Mechanism of the efflux transport of demethoxycurcumin-O-glucuronides in HeLa cells stably transfected with UDP-glucuronosyltransferase 1A1.

Demethoxycurcumin (DMC) is a safe and natural food-coloring additive, as well as an agent with several therapeutic properties. However, extensive glucuronidation in vivo has resulted in its poor bioavailability. In this study, we aimed to investigate the formation of DMC-O-glucuronides by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) and its transport by breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in HeLa cells stably transfected with UGT1A1 (named HeLa1A1 cells). The chemical inhibitors Ko143 (a selective BCRP inhibitor) and MK571 (a pan-MRP inhibitor) both induced an obvious decrease in the excretion rate of DMC-O-glucuronides and a significant increase in intracellular DMC-O-glucuronide concentrations. Furthermore, BCRP knock-down resulted in a marked reduction in the level of excreted DMC-O-glucuronides (maximal 55.6%), whereas MRP1 and MRP4 silencing significantly decreased the levels of excreted DMC-O-glucuronides (a maximum of 42.9% for MRP1 and a maximum of 29.9% for MRP3), respectively. In contrast, neither the levels of excreted DMC-O-glucuronides nor the accumulation of DMC-O-glucuronides were significantly altered in the MRP4 knock-down HeLa cells. The BCRP, MRP1 and MRP3 transporters were identified as the most important contributors to the excretion of DMC-O-glucuronides. These results may significantly contribute to improving our understanding of mechanisms underlying the cellular disposition of DMC via UGT-mediated metabolism.

Charactering the metabolism of cryptotanshinone by human P450 enzymes and uridine diphosphate glucuronosyltransferases in vitro.

Cryptotanshinone (CT) is the main active component in the root of Salvia miltiorrhiza Bunge (SMB) that displays antibacterial, anti-inflammatory and anticancer activities. In this study, we characterized phase I and phase II metabolism of CT in human liver microsomes in vitro and identified the metabolic enzymes (CYPs and UGTs) involved. The metabolites of CT generated by CYPs were detected using LC-MS/MS and the CYP subtypes involved in the metabolic reactions were identified using chemical inhibitors of CYP enzymes and recombinant human CYP enzymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Glucuronidation of CT was also examined, and the UGT subtypes involved in the metabolic reactions were identified using recombinant human UGT enzymes (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). After adding NADPH to the human liver microsomes incubation system, CT was transformed into 6 main dehydrogenation and hydroxylation metabolites. CYP2A6, CYP3A4 and CYP2C19 were the major contributors to the transformation of its hydroxylation metabolites. CYP2C19, CYP1A2 and CYP3A4 were the major contributors to the transformation of its hydrogenation metabolites in human liver microsomes. This study showed that the metabolites at m/z of 473 were mediated by UGT1A9 and that the metabolites at m/z of 489 were mediated by UGT2B7 and UGT2B4. CT was extensively metabolized by UGTs following metabolism by CYPs in the liver.

Metabolism of Flavone-8-acetic Acid in Mice.

Flavone-8-acetic acid (FAA) is a potent antivascular agent in mice but not in humans. Assuming that FAA was bioactivated in mice, we previously demonstrated that 6-OH-FAA was formed from FAA by mouse microsomes but not by human microsomes; its antivascular activity was 2.1- to 15.9-fold stronger than that of FAA, and its antivascular activity was mediated through the Ras homolog gene family (Rho) protein kinase A (RhoA) pathway. The present work aimed to study FAA metabolism in order to verify if 6-OH-FAA is formed in mice. Using synthesized standards and high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) analysis, we herein demonstrated, for the first time, that in vitro FAA and its monohydroxylated derivatives could directly undergo phase II metabolism forming glucuronides, and two FAA epoxides were mostly scavenged by NAC and GSH forming corresponding adducts. FAA was metabolized in mice. Several metabolites were formed, in particular 6-OHFAA. The antitumor activity of 6-OH-FAA in vivo is worthy of investigation.

Improved one-pot multienzyme (OPME) systems for synthesizing UDP-uronic acids and glucuronides.

Arabidopsis thaliana glucuronokinase (AtGlcAK) was cloned and shown to be able to use various uronic acids as substrates to produce the corresponding uronic acid-1-phosphates. AtGlcAK or Bifidobacterium infantis galactokinase (BiGalK) was used with a UDP-sugar pyrophosphorylase, an inorganic pyrophosphatase, with or without a glycosyltransferase for highly efficient synthesis of UDP-uronic acids and glucuronides. These improved cost-effective one-pot multienzyme (OPME) systems avoid the use of nicotinamide adenine dinucleotide (NAD(+))-cofactor in dehydrogenase-dependent UDP-glucuronic acid production processes and can be broadly applied for synthesizing various glucuronic acid-containing molecules.

Enzymatic synthesis of uracil glucuronide, labeling with 125/131I, and in vitro evaluation on adenocarcinoma cells.

Human UDP-glucuronosyltransferases (UGTs) are a family of membrane-bound enzymes of the endoplasmic reticulum. They catalyze the glucuronidation of various endogenous and exogenous compounds, converting them into more polar glucuronides. In this study, uracil glucuronide was enzymatically synthesized using a UGT-rich microsome preparate, which was separated from Hutu-80 cells. Two different glucuronide derivatives were obtained, with a total reaction yield of 22.95% +/- 2.4% (n = 4). The glucuronide ligands were defined as uracil-n-glucuronide (UNG) and uracil-o-glucuronide (UOG). These were then analyzed by high-performance liquid chromatography-mass spectrometry and labeled with I-125 and I-131, separately. The radiolabeled (125/131)I-UNG and (125/131)I-UOG presented good incorporation ratios for Hutu-80, Caco-2, Detroit 562, and ACBRI 519 cells. The incorporation ratios of (125/131)I-UOG were higher than those of (125/131)I-UNG and of other labeled components for all cell types, and were also statistically significant compared to the values of (125/131)I-UNG for primary human intestinal epithelial cells (ACBRI 519) and human intestinal adenocarcinoma cells. Cell incorporation rates of n-glucuronides and o-glucuronides were higher compared to uracil, with o-glucuronides being more selective. The results suggest that both I-125- and I-131-labeled glucuronides can be used in imaging and therapy, and further research should be done in preclinical stages.

Alternative-splicing forms of the major phase II conjugating UGT1A gene negatively regulate glucuronidation in human carcinoma cell lines.

The UDP-glucuronosyltransferase UGT1A gene is a major biotransformation gene involved in the metabolism of a vast array of molecules. Recently, we uncovered a new series of alternative spliced isoforms referred to as isoforms 2 or UGT1As_i2 that use an alternative exon 5 (5b). The function of such mRNAs and the corresponding 45 kDa proteins still remains unclear. Although devoid of glucuronosyltransferase activity, UGT1As_i2 are widely co-expressed with the enzymatically active and classical UGT1A isoforms (UGT1As_i1). In this study, we observed abundant signal in human colon tissue samples, predominantly along intestinal crypts. In human cells, UGT1A_i2 proteins are expressed in similar subcellular compartments as UGT1As_i1. Cellular properties of i2-spliced forms were then studied using synthetic small-interfering RNA (siRNA) in two human colon cancer cell lines that show a significant amount of exon 5a- and exon 5b-containing mRNAs and that display enzymatic activities for UGT1As substrates. We observed that siRNA-mediated knockdown of endogenous i2 upregulates cellular glucuronidation activities by 120-170% (P<0.01) for all substrates tested. Functional data support a dominant-negative function for endogenous exon 5b-spliced forms of UGT1A, hence potentially affecting in vivo glucuronidation capacity. This new regulatory strategy may ensure an additional mean to modulate cellular response to endo/xeno stimulus.

The human UDP-glucuronosyltransferase UGT1A3 is highly selective towards N2 in the tetrazole ring of losartan, candesartan, and zolarsartan.

Losartan, candesartan, and zolarsartan are AT(1) receptor antagonists that inhibit the effect of angiotensin II. We have examined their glucuronidation by liver microsomes from several animals and by recombinant human UDP-glucuronosyltransferases (UGTs). Large differences in the production of different glucuronide regioisomers of the three sartans were observed among liver microsomes from human (HLM), rabbit, rat, pig, moose, and bovine. However, all the liver microsomes produced one or two N-glucuronides in which either N1 or N2 of the tetrazole ring were conjugated. O-Glucuronides were also detected, including acyl glucuronides of zolarsartan and candesartan. Examination of individual human UGTs of subfamilies 1A and 2B revealed that N-glucuronidation activity is widespread, along with variable regioselectivity with respect to the tetrazole nitrogens of these sartans. Interestingly, UGT1A3 exhibited a strong regioselectivity towards the N2 position of the tetrazole ring in all three sartans. Moreover, the tetrazole-N2 of zolarsartan was only conjugated by UGT1A3, whereas the tetrazole-N1 of this aglycone was accessible to other enzymes, including UGT1A5. Zolarsartan O-glucuronide was mainly produced by UGTs 1A10 and 2B7. UGT2B7, alongside UGT1A3, glucuronidated candesartan at the tetrazole-N2 position, whereas UGTs 1A7-1A10 mainly yielded candesartan O-glucuronide. In the case of losartan, no O-glucuronide was generated by any tested human enzyme. Nevertheless, UGTs 1A1, 1A3, 1A10, 2B7, and 2B17 glucuronidated losartan at the tetrazole-N2, while UGT1A10 also yielded the respective N1-glucuronide. Kinetic analyses revealed that the main contributors to losartan glucuronidation in HLM are UGT1A1 and UGT2B7. The results provide ample new data on substrate specificity in drug glucuronidation.

Glucuronidation of nicotine and cotinine by UGT2B10: loss of function by the UGT2B10 Codon 67 (Asp>Tyr) polymorphism.

Nicotine, the major addicting agent in tobacco and tobacco smoke, undergoes a complex metabolic pathway, with approximately 22% of nicotine urinary metabolites in the form of phase II N-glucuronidated compounds. Recent studies have shown that UGT2B10 is a major enzyme involved in the N-glucuronidation of several tobacco-specific nitrosamines. In the present study, microsomes of UGT2B10-overexpressing HEK293 cells exhibited high N-glucuronidation activity against both nicotine and cotinine with apparent KM's that were 37- and 3-fold lower than that observed for microsomes of UGT1A4-overexpressing cells against nicotine and cotinine, respectively. The KM of microsomes from wild-type (WT) UGT2B10-overexpressing cells for nicotine and cotinine was similar to that observed for human liver microsomes (HLM) against both substrates. The level of glucuronidated nicotine or cotinine in 112 HLM samples was correlated with UGT2B10 genotype; the levels of nicotine- and cotinine-glucuronide were 21% to 30% lower in specimens from subjects with the UGT2B10 (*1/*2) genotype compared with specimens from subjects with the WT UGT2B10 (*1/*1) genotype; a 5- and 16-fold lower level of nicotine- and cotinine-glucuronide formation, respectively, was observed in HLM from subjects with the UGT2B10 (*2/*2) genotype. In contrast to the relatively high activity observed for cells overexpressing WT UGT2B10 in vitro, little or no glucuronidation was observed for microsomes from cells overexpressing the UGT2B10*2 variant against either nicotine or cotinine. These data suggest that UGT2B10 is the major hepatic enzyme involved in nicotine/cotinine glucuronidation and that the UGT2B10*2 variant significantly reduces nicotine- and cotinine-N-glucuronidation formation and plays an important role in nicotine metabolism and elimination.

Metabolic activation of indole-containing prostaglandin D2 receptor 1 antagonists: impacts of glutathione trapping and glucuronide conjugation on covalent binding.

Some DP1 receptor antagonists from an indole-containing series were shown to cause in vitro covalent binding to protein in rat and human liver microsomes. Glutathione trapping experiments along with in vitro labeling assays confirmed that the presence of a strong electron withdrawing group was necessary to abrogate in vitro covalent binding, leading to the discovery of MK-0524. Hepatocyte incubations and in vivo studies showed that acyl-glucuronide formation did not translate into covalent binding.

UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for etoposide glucuronidation in human liver and intestinal microsomes: structural characterization of phenolic and alcoholic glucuronides of etoposide and estimation of enzyme kinetics.

Etoposide, an important anticancer agent, undergoes glucuronidation both in vitro and in vivo. In this study, three isomeric glucuronides of etoposide, including one phenolic (EPG) and two alcoholic glucuronides (EAG1 and EAG2), were biosynthesized in vitro with human liver microsomes (HLMs), and identified by liquid chromatography-electrospray ionization-mass spectrometry and confirmed by beta-glucuronidase cleavage. In vitro UDP-glucuronosyltransferase (UGT) reaction screening with 12 recombinant human UGTs demonstrated that etoposide glucuronidation is mainly catalyzed by UGT1A1. Although UGT1A8 and 1A3 also catalyzed the glucuronidation of etoposide, their activities were approximately 10 and 1% of UGT1A1. Enzyme kinetic study indicated that the predominant form of etoposide glucuronide in HLMs and human intestinal microsomes (HIMs) was EPG, whereas EAG1 and EAG2 were the minor metabolites, with approximately an 8 to 10% glucuronidation rate of EPG. For the formation of EPG, the V(max) of HLMs (110 pmol/min/mg protein) was very similar to that of recombinant UGT1A1 (124 pmol/min/mg protein), whereas the V(max) of HIMs (54.4 pmol/min/mg protein) was 2-fold lower than those of HIMs and UGT1A1. The K(m) values of HLMs (530 microM) and HIMs (608 microM) were 2-fold higher than that of UGT1A1 (285 microM). The V(max)/K(m) values for the formation of EPG were 0.21 and 0.09 microl/min/mg protein for HLMs and HIMs, respectively. The data indicated that UGT1A1 is principally responsible for the formation of etoposide glucuronides, mainly in the form of phenolic glucuronide, suggesting that etoposide can be used as a highly selective probe substrate for human UGT1A1 in vitro.

Glucuronidation of SN-38 and NU/ICRF 505 in human colon cancer and adjacent normal colon.

BACKGROUND: Glucuronidation represents a novel mechanism of intrinsic drug resistance in colon cancer cells. To safely reverse this mechanism in vivo, it is essential to identify which isoforms of UDP-glucuronosyltransferases are responsible for catalysing this drug metabolism in tumour tissue. MATERIALS AND METHODS: LC-MS was applied to measure rates of glucuronidation of two anticancer compounds (SN-38 and NU/ICRF 505) by patient colon cancer biopsies and paired normal colon. RESULTS: Three independent lines of enquiry indicated that, in the tumour specimens, SN-38 was glucuronidated primarily by UGT1A1, the isozyme generally recognised as being responsible for hepatic detoxification of this compound, while with NU/ICRF 505 two candidate isoforms emerged - UGT1A8 and/or UGT1A10 - both of which are not normally expressed in the liver. CONCLUSION: These data suggest that tumour selective modulation of this drug resistance mechanism in patients may be feasible with NU/ICRF 505 but more difficult to realise with SN-38. De novo drug resistance is recognised as contributing significantly to the poor response rates of colorectal cancer (CRC) to chemotherapy (1). Nonetheless, the underlying mechanisms responsible for drug insensitivity remain

Prominent but reverse stereoselectivity in propranolol glucuronidation by human UDP-glucuronosyltransferases 1A9 and 1A10.

Propranolol is a nonselective beta-adrenergic blocker used as a racemic mixture in the treatment of hypertension, cardiac arrhythmias, and angina pectoris. For study of the stereoselective glucuronidation of this drug, the two propranolol glucuronide diastereomers were biosynthesized, purified, and characterized. A screen of 15 recombinant human UDP-glucuronosyltransferases (UGTs) indicated that only a few isoforms catalyze propranolol glucuronidation. Analysis of UGT2B4 and UGT2B7 revealed no significant stereoselectivity, but these two enzymes differed in glucuronidation kinetics. The glucuronidation kinetics of R-propranolol by UGT2B4 exhibited a sigmoid curve, whereas the glucuronidation of the same substrate by UGT2B7 was inhibited by substrate concentrations above 1 mM. Among the UGTs of subfamily 1A, UGT1A9 and UGT1A10 displayed high and, surprisingly, opposite stereoselectivity in the glucuronidation of propranolol enantiomers. UGT1A9 glucuronidated S-propranolol much faster than R-propranolol, whereas UGT1A10 exhibited the opposite enantiomer preference. Nonetheless, the Km values for the two enantiomers, both for UGT1A9 and for UGT1A10, were in the same range, suggesting similar affinities for the two enantiomers. Unlike UGT1A9, the expression of UGT1A10 is extrahepatic. Hence, the reverse stereoselectivity of these two UGTs may signify specific differences in the glucuronidation of propranolol enantiomers between intestine and liver microsomes. Subsequent experiments confirmed this hypothesis: human liver microsomes glucuronidated S-propranolol faster than R-propranolol, whereas human intestine microsomes glucuronidated S-propranolol faster. These findings suggest a contribution of intestinal UGTs to drug metabolism, at least for UGT1A10 substrates.

UGT1A10 is responsible for SN-38 glucuronidation and its expression in human lung cancers.

BACKGROUND: We previously reported that upregulation of glucuronidation activity catalyzed by uridine 5'diphosphoglucuronosyltransferase (UGT) is one of the mechanisms associated with irinotecan hydrochloride/7-ethyl-10-hydroaxycamptothecin (CPT-11/SN-38) resistance. In order to extend this result to the clinical setting, it is important to elucidate the role of SN-38 glucuronidation by UGT1A isoforms in CPT-11/SN-38 resistance in vivo. MATERIALS AND METHODS: We examined SN-38 glucuronidation activity in COS-7 cells transfected with full-length cDNAs for human UGT isoforms (UGT1A1, UGT1A3, UGT1A6 and UGT1A10). The gene expression levels of UGT isoforms were examined in lung cancer cell lines and 14 lung cancer samples by semi, quantitative RT-PCR. RESULTS: Our HPLC assay results showed that both UGT1A1 and UGT1A10 are responsible for SN-38 glucuronidation. The levels of UGT1A1 and UGT1A10 expression in a CPT-11/SN-38-resistant cell line were increased compared to levels in the parent cell line. Furthermore, there was considerable intersubject variability in 14 lung cancer samples, but UGT1A1 and UGT1A10 expression levels were significantly correlated (r=0. 70, p=0.004). Our results suggest that not only UGT 1A1, but also UGT 1A10, plays an important role in detoxifying CPT-11/SN-38, leading to resistance to CPT-11/SN-38 in lung cancer.

The functional UGT1A1 promoter polymorphism decreases endometrial cancer risk.

UDP-glucuronosyltransferase (UGT) 1A1 is involved in the inactivation of estradiol (E(2)) and its oxidized metabolites. These metabolites have been shown to contribute to the development of endometrial cancer in animal studies. Thus UGT1A1 represents a candidate gene in endometrial carcinogenesis. In this study, we established the substrate specificity of UGT1A1 for E(2) and its 2- and 4-hydroxylated metabolites. Intrinsic clearances indicated that UGT1A1 had a preference for the glucuronidation of 2-hydroxyestradiol, a metabolite associated with antiproliferative activity. Expression analysis demonstrated that UGT1A1 is present in the nonmalignant endometrium. Subsequently, we sought to determine whether the common UGT1A1 promoter allele, UGT1A1*28 [A(TA)(7)TAA], which decreases gene transcription, was associated with endometrial cancer risk in a case-control study nested within the Nurses' Health Study (222 cases, 666 matched controls). Conditional logistic regression demonstrated a significant inverse association with the UGT1A1*28 allele and endometrial cancer risk. Compared with women homozygous for the UGT1A1*1 [A(TA)(6)TAA] allele, the adjusted odds ratio (OR) was 0.81 [95% confidence interval (CI), 0.56-1.16] for the UGT1A1*1/*28 genotype and 0.40 (95% CI, 0.21-0.75) for the homozygous UGT1A1*28 genotype (P(trend) = 0.007). There was a suggestion of an interaction by menopausal status [OR = 0.39 (95% CI, 0.18-0.85) for premenopausal women and OR = 0.79 (95% CI, 0.55-1.13) for postmenopausal women who carry the UGT1A1*28 allele (P(interaction) = 0.05)]. These observations suggest that lower expression of UGT1A1 decreases the risk of endometrial cancer by reducing the excretion of 2-hydroxyestradiol, the antiproliferative metabolite of E(2), in the endometrium.

Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate.

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are the major polyphenolic constituents in green tea. In this study, we characterized the enzymology of cytosolic catechol-O-methyltransferase (COMT)-catalyzed methylation of EGCG and EGC in humans, mice, and rats. At 1 microM, EGCG was readily methylated by liver cytosolic COMT to 4"-O-methyl-EGCG and then to 4',4"-di-O-methyl-EGCG; EGC was methylated to 4'-O-methyl-EGC. The K(m) and V(max) values for EGC methylation were higher than EGCG; for example, with human liver cytosol, the K(m) were 4.0 versus 0.16 microM and V(max) were 1.28 versus 0.16 nmol/mg/min. Rat liver cytosol had higher COMT activity than that of humans or mice. The small intestine had lower specific activity than the liver in the methylation of EGCG and EGC. Glucuronidation on the B-ring or the D-ring of EGCG greatly inhibited the methylation on the same ring, but glucuronidation on the A-ring of EGCG or EGC did not affect their methylation. Using EGC and 3,4-dihydroxy-L-phenylalanine as substrates, EGCG, 4"-O-methyl-EGCG, and 4',4"-di-O-methyl-EGCG were all potent inhibitors (IC(50) approximately 0.2 microM) of COMT. The COMT-inhibiting activity of (-)-EGCG-3'-O-glucuronide was similar to EGCG, but (-)-EGCG-4"-O-glucuronide was less potent. The present work provides basic information on the methylation of EGCG and suggests that EGCG may inhibit COMT-catalyzed methylation of endogenous and exogenous compounds.

Glucuronides of tea catechins: enzymology of biosynthesis and biological activities.

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are major green tea catechins with antioxidant and anticancer activities. In this study, we characterized the glucuronidation of EGCG and EGC in human, mouse, and rat microsomes and by nine different human UGT 1A and 2B isozymes expressed in insect cells. Six EGCG and EGC glucuronides were biosynthesized, and their structures were identified for the first time. (-)-EGCG-4"-O-glucuronide was the major EGCG glucuronide formed in all incubations. The catalytic efficiency (V(max)/K(m)) for (-)-EGCG-4"-O-glucuronide formation followed the order: mouse intestine > mouse liver > human liver > rat liver >> rat small intestine. The UGT-catalyzed glucuronidation of EGC was much lower than that of EGCG. The V(max)/K(m) for (-)-EGC-3'-O-glucuronide followed the following order: mouse liver > human liver > rat liver > rat and mouse small intestine. Human UGT1A1, 1A8, and 1A9 had high activities with EGCG. UGT1A8, an intestine-specific UGT, had the highest V(max)/K(m) for EGCG but low activity with EGC. Mice appeared to be more similar to humans than rats to humans in the glucuronidation of EGCG and EGC. Some of these catechin glucuronides retained the activities of their parent compounds in radical scavenging and in inhibiting the release of arachidonic acid from HT-29 human colon cancer cells. These results provide foundations for understanding the biotransformation and biological activities of tea catechins.

Glucuronidation: an important mechanism for detoxification of benzo[a]pyrene metabolites in aerodigestive tract tissues.

UDP-glucuronosyltransferases (UGTs) have been implicated as important detoxifying enzymes for several major tobacco carcinogens. Because the aerodigestive tract is a primary target for exposure to tobacco smoke carcinogens, the major goal of the present study was to determine whether aerodigestive tract tissues exhibit glucuronidating activity against metabolites of benzo[a]pyrene (BaP) and to explore the pattern of expression of UGT genes in a series of aerodigestive tract tissue specimens. Glucuronidation of the phenolic BaP metabolites 3-, 7-, and 9-hydroxy-BaP was observed in all upper aerodigestive tract tissue microsome specimens tested, as determined by high-pressure liquid chromatography analysis. Glucuronidating activity toward the procarcinogenic BaP metabolite trans-BaP-7,8-dihydrodiol(+/-) was also detected in aerodigestive tract tissues. By semiquantitative duplex reverse transcription-polymerase chain reaction analysis, UGT1A7 and UGT1A10 were shown to be well expressed in all aerodigestive tract tissues examined, including tongue, tonsil, floor of mouth, larynx, and esophagus. UGT1A8 and UGT1A6 were expressed primarily in larynx; no expression was observed for UGTs 1A1, 1A3, 1A4, 1A5, 1A9. Of the family 2B UGTs, only UGT2B4 and UGT2B17 exhibited significant levels of expression in aerodigestive tract tissues. Of the aerodigestive tract-expressing UGTs, only UGTs 1A7, 1A8, and 1A10 exhibited glucuronidating activity against 7-hydroxy-BaP, with UGT1A10 exhibiting the highest affinity as determined by kinetic analysis (K(m) = 49 microM). No UGT expression or glucuronidating activity was observed for any of the lung specimens analyzed in this study. These results suggest that several family 1 UGTs may potentially play an important role in BaP detoxification in the aerodigestive tract.

The hepatic glycogenoreticular system.

One of the major liver functions is the ability of hepatocytes to store glucose in the form of glycogen for various purposes. Beside glucose production and secretion, the synthesis of glucuronides and ascorbate has been reported to be dependent on the extent of the glycogen stores and on the rate of glycogenolysis in the liver. It is common that the final steps of these pathways are catalysed by intraluminally orientated enzymes of the endoplasmic reticulum, which are supported by transporters for the permeation of substrates and products. On the basis of the close morphological and functional proximity of glycogen, glycogen-dependent pathways and the (smooth) endoplasmic reticulum we propose to use the term glycogenoreticular system for the description of this export-orientated hepatocyte-specific metabolic unit.

Characterization of autoantibodies against uridine-diphosphate glucuronosyltransferase in patients with inflammatory liver diseases.

Uridine diphosphate glucuronosyltransferase (UGT) was identified as an antigenic target in a subgroup of liver-kidney microsomal autoantibodies and was termed LKM-3. To evaluate the nature of LKM-3 antibodies, we screened sera from 80 untreated patients with autoimmune hepatitis (AIH) type 1 and 2, primary biliary cirrhosis (PBC), AIH/PBC, hepatitis C virus (HCV) infection, and 12 healthy individuals (controls) against 7 recombinant human UGT isoenzymes (UGT1A1, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, and UGT2B7). Autoantibodies reacting against various UGT isoenzymes were observed in sera from 3 of 18 AIH type 2 and 1 of 27 of the HCV patients. The anti-UGT-positive sera from AIH type 2 patients revealed the strongest immunoreactivity against UGT1A1, the main UGT-isoform involved in the bilirubin glucuronidation. Additionally, these sera were able to block UGT-mediated substrate glucuronidation in vitro. The prevalence for UGT1A1 was shown by 2 independent techniques: (1) UGT1A1 was identified as the main antigen by Western blotting. Preabsorption of sera with UGT1A1 prevented reaction against all tested UGT-isoforms. (2) In vitro immunoinhibition experiments showed that glucuronidation of the anticancer drug flavopiridol by UGT1A1 was more strongly inhibited than its UGT1A9-mediated biotransformation. In contrast, the serum from the HCV-patient reacted predominately with UGT1A6, and moreover, the immunoreactivity pattern was different from that of the AIH group. To summarize, we show the subtype preference of antibodies against UGT1A1 in a subgroup of AIH type 2 patients. These autoantibodies inhibit UGT-mediated glucuronidation in vitro, but it is unlikely that anti-UGT antibodies will have a marked effect on the patients capacity for drug biotransformation, as serum bilirubin levels in patients remained within the normal range.

The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines.

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.