Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7.

In this work, UDP-glucuronosyltransferases (UGTs), UGT1A3, 2B7(H268) and 2B7(Y268), stably expressed in human embryonic kidney cells (HK293) were used to assess glucuronidation activities with a variety of steroid hormone and bile acid substrates. The rate of synthesis of carboxyl- and hydroxyl-linked glucuronides was determined under optimal reaction conditions. Expressed UGT1A3 catalyzed bile acid glucuronidation at high rates exclusively at the carboxyl moiety for all compounds tested. In contrast, UGT1A4 catalyzed bile acid glucuronidation at very low rates exclusively at the 3alpha-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3alpha-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3alpha-hydroxylated androgenic steroid, was glucuronidated at very high rates by expressed UGT2B7. Of the estrogenic compounds tested, UGT2B7 catalyzed the glucuronidation of estriol at rates comparable to those determined for androsterone. Other structural discrimination was found with UGT2B7 which had activity toward estriol and estradiol exclusively at the 17beta-OH position, yielding the cholestatic steroid D-ring glucuronides.

Glucuronidation of catechol estrogens by expressed human UDP-glucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7.

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

Characterization of two UDP glucuronosyltransferases that are predominantly expressed in human colon.

The liver and gastrointestinal tract are major sites of drug metabolism. However, although the UDP glucuronosyltransferase family of drug-metabolizing enzymes has been extensively characterized in the liver, little is known about this family in the gastrointestinal tract. In this work, an analysis of human colon RNA samples revealed the presence of two UDP glucuronosyltransferase forms that could not be detected in human liver. The cDNA encoding these two forms, UGT1A8 and UGT1A10, was synthesized and expressed in COS-7 cells. Both proteins have molecular masses of 56 kDa and are active towards hydroxylated metabolites of the carcinogens, benzo(alpha)pyrene and 2-acetylaminofluorene. UGT1A8 was most active towards the 10- and 11-hydroxy benzo(alpha)pyrenes and the preferred 2-acetylaminofluorene metabolites were the 1-, 2-, and 8-hydroxy derivatives. UGT1A10 was most active towards the 11- and 12-hydroxybenzo(alpha)pyrenes and the 1- and 3-hydroxy derivatives of 2-acetylaminofluorene. Both enzymes were inactive towards the benzo(alpha)pyrene trans 4, 5 and 7, 8 dihydrodiols. In addition, these UDP glucuronosyltransferases displayed differential activity towards several phenolic substrates. A survey of human tissues indicated that UGT1A8 and UGT1A10 transcripts are predominantly expressed in the gastrointestinal tract, in contrast to most other UDP glucuronosyltransferase forms which are expressed in the liver and other tissues. These results suggest that UGT1A8 and UGT1A10 may play an important role in the metabolism of dietary xenobiotics.

Glucuronidation of amines and other xenobiotics catalyzed by expressed human UDP-glucuronosyltransferase 1A3.

Glucuronide conjugation of xenobiotics containing a tertiary amine moiety represents a unique and important metabolic pathway for these compounds in humans. Previously, human UDP-glucuronosyltransferase (UGT) 1A4 was shown to be an important enzyme for the formation of quaternary ammonium-linked glucuronides. UGT1A3 is 93% identical to UGT1A4 in primary amino acid sequence. We show that human UGT1A3, transiently expressed in human embryonic kidney 293 cells, also catalyzes the N-glucuronidation of primary, secondary, and tertiary amine substrates, such as 4-aminobiphenyl, diphenylamine, and cyproheptadine. In contrast to expressed human UGT1A4, which catalyzes the glucuronidation of amines with high efficiency, glucuronidation of amines catalyzed by UGT1A3 exhibited low efficiency, suggesting that UGT1A3 makes only a limited contribution to the metabolic elimination of these compounds. The reactivity of expressed human UGT1A3 toward hydroxylated and carboxylic acid-containing compounds was also examined. In addition to amines, expressed human UGT1A3 catalyzed the glucuronidation of opioids (e.g. morphine and buprenorphine), coumarins, flavonoids (e.g. naringenin and quercetin), anthraquinones, and small phenolic compounds (e.g. 4-nitrophenol). Drugs containing a carboxylic acid moiety, such as nonsteroidal anti-inflammatory agents (e.g. naproxen and ibuprofen) and fibrates (e.g. ciprofibrate), were substrates for human UGT1A3. In contrast, compounds containing an aliphatic hydroxyl group, such as sapogenins, monoterpenoid alcohols (e.g. menthol and borneol), and androgens, were not conjugated by expressed human UGT1A3. Of the compounds tested, scopoletin, naringenin, and norbuprenorphine appeared to be the best xenobiotic substrates for human UGT1A3.

The human UDP glucuronosyltransferase, UGT1A10, glucuronidates mycophenolic acid.

The cDNA encoding the UDP glucuronosyltransferase, UGT1A10, has been cloned from human colon. The deduced amino acid sequence of the cDNA is 90% similar in sequence to that of a previously characterized form, UGT1A9 (Hlug P4), and contains a signal peptide and carboxyl-terminal hydrophobic domain characteristic of all UDP glucuronosyltransferases isolated to date. The enzyme synthesized in UGT1A10 cDNA-transfected COS-7 cells has a relative molecular mass of 56 kDa and is very active in the glucuronidation of mycophenolic acid (apparent Km of 34 microM and Vmax of 0.6 nmoles/min/mg protein). Other UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10 and 2B11) synthesized in COS cells had relatively little activity towards mycophenolic acid, suggesting that UGT1A10 may have a significant role in the elimination of this antineoplastic and immunosuppressive agent in vivo.

Steroid UDP glucuronosyltransferases: characterization and regulation.

Under normal physiological conditions, glucuronidation generally terminates the biological activities of steroids and leads to their elimination in the bile and urine. This process is postulated to play a role in homeostasis by regulating the intracellular steady-state levels of these effector ligands. Indeed, the duration of response to specific steroid signals may be partly determined by the capacity of the cell or tissue to eliminate the steroids as unreactive glucuronides. Under pathophysiological conditions or during steroid therapies, glucuronidation may sometimes result in the formation of more biologically active or toxic metabolites as exemplified by the steroid D ring glucuronides. The degree of toxicity or biological effect in the cell exposed to these steroids will also depend on its complement of UGTs. To investigate these processes in more detail, the steroid specificities and distribution of individual UGTs in various target organs require elucidation. In this review, our current knowledge of the steroid specificities of various rat and human UGTs is described and preliminary investigations on the mechanisms governing tissue specificity are presented.

cDNA cloning and characterization of the human UDP glucuronosyltransferase, UGT1A3.

The cDNA encoding the UDP glucuronosyltransferase, UGT1A3, has been cloned and expressed in cell culture. The deduced amino acid sequence of the cDNA is 90% similar in sequence to that of a previously characterized form, UGT1A4 and to that of a third form, UGT1A5 whose function in unknown. UGT1A3, when expressed in COS cells has a relative molecular mass of 55 kDa and is active in the glucuronidation of estrone and 2-hydroxyestrone. Activity towards other polyhydroxylated estrogens including 4-hydroxyestrogen and estriol and its metabolites was not detected. UGT1A3 was also inactive towards androgens and their metabolites. The enzyme preferentially glucuronidated the N-hydroxy metabolite of 2-acetylaminofluorence and was more active towards the 6- and 12-hydroxylated metabolites of benzo[alpha]pyrene. RNA encoding UGT1A3 was detected in human liver and colon tissue.

cDNA expression studies of rat liver aryl sulphotransferase.

A cDNA encoding an isoenzyme of rat liver aryl sulphotransferase was isolated from a rat liver bacteriophage Lambda gt 11 library by the polymerase chain reaction technique. The resulting cDNA was functionally expressed in COS-7 cells and characterised by determining the sulphating capacity of the cells with a range of substrates. The COS-expressed enzyme catalysed the sulphation of both phenol and dopamine with Kms of the same order as those obtained for the high affinity isozyme in rat liver cytosol, while low activity was observed with tyrosine methyl ester. The common food additive vanillin was also a good substrate for sulphate conjugation. The sulphation of vanillin catalysed by the COS-expressed enzyme was consistent with a single enzyme system, in contrast, the kinetics of the reaction catalysed by cytosolic sulphotransferase indicated that vanillin was sulphated by more than one isozyme.

N-and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells.

Human monomorphic and polymorphic arylamine acetyltransferases (EC 2.3.1.5) were expressed in monkey kidney COS-1 cells and used to study the N- and O-acetylation of a number of carcinogenic amines and their N-hydroxy metabolites. The monomorphic enzyme N-acetylated the aromatic amines, 2-aminofluorene and 4-aminobiphenyl, and also O-acetylated their N-hydroxy derivatives. None of the food-derived heterocyclic amines (Glu-P-1, PhIP, IQ, MeIQx) were substrates and their N-hydroxy metabolites were poorly O-acetylated by this isozyme. By contrast, the polymorphic acetyltransferase catalyzed the N-acetylation of both aromatic amines, and to a lesser extent, Glu-P-1 and PhIP. However, all six N-hydroxy amine substrates were readily O-acetylated to form DNA-bound adducts by the polymorphic isozyme. These data suggest that, for the heterocyclic amine carcinogens, rapid acetylator individuals will be predisposed to their genotoxicity.