28) genotype compared with subjects having the wild-type UGT1A1(*1/*1) genotype in assays of liver microsomes that included 0.1 mM alpha-naphthylamine, a competitive inhibitor of UGT1A9 and not UGT1A1. Similar phenotype:genotype correlations were observed when we compared subjects with the UGT1A1(*28/*28) genotype with subjects having the UGT1A1(*1/*28) genotype. In assays with alpha-naphthylamine, the K(m) of liver microsomes against BPD(-) was similar to that reported for UGT1A1-overexpressing baculosomes (319 micro M versus 290 micro M; Fang et al., Cancer Res., 62: 1978-1986, 2002). These data suggest that the UGT1A1 TATAA box polymorphism plays a role in an individual's overall ability to detoxify benzo(a)pyrene and in cancer risk.

03/02605 Predicting the heating value of MSW with a feed forward neural network: Dong, C. et al. Waste Management, 2003, 23, (2), 103–106

PCB 180 (2,2′,3,4,4′,5,5′-heptachlorobiphenyl) is a persistent and accumulating polychlorinated biphenyl abundantly present in food and the environment. In this study, we used highly purified PCB 180 (dioxinlike impurities: 2.7 ng TEQWHO/g PCB 180) in a 28-day toxicity study in young adult Sprague-Dawley rats. Male and female rats were given total doses of 3, 10, 30, 100, 300, 1000 or 1700 mg/kg b.w. PCB 180 by gavage. Increased liver weights were observed at ≥300 mg/kg b.w. in males and females. No increases in serum ALT or ALP activities were found. A significant increase in liver pentoxyresorufin O-dealkylase (PROD) activity was found in males at ≥10 mg/kg b.w. and in females at ≥30 mg/kg b.w. In both genders, a significant induction of hepatic 7-ethoxyresorufin O-deethylase (EROD) activity was also observed in males at ≥10 mg/kg b.w. and in females at ≥300 mg/kg b.w. Western blotting showed that mainly cytochromes P450 (CYPs) 2B1/2 and 3A1 were induced while slight effects were seen on CYP1A1, CYP1A2 and CYP1B1. However, no induction of CYP1A1, 1A2 and 1B1 was found on the mRNA level, except for a slight effect in females at 1000 mg/kg b.w. Furthermore, hepatic UDP-glucuronosyltransferases (UGTs) 1A1 and 1A6 were markedly induced in males and slightly induced in females. The hepatic concentrations of apolar retinoids were decreased in males at ≥30 mg/kg b.w. and in females at ≥300 mg/kg b.w. Taken together our findings show that pure PCB 180 leads to hepatic changes in a dose range which did not cause CYP1A1 induction but causes centrilobular liver hypertrophy, affects drug-metabolizing enzymes involved in the metabolism of exogenous and endogenous substrates and leads to changes in liver retinoid levels. A benchmark dose (BMD) approach is presented in order to model lowest effective dose levels for these effects. Comparison of PCB 180 liver level related to BMDL5 for hepatic hypertrophy in rats with human data on ‘total′ hepatic PCB levels in individuals without history of specific exposure suggests a relatively small margin of tissue burden in the range of 37-fold. Our results show that the highly pure non dioxin-like PCB 180 exerted strong effects different to dioxin-like compounds and that the low TEQ contamination allowed a characterization of the PCB as non-dioxinlike.

03/02601 Occupational hygiene and health of workers at South Kuzbass open-pit coal mine: Oleshchenko, A. M. et al. Meditsina Truda i Promyshlennaya Ekologiya, 2002, (10), 40–43. (In Russian)

PCB 180 (2,2′,3,4,4′,5,5′-heptachlorobiphenyl) is a persistent and accumulating polychlorinated biphenyl abundantly present in food and the environment. In this study, we used highly purified PCB 180 (dioxinlike impurities: 2.7 ng TEQWHO/g PCB 180) in a 28-day toxicity study in young adult Sprague-Dawley rats. Male and female rats were given total doses of 3, 10, 30, 100, 300, 1000 or 1700 mg/kg b.w. PCB 180 by gavage. Increased liver weights were observed at ≥300 mg/kg b.w. in males and females. No increases in serum ALT or ALP activities were found. A significant increase in liver pentoxyresorufin O-dealkylase (PROD) activity was found in males at ≥10 mg/kg b.w. and in females at ≥30 mg/kg b.w. In both genders, a significant induction of hepatic 7-ethoxyresorufin O-deethylase (EROD) activity was also observed in males at ≥10 mg/kg b.w. and in females at ≥300 mg/kg b.w. Western blotting showed that mainly cytochromes P450 (CYPs) 2B1/2 and 3A1 were induced while slight effects were seen on CYP1A1, CYP1A2 and CYP1B1. However, no induction of CYP1A1, 1A2 and 1B1 was found on the mRNA level, except for a slight effect in females at 1000 mg/kg b.w. Furthermore, hepatic UDP-glucuronosyltransferases (UGTs) 1A1 and 1A6 were markedly induced in males and slightly induced in females. The hepatic concentrations of apolar retinoids were decreased in males at ≥30 mg/kg b.w. and in females at ≥300 mg/kg b.w. Taken together our findings show that pure PCB 180 leads to hepatic changes in a dose range which did not cause CYP1A1 induction but causes centrilobular liver hypertrophy, affects drug-metabolizing enzymes involved in the metabolism of exogenous and endogenous substrates and leads to changes in liver retinoid levels. A benchmark dose (BMD) approach is presented in order to model lowest effective dose levels for these effects. Comparison of PCB 180 liver level related to BMDL5 for hepatic hypertrophy in rats with human data on ‘total′ hepatic PCB levels in individuals without history of specific exposure suggests a relatively small margin of tissue burden in the range of 37-fold. Our results show that the highly pure non dioxin-like PCB 180 exerted strong effects different to dioxin-like compounds and that the low TEQ contamination allowed a characterization of the PCB as non-dioxinlike.

Effect of spironolactone on the expression of rat hepatic UDP-glucuronosyltransferase

Spironolactone (SL) increases the glucuronidation rate of several compounds. We analyzed the molecular basis of changes occurring in major rat liver UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform of family 2, in response to SL. UGT activity toward bilirubin, ethynylestradiol and p-nitrophenol was assayed in native and activated microsomes. Protein and mRNA levels were determined by Western and Northern blotting. The lipid composition and physicochemical properties of the microsomal membrane were also analyzed. Glucuronidation rates of bilirubin and ethynylestradiol (at both 3-OH and 17β-OH positions), determined in UDP- N-acetylglucosamine-activated membranes, were increased in SL group. Western blot analysis revealed increased levels of UGT1A1 and 1A5 (bilirubin and 3-OH ethynylestradiol conjugation), and 2B1 (17β-OH ethynylestradiol conjugation). Northern blot studies suggested transcriptional regulation by the steroid. Analysis of UGT activity in native vs. alamethicin-activated microsomes indicated increased latency, which was not associated to changes in physicochemical properties of the microsomal membrane. p-Nitrophenol glucuronidation rate and mRNA and protein levels of UGT1A6, the main isoform conjugating planar phenols, were not affected by the inducer. The data suggest transcriptional regulation of specific isoforms of hepatic UGT by SL, thus explaining previously reported increases in UGT activity toward selective substrates.

Functional characterization of human UDP-glucuronosyltransferase 1A9 variant, D256N, found in Japanese cancer patients.

SN-38 (7-ethyl-10-hydroxycamptothecin), an active metabolite of the antitumor prodrug irinotecan, is conjugated and detoxified to SN-38 10-O-beta-d-glucuronide by hepatic UDP-glucuronosyltransferase (UGT) 1A1. Recent studies have revealed that other UGT1A isoforms, UGT1A7 and UGT1A9, also participate in SN-38 glucuronidation. Although several genetic polymorphisms are reported for UGT1A1 and UGT1A7 that affect the SN-38 glucuronidation activities, no such polymorphisms have been identified for UGT1A9. In the present study, UGT1A9 exon 1 and its flanking regions were sequenced from 61 Japanese cancer patients who were all treated with irinotecan. A novel nonsynonymous single nucleotide polymorphism was identified in UGT1A9 exon 1, heterozygous 766G>A resulting in the amino acid substitution of D256N. The wild-type and D256N UGT1A9s were transiently expressed at similar protein levels in COS-1 cells, and their membrane fractions were characterized in vitro for the glucuronidation activities toward SN-38. The apparent Km values were 19.3 and 44.4 microM, and the Vmax values were 2.94 and 0.24 pmol/min/mg of membrane protein for the wild-type and D256N variant, respectively. The SN-38 glucuronidation efficiency (normalized Vmax/Km) of D256N was less than 5% that of wild-type UGT1A9. These results clearly indicate that the D256N variant is essentially nonfunctional with regard to SN-38 glucuronidation. These findings highlight the importance of further studies into the potential influence of UGT1A9 D256N variant to irinotecan metabolism in vivo.