Sulfation of the isoflavones genistein and daidzein in human and rat liver and gastrointestinal tract.

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.

Carboxyl nonsteroidal anti-inflammatory drugs are efficiently glucuronidated by microsomes of the human gastrointestinal tract.

Limited studies have been carried out on the biotransformation of carboxyl nonsteroidal anti-inflammatory drugs (NSAIDs) in the liver. However, the role of the intestine in NSAID metabolism has not been investigated. In this report, the contribution of UDP-glucuronosyltransferases (UGTs) in the human gastrointestinal (GI) tract from five donors to the glucuronidation of the NSAIDs, RS-ketoprofen, S-naproxen, RS- and S-etodolac, was investigated. UGT activity and, for some donors, mRNA levels were evaluated. All NSAIDs were glucuronidated throughout the GI tract; however, glucuronidation was low in stomach and duodenum as compared to the remainder of the intestine. RT-PCR analysis demonstrated that the UGT1A isoforms, UGT1A3, 1A8, and 1A10, and UGT2B7 were expressed in the GI tract. Human recombinant UGT1A3, 1A9, 1A10 and 2B7 were actively involved in the glucuronidation of all NSAIDs while UGT1A7 and the intestine-specific UGT1A8 had no glucuronidating activity towards those compounds. Despite interindividual variations in both the levels of mRNA and the distribution of activity through the intestine, UGTs in the GI tract may contribute significantly to the first pass metabolism of orally administered NSAIDs.

A historical overview of the heterologous expression of mammalian UDP-glucuronosyltransferase isoforms over the past twenty years.

UDP-Glucuronosyltransferases (UGTs) are actively involved in detoxification of xenobiotics and endogenous compounds and are a major source of drug inactivation and drug-drug interactions. UGTs are membrane-bound enzymes mostly localized in the endoplasmic reticulum (ER) and inner and outer nuclear membranes. UGT activities are totally dependent on the phospholipid content of the membrane and, as a result, are usually inactive when isolated from the ER in the presence of detergent. Several UGT expression systems have been described by different laboratories. They include expression in mammalian cells such as COS, V79 and HEK293. Also, baculovirus-infected insect cells systems have been developed and allow the expression of UGT isoforms with or without histidine molecule tags (His-tags). Moreover, as for CYP450, UGT isoforms have been expressed in E.coli. This review concentrates on a detailed description of all these expression systems in terms of their use for substrate specificity studies and the preparation of pure UGT proteins for active site identification and other structural studies. The effect of detergents and alamethicin on UGT catalytic activity in different expression systems will be discussed. Moreover, extensive comparative studies on the characterization of recombinant UGTs in terms of substrate specificity, evaluation of kinetic parameters, and the effect of inhibitors will be presented in this review. An overall picture of the use of different UGT expression systems will help in selecting the best one for identification of the individual UGT isoforms involved in the glucuronidation of drugs, environmental pollutants and physiologically important endogenous compounds. Especially important is an expression system where UGTs are biosynthesized with His-tags. UGTs expressed in this system can be easily purified to homogeneity, which will result in significant development of structure-function relationship studies, including the identification of substrate active sites and eventual crystallization. These are underdeveloped areas of UGT research and the availability of these recombinant UGTs will allow these gaps to be filled.

Glucosidation of hyodeoxycholic acid by UDP-glucuronosyltransferase 2B7.

Previous studies have shown that several endogenous compounds, such as bilirubin and certain bile acids, are glucosidated in human liver. In this work, we have identified human UDP-glucuronosyltransferase 2B7 (UGT2B7) as the isoform that catalyzes the glucosidation of hyodeoxycholic acid (HDCA). The glucosidation by UGT2B7 was specific for HDCA and was not observed with the other bile acids examined, lithocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid. The kinetics of HDCA glucuronidation and glucosidation by UGT2B7 were characterized. The K(m) values for glucuronidation and glucosidation of HDCA were 11.6 and 17.9 microM, respectively, with V(max) values of 4.15 nmol/min/mg protein for glucuronidation and 3.28 nmol/min/mg for glucosidation. At a fixed concentration of HDCA, the apparent K(m) for UDP-glucuronic acid was 89 microM with a V(max) of 3.53 nmol/min/mg. The corresponding parameters for UDP-glucose were 442 microM and 1.98 nmol/min/mg, respectively. UGT2B7 catalyzed the addition of the glucose and glucuronic acid moieties to an hydroxyl group on HDCA and also possessed some capacity to use UDP-xylose as sugar donor. The two polymorphic variants of UGT2B7, UGT2B7(*)1 and UGT2B7(*)2 could both glucosidate HDCA. This is the first report that identifies UGT2B7 as the enzyme responsible for the glucosidation of the bile acid, HDCA.

Glucuronidation of the dietary fatty acids, phytanic acid and docosahexaenoic acid, by human UDP-glucuronosyltransferases.

Linoleic acid has recently been shown to be glucuronidated in vitro by human liver and intestinal microsomes and recombinant UGT2B7. In the present study, the dietary fatty acids (FA), phytanic acid (PA), and docosahexaenoic acid (DHA) have been used as substrates for human UDP-glucuronosyltransferases (UGTs). Both compounds were effectively glucuronidated by human liver microsomes (HLM; 1.25 +/- 0.36 and 1.12 +/- 0.32 nmol/mg x min for PA and DHA, respectively) and UGT2B7 (0.71 and 0.53 nmol/mg x min). Kinetic analysis produced relatively low K(m) values for PA with both HLM and UGT2B7 (149 and 108 microM, respectively). The K(m) for DHA glucuronidation by HLM (460 microM) was considerably higher than that for UGT2B7 (168 microM), suggesting the involvement in microsomes of other UGT isoforms in addition to UGT2B7. Glucuronidation of PA and DHA by gastrointestinal microsomes from 16 human subjects was determined. In general, both PA and DHA were glucuronidated by gastric and intestinal microsomes, and activity toward both substrates was lowest in the stomach, increased in the small intestine, and lower in the colon. However, there were large interindividual variations in UGT activity toward both substrates in all segments of the intestine, as has been seen with other substrates. Thus, PA and DHA are effective in vitro substrates for human liver, gastric and intestinal microsomes, and glucuronidation may play a role in modulating the availability of these FA as ligands for nuclear receptors.

Glucuronidation of catechols by human hepatic, gastric, and intestinal microsomal UDP-glucuronosyltransferases (UGT) and recombinant UGT1A6, UGT1A9, and UGT2B7.

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.

Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by human hepatic and recombinant UDP-glucuronosyltransferases.

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.

Nuclear UDP-glucuronosyltransferases: identification of UGT2B7 and UGT1A6 in human liver nuclear membranes.

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.