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.     

Expression of UDP-glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human brain and identification of 5-hydroxytryptamine as a substrate

The extrahepatic expression of UDP-glucuronosyltransferases (UGTs) is important in the detoxification of a number of endogenous and exogenous compounds, including 5-hydroxytryptamine and morphine. Studies were designed to investigate the extrahepatic expression of human UGTs using RT-PCR techniques and to determine the UGTs involved in the glucuronidation of 5-hydroxytryptamine. Human UGT2B7 expression was found in the human liver, kidney, pancreas, and brain, while UGT1A6 expression is found in the liver, kidney, and brain. This is the first observation of UGTs present in the human central nervous system. Using glucuronidation assays, a significant amount of 5-hydroxytryptamine glucuronide was found to be catalyzed by UGT1A6. These studies suggest that UGT2B7 may play an important role in the overall contribution of morphine analgesia by serving to generate the potent morphine-6-O-glucuronide in situ. UGT1A6 could play an important role in the glucuronidation of 5-hydroxytryptamine in vivo, therefore terminating the actions of the neurotransmitter.     

Glucuronidation of the steroid enantiomers ent-17β-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases

Steroids enantiomers are interesting compounds for detailed exploration of drug metabolizing enzymes, such as the UDP-glucuronosyltransferases (UGTs). We have now studied the glucuronidation of the enantiomers of estradiol, androsterone and etiocholanolone by the 19 human UGTs of subfamilies 1A, 2A and 2B. The results reveal that the pattern of human UGTs of subfamily 2B that glucuronidate ent-17β-estradiol, particularly 2B15 and 2B17, resembles the glucuronidation of epiestradiol (17α-estradiol) rather than 17β-estradiol, the main physiological estrogen. The UGTs of subfamilies 1A and 2A exhibit higher degree of regioselectivity than enantioselectivity in the conjugation of these estradiols, regardless of whether the activity is primarily toward the non-chiral site, 3-OH (UGT1A1, UGT1A3, UGT1A7, UGT1A8 and, above all, UGT1A10), or the 17-OH (UGT1A4). In the cases of etiocholanolone and androsterone, glucuronidation of the ent-androgens, like the conjugation of the natural androgens, is mainly catalyzed by UGTs of subfamilies 2A and 2B. Nevertheless, the glucuronidation of ent-etiocholanolone and ent-androsterone by both UGT2B7 and UGT2B17 differs considerably from their respective activity toward the corresponding endogenous androgens, whereas UGT2A1-catalyzed conjugation is much less affected by the stereochemistry differences. Kinetic analyses reveal that the K(m) value of UGT2A1 for ent-estradiol is much higher than the corresponding value in the other two high activity enzymes, UGT1A10 and UGT2B7. Taken together, the results highlight large enantioselectivity differences between individual UGTs, particularly those of subfamily 2B.     

Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation

Testosterone and epitestosterone are secreted mainly as glucuronide metabolites and the urinary ratio of testosterone glucuronide to epitestosterone glucuronide, often called T/E, serves as a marker for possible anabolic steroids abuse by athletes. UDP-glucuronosyltransferase (UGT) 2B17 is the most important catalyst of testosterone glucuronidation. The T/E might be affected by drugs that interact with UGT2B17, or other enzymes that contribute to testosterone glucuronidation. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used by sportsmen and we have examined the effect of two NSAIDs, diclofenac and ibuprofen, on testosterone and epitestosterone glucuronidation in human liver microsomes. In parallel, we have studied the inhibitory effect of these NSAIDs on recombinant UGT2B17 and UGT2B15, as well as other human hepatic UGTs that revealed low but detectable testosterone glucuronidation activity, namely UGT1A3, UGT1A4, UGT1A9 and UGT2B7. Both diclofenac and ibuprofen inhibited testosterone glucuronidation in microsomes, as well as UGT2B15 and UGT2B17. Interestingly, UGT2B15 was more sensitive than UGT2B17 to the two drugs, particularly to ibuprofen. Human liver microsomes lacking functional UGT2B17 exhibited significantly higher sensitivity to ibuprofen, suggesting that UGT2B15 plays a major role in the residual testosterone glucuronidation activity in UGT2B17-deficient individuals. Nonetheless, a minor contribution of other UGTs, particularly UGT1A9, to testosterone glucuronidation in such individuals cannot be ruled out at this stage. The epitestosterone glucuronidation activity of human liver microsomes was largely insensitive to ibuprofen and diclofenac. Taken together, the results highlight potential interactions between NSAIDs and androgen glucuronidation with possible implications for the validity of doping tests.     

Identification of UGT2B9*2 and UGT2B33 isolated from female rhesus monkey liver

Two UDP-glucuronosyltransferases (UGT2B9(*)2 and UGT2B33) have been isolated from female rhesus monkey liver. Microsomal preparations of the cell lines expressing the UGTs catalyzed the glucuronidation of the general substrate 7-hydroxy-4-(trifluoromethyl)coumarin in addition to selected estrogens (beta-estradiol and estriol) and opioids (morphine, naloxone, and naltrexone). UGT2B9(*)2 displayed highest efficiency for beta-estradiol-17-glucuronide production and did not catalyze the glucuronidation of naltrexone. UGT2B33 displayed highest efficiency for estriol and did not catalyze the glucuronidation of beta-estradiol. UGT2B9(*)2 was found also to catalyze the glucuronidation of 4-hydroxyestrone, 16-epiestriol, and hyodeoxycholic acid, while UGT2B33 was capable of conjugating 4-hydroxyestrone, androsterone, diclofenac, and hyodeoxycholic acid. Three glucocorticoids (cortisone, cortisol, and corticosterone) were not substrates for glucuronidation by liver or kidney microsomes or any expressed UGTs. Our current data suggest the use of beta-estradiol-3-glucuronidation, beta-estradiol-17-glucuronidation, and estriol-17-glucuronidation to assay UGT1A01, UGT2B9(*)2, and UGT2B33 activity in rhesus liver microsomes, respectively.     

UDP-glucuronosyltransferases 1A6 and 1A10 catalyze reduced menadione glucuronidation

Menadione (2-methyl-1,4-naphthoquine), also known as vitamin K3, has been widely used as a model compound in the field of oxidative stress-related research. The metabolism of menadione has been studied, and it is known that menadione undergoes a two-electron reduction by NAD(P)H:Quinone oxidoreductase 1 (NQO1) after which the reduced form of menadione (2-methyl-1,4-naphthalenediol, menadiol) is glucuronidated and excreted in urine. To investigate which human UDP-glucuronosyltransferase (UGT) isoforms participate in the glucuronidation of menadiol reduced by NQO1 from menadione, we first constructed heterologously expressed NQO1 in Sf9 cells and tested the menadiol glucuronidating activity of 16 human recombinant UGT isoforms. Of the 16 UGT isoforms, UGTs 1A6, 1A7, 1A8, 1A9, and 1A10 catalyzed menadiol glucuronidation, and, of these, UGTs 1A6 and 1A10 catalyzed menadiol glucuronidation at much higher rates than the other UGTs. Menadiol was regioselectively glucuronidated in the manner of 4-position > 1-position by UGTs 1A7, 1A8, 1A9, and 1A10. In contrast to these UGTs, only UGT1A6 exhibited 1-menadiol-preferential glucuronidating activity. The results suggest possible detoxification pathways for quinones via NQO1 reduction followed by UGT glucuronidation.     

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 linoleic acid diols by human microsomal and recombinant UDP-glucuronosyltransferases: identification of UGT2B7 as the major isoform involved

Recent reports suggest that linoleic acid (LA) epoxides and diols are associated with important physiological, pharmacological, and pathological events in vivo. We have shown recently that LA-diols are excellent substrates for human liver microsomal UDP-glucuronosyltransferases (UGTs); however, it is not known if other human tissues glucuronidate LA-diols or which UGT isozyme(s) is involved. The present studies with human intestinal microsomes indicate that glucuronidation of LA-diols occurs throughout the gastrointestinal tract, with the highest activity in the small intestine. LA-diols yielded exclusively hydroxyl-linked glucuronides, whereas LA yielded the carboxyl-linked glucuronide. Studies with human recombinant UGTs demonstrated that only UGT2B7 glucuronidated LA and LA-diols. Kinetic analysis with UGT2B7 yielded apparent K(m) values in the range of 40-70 microM and V(max) values from 4.5 to 5.4 nmol/mg x min. These studies indicate that LA and LA-diols are excellent substrates for intestinal UGTs and provide the first evidence for UGT2B7 being the major isoform involved.     

Glucuronidation of carboxylic acid containing compounds by UDP-glucuronosyltransferase isoforms

Glucuronide conjugation of xenobiotics containing a carboxylic acid moiety represents an important metabolic pathway for these compounds in humans. Several human UDP-glucuronosyltransferases (UGTs) have been shown to catalyze the formation of acyl-glucuronides, including UGT2B7, UGT1A3, and UGT1A9. In this study, recombinant expressed UGT isoforms were investigated with many structurally related carboxylic acid analogues, and the UGT rank order for catalyzing the glucuronidation of carboxylic acids was UGT2B7?UGT1A3 approximately UGT1A9. Despite being a poor substrate with UGT1A3, coumarin-3-carboxylic acid was not a substrate for any other UGT isoform tested in this study, suggesting that it could be a specific substrate for UGT1A3. Interestingly, UGT1A7 and UGT1A10 also react with several carboxylic acid aglycones. Kinetic analysis showed that UGT2B7 exhibits much higher glucuronidation efficiency (Vmax/Km) with ibuprofen, ketoprofen, and others, compared to UGT1A3. These data indicate that UGT2B7 could be the major isoform involved in the glucuronidation of carboxylic acid compounds in humans.     

Determination of major UDP-glucuronosyltransferase enzymes and their genotypes responsible for 20-HETE glucuronidation

The compound 20-HETE is involved in numerous physiological functions, including blood pressure and platelet aggregation. Glucuronidation of 20-HETE by UDP-glucuronosyltransferases (UGTs) is thought to be a primary pathway of 20-HETE elimination in humans. The present study identified major UGT enzymes responsible for 20-HETE glucuronidation and investigated their genetic influence on the glucuronidation reaction using human livers (n = 44). Twelve recombinant UGTs were screened to identify major contributors to 20-HETE glucuronidation. Based on these results, UGT2B7, UGT1A9, and UGT1A3 exhibited as major contributors to 20-HETE glucuronidation. The K_m values of 20-HETE glucuronidation by UGT1A3, UGT1A9, and UGT2B7 were 78.4, 22.2, and 14.8 μM, respectively, while V_max values were 1.33, 1.78, and 1.62 nmol/min/mg protein, respectively. Protein expression levels and genetic variants of UGT1A3, UGT1A9, and UGT2B7 were analyzed in human livers using Western blotting and genotyping, respectively. Glucuronidation of 20-HETE was significantly correlated with the protein levels of UGT2B7 (r² = 0.33, P < 0.001) and UGT1A9 (r² = 0.31, P < 0.001), but not UGT1A3 (r² = 0.02, P > 0.05). A correlation between genotype and 20-HETE glucuronidation revealed that UGT2B7 802C>T, UGT1A9 −118T₉>T₁₀, and UGT1A9 1399T>C significantly altered 20-HETE glucuronide formation (P < 0.05–0.001). Increased levels of 20-HETE comprise a risk factor for cardiovascular diseases, and the present data may increase our understanding of 20-HETE metabolism and cardiovascular complications.     

Anti-Cancer Drugs Elicit Re-Expression of UDP-Glucuronosyltransferases in Melanoma Cells

The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the detoxification of carcinogens as well as clearance of anti-cancer drugs. In humans, 19 UGT family members have been identified and are expressed in a tissue specific manner throughout the body. However, the UGTs have not been previously characterized in melanocytes or melanoma. In the present study, UGT2B7, UGT2B10, and UGT2B15 were identified as being normally expressed in human melanocytes. The same three UGT family members were also expressed in the primary melanoma cell line WM115. No UGT expression was detected in another primary melanoma cell line, WM3211, or in any metastatic melanoma cell line examined. These results suggest that UGT expression is lost during melanoma progression. Treatment of WM3211 or metastatic melanoma cell lines with anti-cancer agents (including vemurafenib) induced expression of UGT2B7, UGT2B10 and UGT2B15 demonstrating that melanoma cells retain the ability to re-express these same three UGTs. The corresponding increase in glucuronidation activity in melanoma cells following anti-cancer treatment was also observed. Furthermore, knockdown of UGT2B7 in WM115 cells sensitized these cells to treatment by adriamycin and epirubicin indicating that UGT2B7 is involved in resistance to these drugs. However, knockdown of UGT2B7 had no effect on temozolomide toxicity. Taken together, these results clearly demonstrate a role for UGTs in melanoma etiology. Since the UGTs are drug metabolism enzymes, we propose that re-expression of the UGTs constitutes a previously unsuspected mechanism for intratumoral drug resistance in melanoma.     

Tissue-specific, inducible, and hormonal control of the human UDP-glucuronosyltransferase-1 (UGT1) locus

The human UDP-glucuronosyltransferase 1 (UGT1) locus spans nearly 200 kb on chromosome 2 and encodes nine UGT1A proteins that play a prominent role in drug and xenobiotic metabolism. Transgenic UGT1 (Tg-UGT1) mice have been created, and it has been demonstrated that tissue-specific and xenobiotic receptor control of the UGT1A genes is influenced through circulating humoral factors. In Tg-UGT1 mice, the UGT1A proteins are differentially expressed in the liver and gastrointestinal tract. Gene expression profiles confirmed that all of the UGT1A genes can be targeted for regulation by the pregnane X receptor activator pregnenolone-16alpha-carbonitrile (PCN) or the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, the selective induction of glucuronidation activity toward lamotrigine, ethinyl estradiol, chenodeoxycholic acid, and lithocholic acid by either PCN or TCDD in small intestine from Tg-UGT1 mice corresponded to expression of the locus in this tissue. Induction of UGT1A1 by PCN and TCDD is believed to be highly dependent upon glucocorticoids, because submicromolar concentrations of dexamethasone actively promote PCN and TCDD induction of UGT1A1 in Tg-UGT1 primary hepatocytes. The role of hormonal control of the UGT1 locus was further verified in pregnant and nursing Tg-UGT1 mice. In maternal 14-day post-conception Tg-UGT1mice, liver UGT1A1, UGT1A4, and UGT1A6 were induced, with the levels returning to near normal by birth. However, maternal liver UGT1A4 and UGT1A6 were dramatically elevated and maintained after birth, indicating that these proteins may play a critical role in maternal metabolism during lactation. With expression of the UGT1 locus confirmed in a variety of mouse tissues, these results suggested that the Tg-UGT1 mice will be a useful model to examine the regulatory and functional properties of human glucuronidation.     

Biotransformation of Bisphenol AF to Its Major Glucuronide Metabolite Reduces Estrogenic Activity

Bisphenol AF (BPAF), an endocrine disrupting chemical, can induce estrogenic activity through binding to estrogen receptor (ER). However, the metabolism of BPAF in vivo and the estrogenic activity of its metabolites remain unknown. In the present study, we identified four metabolites including BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate in the urine of Sprague-Dawley (SD) rats. BPAF-G was further characterized by nuclear magnetic resonance (NMR). After treatment with a single dose of BPAF, BPAF was metabolized rapidly to BPAF-G, as detected in the plasma of SD rats. Biotransformation of BPAF to BPAF-G was confirmed with human liver microsomes (HLM), and V_max of glucuronidation for HLM was 11.6 nmol/min/mg. We also found that BPAF glucuronidation could be mediated through several human recombinant UDP-glucuronosyltransferases (UGTs) including UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, among which UGT2B7 showed the highest efficiency of glucuronidation. To explain the biological function of BPAF biotransformation, the estrogenic activities of BPAF and BPAF-G were evaluated in ER-positive breast cancer T47D and MCF7 cells. BPAF significantly stimulates ER-regulated gene expression and cell proliferation at the dose of 100 nM and 1 μM in breast cancer cells. However, BPAF-G did not show any induction of estrogenic activity at the same dosages, implying that formation of BPAF-G is a potential host defense mechanism against BPAF. Based on our study, biotransformation of BPAF to BPAF-G can eliminate BPAF-induced estrogenic activity, which is therefore considered as reducing the potential threat to human beings.     

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 microM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.