Glucuronidation of the Nonsteroidal Antiestrogen EM-652 (SCH 57068), by Human and Monkey Steroid Conjugating UDP-Glucuronosyltransferase Enzymes

Barbier, Olivier; Albert, Caroline; Martineau, Isabelle; Vallée, Michel; High, Kim A.; Labrie, Fernand; Hum, Dean W.; Labrie, Claude; Bélanger, Alain

doi:10.1124/mol.59.3.636

Cited by 16 publications

(28 citation statements)

References 37 publications

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“…To ascertain the expression of the wild-type and mutant UGT1A10 proteins, 10 g of microsomal protein from HEK293 cells stably expressing human wild-type UGT1A10 or mutant UGT1A10 were separated by 10% SDS-polyacrylamide gel electrophoresis. The gel was transferred onto a nitrocellulose membrane and probed with the antihuman UGT1 common carboxyl terminus region antiserum RC-71 (1:2000 dilution) (Barbier et al, 2001). To show the relative amount of proteins loaded in each lane of the Western blot, the same blot was subsequently probed with an anticalnexin CT antibody (StressGen Biotechnologies, Victoria, British Columbia, Canada; 1:1000 dilution).…”

Section: Methodsmentioning

confidence: 99%

Amino Acid Residue Ile211 Is Essential for the Enzymatic Activity of Human Udp-Glucuronosyltransferase 1a10 (Ugt1a10)

Martineau

Tchernof²,

Bélanger³

2004

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Conjugation of exogenous and endogenous compounds by uridine diphosphoglucuronosyltransferases (UGTs) is a pathway catalyzing the transfer of a glucuronic acid molecule from UDP glucuronic acid to lipophilic aglycones, which become more polar and more easily excretable in the bile or urine. UGTs are divided into two major families, UGT1 and UGT2. The isoform UGT1A10, along with UGT1A7 and UGT1A8, is expressed exclusively in extrahepatic tissues, notably in the gastrointestinal tract. Here, we report the isolation of a mutant clone of the human UGT1A10, at position 211 of the protein, where a threonine residue replaces an isoleucine residue (allele Thr211). Because the isoleucine is conserved among many UGT1A isoforms, we proceeded to the analysis of the activity of the wild-type UGT1A10 (T211I) and compared it with that of the variant enzyme (I211T*). In vitro assays with microsomal extracts from stably expressing human embryonic kidney 293 (HEK293) cells showed that the mutant enzyme lost all detectable activity toward major substrates, which demonstrate that the residue isoleucine at position 211 is essential for UGT1A10 activity. Mutant UGT1A10 (I211T*) also lost all detectable activity toward mycophenolic acid. Genomic DNA from 103 unrelated individuals was sequenced for this mutation, and two heterozygous genotypes were detected for this mutation (frequency: 2 per 100 individuals). Because UGT1A10 appears to be expressed in all gastrointestinal tissues and is active toward a wide range of substrates, lack of activity of this isoform may have an impact on individual glucuronidation efficiency.

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Section: Methodsmentioning

confidence: 99%

Amino Acid Residue Ile211 Is Essential for the Enzymatic Activity of Human Udp-Glucuronosyltransferase 1a10 (Ugt1a10)

Martineau

Tchernof²,

Bélanger³

2004

Drug Metab Dispos

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“…22 For Western blot experiments, UGT1A3-HK293 microsomes (2 g) or total proteins from hepatocytes (5 g) were immunoblotted with an anti-UGT1A (1:2,000) antibody as previously described. 21 Glucuronidation Assays and Determination of Bile Acid Glucuronides in Culture Media and Plasma. In vitro glucuronidation assays were performed for 1 hour in the presence of CDCA or LCA (100 mol/L) and 10 g microsomes in a glucuronidation assay buffer as described.…”

Section: Methodsmentioning

confidence: 99%

“…UGT1A3-HK293 cells were obtained and cultured as described. 21 For RNA and protein analyses, 350,000 hepatocytes/well were seeded in 24-well plates and cultured in the Invitro Gro CP medium for 48 hours. Cells were then treated with vehicle [dimethylsulfoxide (DMSO), 0.1% v/v] or T0901317 (0.1 and 1 mol/L) for 24 hours in the Invitro Gro Hi medium.…”

Section: Methodsmentioning

confidence: 99%

The liver X-receptor alpha controls hepatic expression of the human bile acid–glucuronidating UGT1A3 enzyme in human cells and transgenic mice

et al. 2006

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Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24-glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol-activated nuclear receptor liver X-receptor alpha (LXR␣). In human hepatic cells and human UGT1A transgenic mice, LXR␣ activators induce UGT1A3 mRNA levels and the formation of CDCA-24glucuronide (24G) and LCA-24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site-directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXR␣ is found to interact with the SRC-1␣ and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC-1␤. In conclusion, these observations establish LXR␣ as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively. B ile acids (BAs) are biological detergents that serve a number of important functions, including the hepatic generation of bile flow and the uptake of cholesterol, fat-soluble vitamins, and other lipids in the intestine. 1 However, because of their detergent properties, BAs are inherently cytotoxic, and perturbations in their normal synthesis, transport or secretion can result in a variety of pathophysiological conditions, including intrahepatic cholestasis. 2 The primary chenodeoxycholic (CDCA) and cholic bile acids (CA) are formed in the liver and subsequently converted into secondary lithocholic (LCA) and deoxycholic acids in the intestine by bacterial dehydroxylases before reabsorption and return to the liver (see Chiang 1 ). During this enterohepatic circulation, BAs undergo several metabolic alterations, including glucuronide conjugation. 3 The most abundant glucuronide conjugate reported in human plasma is CDCA-glucuronide, followed by LCA-glucuronide, 4,5 the concentrations of which are respectively increased by 50-and 30-fold in cholestatic patients. 5 BA glucuronidation allows their transport by transporters at the basolateral membrane of hepatocytes, 3 thus favoring urinary rather than biliary excretion during cholestasis. 3 Glucuronide conjugation results in the formation of ether-type or acyl-type glucuronides in which the glucuronosyl group is added to the 3␣-hydroxyl group or the 24-carboxyl group of the steroid nucleus of primary and secondary BAs, respectively. 6,7

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“…In aerobic environment, aerobic bacteria were several folds more active than anaerobic ones [70]. A recent study has isolated bacteria that transformed fE1 into fE2, resulting in further increase in manures' estrogenic activity since a transformation of fE1 to fE2 results in two folds increase in estrogenic activity [71]. In addition, estrogen glucuronates and sulfates both exhibited comparable de-conjugation of conjugated estrogens in manure in oxidative and reductive environment, although D'Ascenzo et al [72] and Hutchins et al [73] have shown that sulfate-conjugated estrogen are more recalcitrant to biodegradation.…”

Section: Effects Of Aerobic and Anaerobic Digestions On Estrogen Concmentioning

confidence: 99%

Aerobic and Anaerobic Transformations in Estrogens and Nutrients in Swine Manure: Environmental Consequences

Singh

2015

Agriculture

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Pig manure is an excellent fertilizer and rich source of organic carbon and nitrogen compounds such organic nitrogen (O-N) (95% of total nitrogen) that is plant-unavailable-nitrogen (PUN) and mineralized nitrogen (about 1% of total nitrogen) such as ammonium (NH4 + ) and nitrate (NO3) that are plant-available-nitrogen (PAN). In addition, manure also contains two forms of estrogens: (i) poorly estrogenic thus essentially nontoxic conjugated estrogens (cEs) such as estrone (cE1), estradiol (cE2) and estriol (cE3); and (ii) highly estrogenic and toxic free estrogens (fEs) such as fE2, fE1 and fE3. This study showed that aerobic processing reduced concentrations of total carbon (TC), O-N, PAN and NH4 + /NH3 ratio, transiently hydrolyzed cEs (cE2 > cE1 > cE3) into corresponding fEs, transiently increased estrogenic activity and potential toxicity, and rapidly degraded fEs (fE2, fE1 > fE3), thus reducing the estrogenic activity in manure. Unlike aerobic processing, anaerobic processing stabilized and increased PAN and NH4 + /NH3 ratio, thus increasing the manure's fertilizer value. However, anaerobic processing, relative to aerobic processing, poorly hydrolyzed cEs (reducing transient toxicity and increasing reserve toxicity potential) and poorly degraded fEs (increasing toxicity) in manure. Thus, aerobic and anaerobic environments have distinct effects on manures' PAN and estrogenic activity, presenting an interesting dilemma: anaerobic incubation that increases manures' PAN does not effectively degrade estrogens, while aerobic incubation that effectively degrades estrogens (after transiently increasing their estrogenic activity) also decreases PAN, thus making manure less profitable. New techniques are need to fully use manure as organic fertilizer. OPEN ACCESSAgriculture 2015, 5 698

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Glucuronidation of the Nonsteroidal Antiestrogen EM-652 (SCH 57068), by Human and Monkey Steroid Conjugating UDP-Glucuronosyltransferase Enzymes

Cited by 16 publications

References 37 publications

Amino Acid Residue Ile211 Is Essential for the Enzymatic Activity of Human Udp-Glucuronosyltransferase 1a10 (Ugt1a10)

Amino Acid Residue Ile211 Is Essential for the Enzymatic Activity of Human Udp-Glucuronosyltransferase 1a10 (Ugt1a10)

The liver X-receptor alpha controls hepatic expression of the human bile acid–glucuronidating UGT1A3 enzyme in human cells and transgenic mice

Aerobic and Anaerobic Transformations in Estrogens and Nutrients in Swine Manure: Environmental Consequences

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