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Trans-Resveratrol (tRes) has been shown to have powerful antioxidant, anti-inflammatory, anticarcinogenic, and antiaging properties; however, its use as a therapeutic agent is limited by its rapid metabolism into its conjugated forms by UDP-glucuronosyltransferases (UGTs). The aim of the current study was to test the hypothesis that the limited bioavailability of tRes can be improved by modifying its structure to create analogs which would be glucuronidated at a lower rate than tRes itself. In this work, three synthetic stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore, each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using HPLC and verified by β-glucuronidase hydrolysis and LC-MS/MS analysis. NI-12a was glucuronidated at both the -COOH and -OH functions, NI-ST-05 formed a novel N-O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme, UGT1A10, with apparent Km values of 240 and 6.2 μM, respectively. The involvement of hepatic and intestinal UGTs in the metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high individual variation in activity was demonstrated between donors. In summary, these studies clearly establish that modified, tRes-based stilbenoids may be preferable alternatives to tRes itself due to increased bioavailability via altered conjugation.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (NSC-645809), is an antitumor agent shown to be effective against breast cancer in phase II clinical trials. A similar compound, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, shows high activity against experimental tumors and is expected to have even more beneficial pharmacological properties than C-1311. Previously published studies showed that these compounds are not substrates for cytochrome P450s; however, they do contain functional groups that are common targets for glucuronidation. Therefore, the aim of this work was to identify the human UDP-glucuronosyltransferases (UGTs) able to glucuronidate these two compounds. High-performance liquid chromatography analysis was used to examine the activities of human recombinant UGT1A and UGT2B isoforms and microsomes from human liver [human liver microsomes (HLM)], whole human intestinal mucosa [human intestinal microsomes (HIM)], and seven isolated segments of human gastrointestinal tract. Recombinant extrahepatic UGT1A10 glucuronidated 8-hydroxyl groups with the highest catalytic efficiency compared with other recombinant UGTs, V(max)/K(m) = 27.2 and 8.8 μl · min⁻¹ · mg protein⁻¹, for C-1305 and C-1311, respectively. In human hepatic and intestinal microsomes (HLM and HIM, respectively), high variability in UGT activities was observed among donors and for different regions of intestinal tract. However, both compounds underwent UGT-mediated metabolism to 8-O-glucuronides by microsomes from both sources with comparable efficiency; V(max)/K(m) values were from 4.0 to 5.5 μl · min⁻¹ · mg protein⁻¹. In summary, these studies suggest that imid azoacridinone and triazoloacridinone drugs are glucuronidated in human liver and intestine in vivo and may form the basis for future translational studies of the potential role of UGTs in resistance to these drugs.

Phytoestrogens, in particular the isoflavone aglycones genistein and daidzein, are thought to be the bioactive components of soy. Like estrogens, isoflavones can be sulfur-conjugated. However, although isoflavones in the serum are found largely in the form of glucuronide and sulfur conjugates following soy consumption, little is known regarding the relative contributions of sulfotransferases in the liver and small intestine to isoflavone sulfation. Since the sulfates may be deconjugated in target tissues, circulating isoflavone sulfates may act as a source of tissue aglycones. In the current study genistein and daidzein sulfotransferase activities were measured in cytosol from human and rat liver and gastrointestinal tract. Isoflavone sulfation in the human gastrointestinal (GI) tract was correlated with activities towards substrates for previously characterized human sulfotransferases. Western blots of human cytosols were also conducted using antisera towards human sulfotransferases SULT1E1 and SULT2A1. Whereas rat liver was almost fourfold more active than small intestine in sulfation of genistein, in the human, activities in the two tissues were comparable. In contrast, intestinal sulfation of daidzein was comparable to hepatic sulfation in the rat and significantly greater in the human. Genistein and daidzein sulfation occurred throughout the human GI tract, but with a different distribution and different interindividual variability. Whereas genistein sulfation in the human GI tract correlated significantly with sulfation of the prototypical human phenolic sulfotransferase SULT1A family substrate 2-naphthol (r2 = 0.71), daidzein sulfotransferase activity did not correlate with activities towards any prototypical sulfotransferase substrate or with genistein sulfation. Our results suggest that metabolism in the human GI tract has an important role in the generation of potentially bioactive isoflavone sulfates and a major role for the human phenolic sulfotransferase SULT1A family in metabolism of genistein in the gut. However, human intestinal daidzein sulfation appears to be catalyzed by a separate enzyme.

Arachidonic acid (AA) can be metabolized to various metabolites, which can act as mediators of cellular processes. The objective of this work was to identify whether AA, prostaglandin (PG) B1 and E2, and 15- and 20-hydroxyeicosatetraenoic acids (15- and 20-HETE) are metabolized via glucuronidation. Assays with human recombinant UDP-glucuronosyltransferase 1A (UGT1A) isoforms revealed that AA and 15-HETE were glucuronidated by UGT1A1, 1A3, 1A4, 1A9, and 1A10, whereas 20-HETE was glucuronidated by UGT1A1 and 1A4 and PGB1 was glucuronidated by UGT1A1, 1A9, and 1A10. All substrates were glucuronidated by recombinant UGT2B7, with AA and 20-HETE being the best substrates. Kinetic analysis of UGT1A1 and 1A9 with AA resulted in Km values of 37.9 and 45.8 microM, respectively. PGB1 was glucuronidated by UGT1A1 with a Km of 26.3 microM. The Km values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB1 and 15-HETE showed that hydroxyl groups were the major target of glucuronidation. This work demonstrates a novel metabolic pathway for HETEs and PGs and the role of UGT1A isoforms in this process. These results indicate that glucuronidation may play a significant role in modulation of the availability of these fatty acid derivatives for cellular processes., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

The substrate specificity of human gastric and intestinal UDP-glucuronosyltransferases (UGTs) toward catechols was investigated and compared to that of liver UGTs. Small catechols were efficiently glucuronidated by stomach (0.8-10.2 nmol/mgprotein x min) and intestine (0.9-7.7 nmol/mgprotein x min) with activities in a range similar to those found in liver (2.9-19 nmol/mgprotein x min). Large interindividual variations were observed among the samples. Immunoblot analysis demonstrated the presence of UGT1A6 and UGT2B7 in stomach and throughout the intestine. Recombinant human UGT1A6, 1A9, and 2B7, stably expressed in mammalian cells, all effectively catalyzed catechol glucuronidation. K(m) values (0.09-13.6mM) indicated low affinity for UGTs and V(max) values ranged from 0.51 to 64.0 nmol/mgprotein x min. These results demonstrate for the first time glucuronidation of catechols by gastric and intestinal microsomal UGTs and three human recombinant UGT isoforms.

We have demonstrated the subcellular localization of the human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A6, in endoplasmic reticulum (ER) and nuclear membrane from human hepatocytes and cell lines, by in situ immunostaining and Western blot. Double immunostaining for UGT2B7 and calnexin, an ER resident protein, showed that UGT2B7 was equally present in ER and nuclear membrane whereas calnexin was present almost exclusively in ER. Immunogold labeling of HK293 cells expressing UGT2B7 established the presence of UGT2B7 in both nuclear membranes. Enzymatic assays with UGT2B7 substrates confirmed the presence of functional UGT2B7 protein in ER, whole nuclei, and both outer and inner nuclear membranes. This study has identified, for the first time, the presence of UGT2B7 and UGT1A6 in the nucleus and of UGT2B7 in the inner and outer nuclear membranes. This localization may play an important functional role within nuclei: protection from toxic compounds and/or control of steady-state concentrations of nuclear receptor ligands.