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The cytochrome P450 enzymes (CYPs), a group of heme-containing enzymes, catalyze oxidative metabolism of a wide range of drugs and xenobiotics, as well as different endogenous molecules. Strong inhibition of human CYPs is the most common cause of clinically associated pharmacokinetic drug-drug/herb-drug interactions (DDIs/HDIs), which may result in serious adverse drug reactions, even toxicity. Accurate and rapid assessing of the inhibition potentials on CYP activities for therapeutic agents is crucial for the prediction of clinically relevant DDIs/HDIs. Over the past few decades, significant efforts have been invested into developing optical substrates for the human CYPs, generating a variety of powerful tools for high-throughput assays to detect CYP activities in biologic specimens and for screening of CYP inhibitors. This minireview focuses on recent advances in optical substrates developments for human CYPs, as well as their applications in screening CYP inhibitors and DDIs/HDIs studies. The examples for rational design and optimization of highly specific optical substrates for the target CYP enzyme, as well as applications in investigating CYP-mediated DDIs, are illustrated. Finally, the challenges and future perspectives in this field are proposed. Collectively, this review summarizes the reported optical-based biochemical assays for highly efficient CYP activities detection, which strongly facilitated the discovery of CYP inhibitors and the investigations on CYP-mediated DDIs. SIGNIFICANCE STATEMENT: Optical substrates for cytochrome P450 enzymes (CYPs) have emerged as powerful tools for the construction of high-throughput assays for screening of CYP inhibitors. This mini-review covers the advances and challenges in the development of highly specific optical substrates for sensing human CYP isoenzymes, as well as their applications in constructing fluorescence-based high-throughput assays for investigating CYP-mediated drug-drug interactions., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Uridine diphosphate glucuronosyltransferases (UGTs) are key enzymes responsible for the body's ability to process a variety of endogenous and exogenous compounds. Significant gains in understanding UGT function have come from the analysis of variants seen in patients. We cared for a Sudanese child who showed clinical features of type 1 Crigler-Najjar syndrome (CN-1), namely severe unconjugated hyperbilirubinemia leading to liver transplantation. CN-1 is an autosomal recessive disorder caused by damaging mutations in the gene for UGT1A1, the hepatic enzyme responsible for bilirubin conjugation in humans. Clinical genetic testing was unable to identify a known pathogenic UGT1A1 mutation in this child. Instead, a novel homozygous variant resulting in an in-frame deletion, p.Val275del, was noted. Sanger sequencing demonstrated that this variant segregated with the disease phenotype in this family. We further performed functional testing using recombinantly expressed UGT1A1 with and without the patient variant, demonstrating that p.Val275del results in a complete lack of glucuronidation activity, a hallmark of CN-1. Sequence analysis of this region shows a high degree of conservation across all known catalytically active human UGTs, further suggesting that it plays a key role in the enzymatic function of UGTs. Finally, we note that the patient's ethnicity likely played a role in his variant being previously undescribed and advocate for greater diversity and inclusion in genomic medicine., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The antiplatelet drug clopidogrel is metabolized to an acyl- β -d-glucuronide, which causes time-dependent inactivation of CYP2C8. Our aim was to characterize the UDP-glucuronosyltransferase (UGT) enzymes that are responsible for the formation of clopidogrel acyl- β -d-glucuronide. Kinetic analyses and targeted inhibition experiments were performed using pooled human liver and intestine microsomes (HLMs and HIMs, respectively) and selected human recombinant UGTs based on preliminary screening. The effects of relevant UGT polymorphisms on the pharmacokinetics of clopidogrel were evaluated in 106 healthy volunteers. UGT2B7 and UGT2B17 exhibited the greatest level of clopidogrel carboxylic acid glucuronidation activities, with a CL int,u of 2.42 and 2.82 µ l⋅min -1 ⋅mg -1 , respectively. Of other enzymes displaying activity (UGT1A3, UGT1A9, UGT1A10-H, and UGT2B4), UGT2B4 (CL int,u 0.51 µ l⋅min -1 ⋅mg -1 ) was estimated to contribute significantly to the hepatic clearance. Nonselective UGT2B inhibitors strongly inhibited clopidogrel acyl- β -d-glucuronide formation in HLMs and HIMs. The UGT2B17 inhibitor imatinib and the UGT2B7 and UGT1A9 inhibitor mefenamic acid inhibited clopidogrel carboxylic acid glucuronidation in HIMs and HLMs, respectively. Incubation of clopidogrel carboxylic acid in HLMs with UDPGA and NADPH resulted in strong inhibition of CYP2C8 activity. In healthy volunteers, the UGT2B17*2 deletion allele was associated with a 10% decrease per copy in the plasma clopidogrel acyl- β -d-glucuronide to clopidogrel carboxylic acid area under the plasma concentration-time curve from 0 to 4 hours (AUC 0-4 ) ratio ( P < 0.05). To conclude, clopidogrel carboxylic acid is metabolized mainly by UGT2B7 and UGT2B4 in the liver and by UGT2B17 in the small intestinal wall. The formation of clopidogrel acyl- β -d-glucuronide is impaired in carriers of the UGT2B17 deletion. These findings may have implications regarding the intracellular mechanisms leading to CYP2C8 inactivation by clopidogrel., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The P24T polymorphic variant of the human UDP-glucuronosyltransferase 1A4 (UGT1A4*2, 70C>A) occurs within the signal peptide, five amino acids upstream of the cleavage site and the start of the mature protein. Bioinformatic analysis of the variant suggested that the signal peptide of part of the translated protein is cleaved two residues upstream of the regular site, whereas the rest is cleaved as usual. To test this, recombinant UGT1A4-P24T, with a C-terminal His-tag, was expressed in sf9 insect cells and affinity-purified for N-terminal protein sequencing. The results were in agreement with the in silico prediction. About half of the mutant protein was cleaved at the regular site, between S28 and G29, whereas the other half was cleaved two amino acids upstream, between A26 and E27. The glucuronidation of two substrates, dexmedetomidine and trifluoperazine, was assayed using membrane-enriched UGT1A4-P24T and wild-type UGT1A4. The variant exhibited much lower glucuronidation rates, but kinetic analyses revealed large differences between them only in the Vmax values. The Km values for both substrates were not affected by the mutation and its consequences. This might suggest that the unusual signal peptide cleavage in UGT1A4-P24T somehow disturbs protein folding. Moreover, it raises the possibility that the effect of UGT1A4-P24T on the glucuronidation rate in mammalian expression systems would be mild since they contain more effective post-translation protein control systems in the endoplasmic reticulum. In summary, our results reveal the effect of a polymorphic mutation on the signal sequence cleavage and thereby also the mature UGT., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Understanding drug glucuronidation in the dog, a preclinical animal, is important but currently poorly characterized at the level of individual enzymes. We have constructed cDNAs for the 10 dog UDP-glucuronosyltransferases of subfamily 1A (dUGT1As), expressed them in insect cells, and assayed their activity as well as the activity of the nine human UGT1As, toward 14 compounds. The goal was to find out whether individual dUGT1As and individual human UGT1As have similar substrate specificities. The results revealed similarities but also many differences. For example, similarly to the human UGT1A10, dUGT1A11 exhibited high glucuronidation activity toward the 3-OH of 17-β-estradiol, 17-α-estradiol, and ethinylestradiol, and also conjugated the drug entacapone. Unlike the human UGT1A10, however, it failed to catalyze considerable rates of R-propranolol, diclofenac, and indomethacin glucuronidation. The estrogen glucuronidation assays revealed that dUGT1A8 and dUGT1A10 have a capacity to catalyze the formation of (linked) diglucuronides, an activity no human UGT1A exhibited. dUGT1A2-dUGT1A4 are homologs of the human UGT1A4, but none of them catalyzed N-glucuronidation of dexmedetomidine. Contrary to the human UGT1A4, however, dUGT1A2-dUGT1A4 catalyzed indomethacin and diclofenac glucuronidation. It may be concluded that, perhaps with the exception of UGT1A6, high similarities in substrate specificity between individual dog and human UGTs of subfamily 1A are rare or partial. Activity assays with liver and intestine microsomes of both dog and human further revealed interspecies differences, particularly in glucuronidation rates. In the dog, the microsomes assays also strongly suggested important roles for dUGTs of other subfamilies, mainly in the liver., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Tamoxifen (Tam) is a selective estrogen receptor modulator used to inhibit breast tumor growth. Tam can be directly N-glucuronidated via the tertiary amine group or O-glucuronidated after cytochrome P450-mediated hydroxylation. In this study, the glucuronidation of Tam and its hydroxylated and/or chlorinated derivatives [4-hydroxytamoxifen (4OHTam), toremifene (Tor), and 4-hydroxytoremifene (4OHTor)] was examined using recombinant human UDP-glucuronosyltransferases (UGTs) from the 1A subfamily and human hepatic microsomes. Recombinant UGT1A4 catalyzed the formation of N-glucuronides of Tam and its derivatives and was the most active UGT enzyme toward these compounds. Therefore, it was hypothesized that single nucleotide polymorphisms (SNPs) in the promoter region of UGT1A4 have the ability to significantly decrease the glucuronidation rates of Tam metabolites in the human liver. In vitro activity of 64 genotyped human liver microsomes was used to determine the association between the UGT1A4 promoter and coding region SNPs and the glucuronidation rates of Tam, 4OHTam, Tor, and 4OHTor. Significant decreases in enzymatic activity were observed in microsomes for individuals heterozygous for -163G/A and -217T/G. These alterations in glucuronidation may lead to prolonged circulating half-lives and may potentially modify the effectiveness of these drugs in the treatment of breast cancer., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Bile acids (BA) are essential modulators of lipid, glucose, and cholesterol homeostasis, but they exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically relevant BA detoxification process. The present study characterized the BA-conjugating activity of the little-studied human UGTs of subfamily 2A: UGT2A1, 2A2, and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of six major bile acids: chenodeoxycholic acid (CDCA), cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA), hyocholic acid (HCA) and hyodeoxycholic acid (HDCA). UGT2A3 exhibited detectable but very low activity with all the tested BA substrates. UGT2A1 was highly efficient in forming LCA-3 and LCA-24G, CDCA-24, DCA-24, HCA-24, and HDCA-24G, whereas UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation and also was able to generate HDCA-6G, HDCA-24G, LCA-24G, and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 µM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 µM range. We demonstrate the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiologic importance of these reactions to BA disposition remains, however, to be clarified in vivo.

Recent observations revealed that human UDP-glucuronosyltransferase (UGT) 2B10 catalyzes N-glucuronidation of amine-containing compounds. Knowledge of the substrate specificity and clinical significance of UGT2B10 is still limited. The purpose of this study was to expand the knowledge of UGT2B10 substrates and to evaluate its significance in drug clearance. Using recombinant UGT2B10, we found that it catalyzes the N-glucuronidation of amitriptyline, imipramine, ketotifen, pizotifen, olanzapine, diphenhydramine, tamoxifen, ketoconazole, and midazolam. These are drugs that were previously reported to be substrates for UGT1A4 or UGT1A3, and that contain in their structure either tertiary aliphatic amines, cyclic amines, or an imidazole group. UGT2B10 was inactive in the glucuronidation of desipramine, nortriptyline, carbamazepine, and afloqualone. This group of drugs contains secondary or primary amines, and these results suggest that UGT2B10 preferably conjugates tertiary amines. This preference is partial because UGT2B10 did not conjugate the tertiary cyclic amine in trifluoperazine. Kinetic analyses revealed that the affinity and clearance of UGT2B10 for amitriptyline, imipramine, and diphenhydramine are significantly higher than the corresponding values of UGT1A4 and UGT1A3, although the Vmax values of UGT1A4 toward these drugs are considerably higher. These findings suggest that UGT2B10 plays a major role in the N-glucuronidation of these drugs at therapeutic concentrations. These results are also supported by inhibition studies with nicotine and hecogenin. In conclusion, this study expands the understanding of the substrate specificity of UGT2B10, highlighting its preference for tertiary amines with higher affinities and clearance values than those of UGT1A4 and UGT1A3.

The glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B was examined. UGT1A10 is highly active in the conjugation of the 3-OH in all these estriols, whereas UGT2B7 is the most active UGT toward one of the ring D hydroxyls, the 16-OH in estriol and 16-epiestriol, but the 17-OH in 17-epiestriol. Kinetic analyses indicated that the 17-OH configuration plays a major role in the affinity of UGT2B7 for estrogens. The glucuronidation of the different estriols by the human liver and intestine microsomes reflects the activity of UGT1A10 and UGT2B7 in combination with the tissues' difference in UGT1A10 expression. The UGT1A10 mutant 1A10-F93G exhibited much higher V(max) values than UGT1A10 in estriol and 17-epiestriol glucuronidation, but a significantly lower value in 16-epiestriol glucuronidation. To this study on estriol glucuronidation we have added experiments with 13-epiestradiol, a synthetic estradiol in which the spatial arrangement of the methyl on C18 and the hydroxyl on C17 is significantly different than in other estrogens. In comparison with estradiol glucuronidation, the C13 configuration change decreases the turnover of UGTs that conjugate the 3-OH, but increases it in UGTs that primarily conjugate the 17-OH. Unexpectedly, UGT2B17 exhibited similar conjugation rates of both the 17-OH and 3-OH of 13-espiestradiol. The combined results reveal the strong preference of UGT1A10 for the 3-OH of physiologic estrogens and the equivalently strong preference of UGT2B7 and UGT2B17 for the hydroxyls on ring D of such steroid hormones.

The presence of bovine serum albumin (BSA) largely modulates the enzyme kinetics parameters of the human UDP-glucuronosyltransferase (UGT) 1A9, increasing both the apparent aglycone substrate affinity of the enzyme and its limiting reaction velocity (Drug Metab Dispos 39:2117-2129, 2011). For a better understanding of the BSA effects and an examination of whether its presence changes the catalytic mechanism, we have studied the enzyme kinetics of 4-methylumbelliferone glucuronidation by UGT1A9 in the presence and absence of 0.1% BSA, using bisubstrate enzyme kinetic experiments, in both the forward and reverse directions, as well as product and dead-end inhibition. The combined results strongly suggest that the reaction mechanism of UGT1A9, and presumably other human UGTs as well, involves the formation of a compulsory-order ternary-complex, with UDP-α-d-glucuronic acid (UDPGA) as the first binding substrate. Based on the enzyme kinetic parameters measured for the forward and reverse reactions, the equilibrium constant of the overall reaction was calculated (Keq = 574) and the relative magnitudes of the reaction rate constants were elucidated. The inclusion of BSA in the bisubstrate kinetic experiments quantitatively changed the apparent enzyme kinetic parameters, presumably by removing internal inhibitors that bind to the binary enzyme-UDPGA (E-UDPGA) complex, as well as to the ternary E-UDPGA-aglycone complex. Nevertheless, the underlying compulsory-order ternary-complex mechanism with UDPGA binding first is the same in both the absence and presence of BSA. The results offer a novel understanding of UGT enzyme kinetic mechanism and BSA effects.

Many laboratories use recombinant UDP-glucuronosyltransferases (UGTs), expressed in baculovirus-infected insect cells, for drug glucuronidation studies. We have infected Sf9 insect cells with increasing amounts of recombinant baculovirus, encoding either UGT1A9 or UGT2B7, and measured both glucuronidation activity and immunodetectable UGT in the resulting cell homogenates. The correlation between glucuronidation rates and degree of infection followed different trends, depending on whether activity was the actual activity measured or was corrected for UGT expression level. Above a certain low level of infection, further increases in infection ratios led to a large decline in normalized activity, presumably due to the presence of full-length but inactive enzyme in the sample. Because immunodetection does not distinguish between active and inactive UGT, comparison of normalized activity between different batches of a recombinant UGT, mutants of a given UGT, or different UGTs is prone to large inaccuracies. Such inaccuracies could be reduced by lowering the degree of infection of the insect cells, in combination with careful monitoring of UGT expression. However, the latter requires suitable antibodies for comparing UGT expression levels among preparations, antibodies that are not always available. Poly-His (His-tag)-containing peptides, fused to the UGT C terminus, allow sensitive immunodetection of expressed enzymes with monoclonal antibodies. We have now carefully examined the effects of two such fusion peptides on enzyme kinetics. A minor increase in the K(m) values has been detected in the His-tagged UGTs, but no changes in parameters such as the kinetic model and the effects of albumin addition.

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.

Magnolol is a food additive that is often found in mints and gums. Human exposure to this compound can reach a high dose; thus, characterization of magnolol disposition in humans is very important. Previous studies indicated that magnolol can undergo extensive glucuronidation in humans in vivo. In this study, in vitro assays were used to characterize the glucuronidation pathway in human liver and intestine. Assays with recombinant human UDP-glucuronosyltransferase enzymes (UGTs) revealed that multiple UGT isoforms were involved in magnolol glucuronidation, including UGT1A1, -1A3, -1A7, -1A8, -1A9, -1A10, and -2B7. Magnolol glucuronidation by human liver microsomes (HLM), human intestine microsomes (HIM), and most recombinant UGTs exhibited strong substrate inhibition kinetics. The degree of substrate inhibition was relatively low in the case of UGT1A10, whereas the reaction catalyzed by UGT1A9 followed biphasic kinetics. Chemical inhibition studies and the relative activity factor (RAF) approach were used to identify the individual UGTs that played important roles in magnolol glucuronidation in HLM and HIM. The results indicate that UGT2B7 is mainly responsible for the reaction in HLM, whereas UGT2B7 and UGT1A10 are significant contributors in HIM. In summary, the current study clarifies the glucuronidation pathway of magnolol and demonstrates that the RAF approach can be used as an efficient method for deciphering the roles of individual UGTs in a given glucuronidation pathway in the native tissue that is catalyzed by multiple isoforms with variable and atypical kinetics.

The human UDP-glucuronosyltransferase (UGT) enzymes UGT1A9 and UGT2B7 play important roles in the hepatic glucuronidation of many drugs. The presence of bovine serum albumin (BSA) during in vitro assays was recently reported to lower the K(m) values of both these UGTs for their aglycone substrates without affecting the corresponding V(max) values. Nonetheless, using the specific substrates entacapone and zidovudine (AZT) for UGT1A9 and UGT2B7, respectively, and using an improved ultrafiltration method for measuring drug binding to BSA and to biological membranes, we found that the presence of BSA during the glucuronidation reaction leads to a large increase in the V(max) value of UGT1A9, in addition to lowering its K(m) value. On the other hand, in the case of UGT2B7, our results agree with the previously described effect of BSA, namely lowering the K(m) value without a large effect on the enzyme's V(max) value. The unexpected BSA effect on UGT1A9 was independent of the expression system because it was found in a recombinant enzyme that was expressed in baculovirus-infected insect cells as well as in the native enzyme in human liver microsomes. Moreover, the effect of BSA on the kinetics of 4-methylumbelliferone glucuronidation by recombinant UGT1A9 was similar to its effect on entacapone glucuronidation. Contrary to the aglycone substrates, the effect of BSA on the apparent K(m) of UGT1A9 for the cosubstrate UDP-α-D-glucuronic acid was nonsignificant. Our findings call for further investigations of the BSA effects on different UGTs and the inhibitors that it may remove.

Cell differentiation increases UDP-glucuronosyltransferase (UGT) gene expression in Caco-2 cells. Glucuronidation of 13 UGT substrates, 1-naphthol, diclofenac, epitestosterone, estradiol, ethinylestradiol, indomethacin, oxazepam, R- and S-propranolol, propofol, testosterone, trifluoperazine, and zidovudine, were studied to derive a broad view on the effect of cell differentiation on the glucuronidation activities of different human UGTs. In parallel, the glucuronidation of these compounds in human liver microsomes (HLM) and human intestinal microsomes (HIM) was analyzed. Because many of the substrates are highly lipophilic, the effects of dimethyl sulfoxide (DMSO) concentrations in the reaction mixture on glucuronidation rates were tested, as well as the effect of alamethicin, a pore-forming peptide. Large differences were observed in the effects of DMSO and alamethicin between recombinant UGTs and Caco-2 cells and HLM and HIM, and, therefore, the activity assays were performed under multiple conditions. Regardless of the assay conditions, however, the results clearly indicated that although differentiation increases glucuronidation activity, the rates in Caco-2 cells are mostly very low, much lower than those in either HLM or HIM. One clear exception was observed: substrates of UGT1A6, such as 1-naphthol, were glucuronidated at very high rates in both undifferentiated and differentiated Caco-2 cells. It may thus be concluded that Caco-2 cells, even differentiated ones, do not provide a good model system to assess first-pass drug glucuronidation in the intestine.

The amino acid sequences of the human UDP-glucuronosyltransferases (UGTs) 1A9 and 1A10 are 93% identical, yet there are large differences in their activity and substrate selectivity. For example, the regioselectivity in propranolol glucuronidation, the regioselectivity in dobutamine glucuronidation, and the glucuronidation rate of alpha- and beta-estradiol differ greatly between UGT1A9 and UGT1A10. To identify the residue responsible for the activity differences, we divided the N-terminal half of the two UGTs into five comparable segments by inserting four unique restriction sites at identical positions in both genes and constructing chimeras in which segments of UGT1A9 were individually replaced by the corresponding segments from UGT1A10. Activity analyses of the resulting mutants, 910A [1A10((1-83))/1A9((84-285))], 910B [1A9((1-83))/1A10((84-147))/1A9((148-285))], 910C [1A9((1-147))/1A10((148-181))/1A9((182-285))], 910D [1A9((1-181))/1A10((182-235))/1A9((236-285))], and 910E [1A9((1-235))/1A10((236-285))] indicated that more than one residue is responsible for the differences between UGT1A9 and UGT1A10. We next prepared four double chimeras, in which two of the above UGT1A9 segments were replaced simultaneously by the corresponding UGT1A10 segments. However, none of the double chimeras glucuronidated either estradiol, propranolol, or dobutamine at rates that resembled those of UGT1A10. On the other hand, studying the kinetics of 1-naphthol glucuronidation yielded more focused results, indicating that residues within segment B (84-147) contribute directly to the K(m) value for this substrate. Further mutagenesis and activity assays suggested that Phe117 of UGT1A9 participates in 1-naphthol binding. In addition, it appears that residues within segment C of the N-terminal domain, mainly at positions 152 and 169, contribute to the higher glucuronidation rates of UGT1A10.

We have examined the glucuronidation of psilocin, a hallucinogenic indole alkaloid, by the 19 recombinant human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B. The glucuronidation of 4-hydroxyindole, a related indole that lacks the N,N-dimethylaminoethyl side chain, was studied as well. UGT1A10 exhibited the highest psilocin glucuronidation activity, whereas the activities of UGTs 1A9, 1A8, 1A7, and 1A6 were significantly lower. On the other hand, UGT1A6 was by far the most active enzyme mediating 4-hydroxyindole glucuronidation, whereas the activities of UGTs 1A7-1A10 toward 4-hydroxyindole resembled their respective psilocin glucuronidation rates. Psilocin glucuronidation by UGT1A10 followed Michaelis-Menten kinetics in which psilocin is a low-affinity high-turnover substrate (K(m) = 3.8 mM; V(max) = 2.5 nmol/min/mg). The kinetics of psilocin glucuronidation by UGT1A9 was more complex and may be best described by biphasic kinetics with both intermediate (K(m1) = 1.0 mM) and very low affinity components. The glucuronidation of 4-hydroxyindole by UGT1A6 exhibited higher affinity (K(m) = 178 microM) and strong substrate inhibition. Experiments with human liver and intestinal microsomes (HLM and HIM, respectively) revealed similar psilocin glucuronidation activity in both samples, but a much higher 4-hydroxyindole glucuronidation rate was found in HLM versus HIM. The expression levels of UGTs 1A6-1A10 in different tissues were studied by quantitative real-time-PCR, and the results, together with the activity assays findings, suggest that whereas psilocin may be subjected to extensive glucuronidation by UGT1A10 in the small intestine, UGT1A9 is likely the main contributor to its glucuronidation once it has been absorbed into the circulation.

We have examined the glucuronidation of androsterone (5alpha-androstane-3alpha-ol-17-one), etiocholanolone (5beta-androstane-3alpha-ol-17-one), 5alpha-androstane-3alpha-,17beta-diol (5alpha-diol), and 5beta-androstane-3alpha-, 17beta-diol (5beta-diol) by 19 recombinant human UDP-glucuronosyltransferases (UGTs). The results reveal large differences in stereo- and regioselectivity between UGT2B7, UGT2B15, and UGT2B17. UGT2B7 conjugated all four androgens at the 3-OH but not at the 17-OH that is available in both diols. UGT2B7 exhibited a higher glucuronidation rate toward the steroids with a flat backbone, androsterone and 5alpha-diol, compared with etiocholanolone and 5beta-diol, which have a bent backbone. UGT2B17 readily glucuronidated androsterone and, particularly, etiocholanolone at the 3-OH, but in the two diols it exhibited high preference for the 17-OH and low glucuronidation rate at the 3-OH. UGT2B15 did not glucuronidate any of the studied four androgens at the 3-OH, but it did conjugate both diols at the 17-OH, with a clear preference for 5alpha-diol. Of the UGT1A subfamily, only UGT1A4 catalyzed the glucuronidation of androsterone and 5alpha-diol at measurable rates, even if low. UGT2A1 and UGT2A2 glucuronidated most compounds in this study, but mostly at rather low rates. An exception was the glucuronidation of etiocholanolone by UGT2A1 that revealed a very low substrate affinity in combination with very high V(max) value. The results shed new light on the substrate selectivity of individual UGTs in steroid glucuronidation. In addition they bear implications for doping analyses and its dependence of genetic polymorphism because testosterone is a precursor in the biosynthesis of these four androgens, whereas the contribution of UGT2B17 to their glucuronidation varies greatly.

An N-terminal domain histidine [corresponding to position 39 of UDP-glucuronosyltransferase (UGT) 1A1] is conserved in all UGT1A and UGT2B subfamily proteins except UGT1A4 (Pro-40) and UGT2B10 (Leu-34). Unlike most UGT1A and UGT2B xenobiotic-metabolizing enzymes, UGT1A4 and UGT2B10 lack the ability to glucuronidate 4-methylumbelliferone (4MU) and 1-naphthol (1NP), both planar phenols, and naproxen (a carboxylic acid). However, only UGT1A4 glucuronidates the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP). In this study, we sought to elucidate the influence of specific N-terminal histidine and proline residues on UGT enzyme substrate selectivity. The conserved N-terminal domain histidine of UGT1A1, UGT1A6, UGT1A9, and UGT2B7 was mutated to proline and leucine 34 of UGT2B10 was substituted with histidine, and the capacity of the wild-type and mutant proteins to glucuronidate 4MU, 1NP, LTG, TFP, and naproxen was characterized. Whereas UGT1A1(H39P), UGT1A6(H38P), and UGT1A9(H37P) lacked the ability to metabolize 4MU, 1NP, and naproxen, all glucuronidated LTG. K(m) values for UGT1A1(H39P) and UGT1A9(H37P) were 774 and 3812 microM, respectively, compared with 1579 microM for UGT1A4. UGT1A1(H39P) also glucuronidated TFP with a V(max)/K(m) value comparable to that of UGT1A4. In contrast to the wild-type enzyme, UGT2B10(L34H) glucuronidated 4MU and 1NP with respective K(m) values of 260 and 118 microM. UGT2B7(H35P) lacked activity toward all substrates. The data confirm a pivotal role for an N-terminal domain proline in the glucuronidation of the tertiary amines LTG and TFP by UGT1A subfamily proteins, whereas glucuronidation reactions involving proton abstraction generally, although not invariably, require a histidine at the equivalent position in both UGT1A and UGT2B enzymes.

Tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450-dependent oxidations, but little work has been done to characterize phase II metabolic pathways. In this study, we test the hypothesis that there are specific human UGTs responsible for classic cannabinoid metabolism. The activities of 12 human recombinant UGTs toward classic cannabinoids [cannabinol (CBN), cannabidiol (CBD), (-)-Delta(8)-THC, (-)-Delta(9)-THC, (+/-)-11-hydroxy-Delta(9)-THC (THC-OH), and (-)-11-nor-9-carboxy-Delta(9)-THC (THC-COOH)] were evaluated using high-performance liquid chromatography-tandem mass spectrometry and labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7 toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate sufficient activity for further characterization of steady-state kinetics. CBN was the most recognized substrate as evidenced by activities from hepatic UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect the introduction of an aromatic ring to Delta(9)-THC, leading to favorable pi stacking with phenylalanines in the UGT active site. Likewise, oxidation of Delta(9)-THC to THC-OH results in UGT1A9 and UGT1A10 activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes play a critical role in the metabolic clearance of cannabinoids. Taken together, glucuronidation of cannabinoids depends on upstream processing including enzymes such as CYP2C9 and CYP3A4.

The purpose of this work was to identify human UDP-glucuronosyltransferases (UGTs) capable of glucuronidating dopamine. Using a sensitive liquid chromatography-tandem mass spectrometry method, we screened all 19 known human UGTs and found that only one enzyme, UGT1A10, catalyzed dopamine glucuronidation at substantial rates, yielding both dopamine-4-O-glucuronide (37.1 pmol/min/mg) and dopamine-3-O-glucuronide (32.7 pmol/min/mg). Much lower (<2 pmol/min/mg) or no dopamine glucuronidation activity was found for all other UGTs tested at 1 mM dopamine. Evaluation of the UGT1A10 expression pattern in human tissues by quantitative reverse transcription-polymerase chain reaction confirmed that it is mainly expressed in small intestine, colon, and adipose tissue, whereas only low levels were found in trachea, stomach, liver, testis, and prostate but not in brain. Dopamine glucuronidation assays using microsomes from human liver and intestine corroborated these findings because activity in intestinal microsomes was markedly higher than that in liver microsomes. Moreover, the glucuronidation regioselectivity in intestinal microsomes was similar to that of recombinant UGT1A10, and both enzyme sources exhibited sigmoidal kinetics with substrate affinity (K(A)) values in the range of 2 to 3 mM. Examination of four UGT1A10 mutants, F90A, F90L, F93A, and F93L, revealed lower dopamine glucuronidation in all of them, particularly in F90A and F93A. Nonetheless, the substrate affinities of the four mutants were similar to that of UGT1A10. It is interesting to note that mutant F93L exhibited regioselectivity, conjugating dopamine at the 4-hydroxyl (OH) position approximately 3 times more efficiently than at the 3-OH position. These results shed new light on the structure and function of UGT1A10 and indicate that dopamine may be a useful probe substrate for this enzyme.

Testosterone and epitestosterone are endogenous steroids that differ in the configuration of the hydroxyl-bearing carbon at C-17. Testosterone is the predominant male sex hormone, whereas the role of epitestosterone is largely unclear. In humans, both androgens are excreted mainly as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone (T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative concentrations of the respective glucuronides. Some male athletes have T/E values greater than the accepted threshold value (4.0), even without testosterone abuse. We have analyzed athletes' urine samples and found that the main reason for such false-positive results in doping tests was a low epitestosterone glucuronide concentration not a high level of testosterone glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected in the different urine samples. Glucuronidation assays with the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A, and UGT2B revealed that UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone with affinity similar to that of testosterone. Epitestosterone glucuronidation is catalyzed mainly by UGT2B7, and the K(m) of this reaction is significantly lower than the K(m) of UGT2B17 for testosterone. Although UGT2B7 and UGT2B17 exhibited high, although converse, stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an extrahepatic enzyme that is expressed mainly in the nasal epithelium, catalyzed the glucuronidation of both steroids at considerable rates and similar kinetics. The results shed new light on the substrate specificity and stereoselectivity of human UGTs.

Recent studies show that the extrahepatic human UDP-glucuronosyltransferase (UGT)1A10 is capable of phase II glucuronidation of several major cytochrome P450 metabolites of warfarin (i.e., 6-, 7-, and 8-hydroxywarfarin). This study expands on this finding by testing the hypothesis that the UGT1A10 F(90)-M(91)-V(92)-F(93) amino acid motif is important for proper recognition and conjugation of hydroxywarfarin derivatives. Site-directed mutagenesis studies demonstrate that F(90) is critical for 6- and 7-hydroxywarfarin glucuronidation based on the complete loss of enzymatic activity toward these substrates. In contrast, V92A and F93A mutants lead to higher rates of substrate turnover, have minimum changes in K(m) values, and demonstrate substrate inhibition kinetics. A completely different activity profile is observed in the presence of 8-hydroxywarfarin. No change in either activity or affinity is observed with F90A when compared with wild type, whereas F93A and V92A mutants show increases in V(max) (3- and 10-fold, respectively) and minimum changes in K(m). Liquid chromatographytandem mass spectrometry studies show that enzymatic products produced by mutants are identical to wild-type products produced in the presence of 6-, 7-, and 8-hydroxywarfarin. Because F(90) is not critical for the glucuronidation of 8-hydroxywarfarin, there is likely another, different amino acid responsible for binding this compound. In addition, an inhibitory binding site may be formed in the presence of 6- and 7-hydroxywarfarin. This new knowledge and continued characterization of the hydroxywarfarin binding site(s) for UGT1A10 will help elucidate the molecular mechanism of hydroxywarfarin glucuronidation and potentially result in more effective anticoagulant therapies.

The glucuronidation of 17beta-estradiol (beta-estradiol) and 17alpha-estradiol (epiestradiol) was studied to elucidate how the orientation of the 17-OH group affects conjugation at the 3-OH or the 17-OH of either diastereomer. Recombinant human UDP-glucuronosyltransferases (UGTs) UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A10 conjugated one or both diastereomers, mainly at the 3-OH. The activity of UGT1A4 was low and unique because it was directed merely toward the 17-OH of both aglycones. UGT1A10 exhibited particularly high estradiol glucuronidation activity, the rate and affinity of which were significantly higher in the case of beta-estradiol than with epiestradiol. UGT1A9 did not catalyze estradiol glucuronidation, but UGT1A9-catalyzed scopoletin glucuronidation was competitively inhibited by beta-estradiol. UGT2B4, UGT2B7, and UGT2B17 exclusively conjugated the estradiols at the 17-OH position in a highly stereoselective fashion. UGT2B4 was specific for epiestradiol; UGT2B7 glucuronidated both diastereomers, with high affinity for epiestradiol, whereas UGT2B17 only glucuronidated beta-estradiol. UGT2B15 glucuronidated both estradiols at the 3-OH, with a strong preference for epiestradiol. Human UGT2A1 and UGT2A2 glucuronidated both diastereoisomers at both hydroxyl groups. Microsomal studies revealed that human liver mainly yielded epiestradiol 17-O-glucuronide, and human intestine primarily yielded beta-estradiol 3-O-glucuronide, whereas rat liver preferentially formed beta-estradiol 17-O-glucuronide. Of the three recombinant rat UGTs that were examined in this study, rUGT2B1 was specific for the 17-OH of beta-estradiol, rUGT2B2 did not catalyze estradiol glucuronidation, whereas rUGT2B3 exhibited high activity toward the 17-OH in both diastereoisomers. The results show that although many UGTs can catalyze estradiol glucuronidation, there are marked differences in their kinetics, regioselectivity, and stereoselectivity.

The human colon carcinoma cell line Caco-2 is often used as a model for intestinal drug absorption. To better understand xenobiotic glucuronidation in Caco-2 cells, we have examined the expression levels of different UDP-glucuronosyltransferases (UGTs) in them. The effects of two main factors were investigated, namely, passage number and cell differentiation. Hence, the mRNA levels of 15 human UGTs of subfamilies 1A and 2B were assessed in both undifferentiated and fully differentiated cells at four passage levels: P31, P37, P43, and P49. Quantitative reverse transcriptase-polymerase chain reaction was used to determine the mRNA levels of individual UGTs, and the values were normalized using beta-actin as a reference gene. The results indicate that although passage number in the tested range exerts a mild effect on the expression level of several UGTs, the contribution of cell differentiation is much larger. The expression of nearly all the UGTs that were examined in this study was significantly, sometimes greatly, increased during cell differentiation. UGT1A6 was a distinct exception to this rule, however, because it was already highly expressed in the undifferentiated cells. The mRNA findings were confirmed at the enzyme activity level by measuring the glucuronidation of 1-naphthol, a very good substrate for UGT1A6, as well as estradiol that is not glucuronidated by this enzyme. The results revealed that 1-naphthol glucuronidation activity was high in both the differentiated and undifferentiated cells, whereas estradiol glucuronidation was only detected in the differentiated cells. Thus, Caco-2 cell differentiation plays a major role in UGT expression and ensuing metabolic reactions.

Medetomidine is a chiral imidazole derivate whose dextroenantiomer is pharmacologically active. The major metabolic pathway of dexmedetomidine [(+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] in humans is N-glucuronidation at the imidazolate nitrogens. We have purified the N3- and N1-glucuronides of dexmedetomidine, termed DG1 and DG2, respectively, according to their elution order in liquid chromatography and determined their structure by 1H nuclear magnetic resonance (NMR). Studying medetomidine glucuronidation by human liver microsomes (HLMs) and recombinant UDP glucuronosyltransferase (UGT) 1A4 indicated that another human UGT plays a major role in these activities. We now demonstrate that this enzyme is UGT2B10. HLMs catalyzed DG1 and DG2 formation, at a ratio of 3:1, with two-enzyme kinetics that contain both a high-affinity component, K(m1) values of 6.6 and 8.7 microM, and a low-affinity component, K(m2) values > 1 mM. The DG1/DG2 ratio in the case of UGT2B10 was lower, 1.4:1, whereas the substrate affinity for both reactions was high, K(m) values of 11 and 16 microM. UGT1A4 produced mainly DG1 (DG1/DG2 ratio of 6.6:1) at low substrate affinities, K(m) values above 0.6 mM, but superior expression-normalized V(max) values. Levomedetomidine [(-)-4-(R)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] glucuronidation by HLMs yielded mostly the N3-glucuronide (LG1, structure determined by NMR), with monophasic kinetics and a K(m) value of 14 microM. The activity of UGT1A4 toward levomedetomide was low and generated both LG1 and LG2, whereas UGT2B10 exhibited relatively high activity and sharp regioselectivity, yielding only LG1, with a K(m) value of 7.4 microM. The results highlight the contribution of UGT2B10 to medetomidine glucuronidation and its potential importance for other N-glucuronidation reactions within the human liver.

All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the (393)DQMDNAK(399) region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q(394),D(396), and K(399) on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D(393) (D(398) in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q(394) (Q(399) in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D(393) is critical for the binding of glucuronic acid and that proximal residues, e.g., Q(394) (Q(399) in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism.

Propranolol is a nonselective beta-adrenergic blocker used as a racemic mixture in the treatment of hypertension, cardiac arrhythmias, and angina pectoris. For study of the stereoselective glucuronidation of this drug, the two propranolol glucuronide diastereomers were biosynthesized, purified, and characterized. A screen of 15 recombinant human UDP-glucuronosyltransferases (UGTs) indicated that only a few isoforms catalyze propranolol glucuronidation. Analysis of UGT2B4 and UGT2B7 revealed no significant stereoselectivity, but these two enzymes differed in glucuronidation kinetics. The glucuronidation kinetics of R-propranolol by UGT2B4 exhibited a sigmoid curve, whereas the glucuronidation of the same substrate by UGT2B7 was inhibited by substrate concentrations above 1 mM. Among the UGTs of subfamily 1A, UGT1A9 and UGT1A10 displayed high and, surprisingly, opposite stereoselectivity in the glucuronidation of propranolol enantiomers. UGT1A9 glucuronidated S-propranolol much faster than R-propranolol, whereas UGT1A10 exhibited the opposite enantiomer preference. Nonetheless, the Km values for the two enantiomers, both for UGT1A9 and for UGT1A10, were in the same range, suggesting similar affinities for the two enantiomers. Unlike UGT1A9, the expression of UGT1A10 is extrahepatic. Hence, the reverse stereoselectivity of these two UGTs may signify specific differences in the glucuronidation of propranolol enantiomers between intestine and liver microsomes. Subsequent experiments confirmed this hypothesis: human liver microsomes glucuronidated S-propranolol faster than R-propranolol, whereas human intestine microsomes glucuronidated S-propranolol faster. These findings suggest a contribution of intestinal UGTs to drug metabolism, at least for UGT1A10 substrates.

The initial glucuronidation rates were determined for eight recombinant human UDP-glucuronosyltransferases (UGTs) of the 1A subfamily, and the bisubstrate kinetics and inhibition patterns were analyzed. At low substrate concentrations, the reactions followed general ternary complex kinetics, whereas at higher concentrations of both substrates, the reactions were mostly characterized by ternary complex kinetics with substrate inhibition. The glucuronidation of entacapone by UGT1A9 was inhibited by 1-naphthol in a competitive fashion, with respect to entacapone, and an uncompetitive fashion, with respect to UDP-glucuronic acid (UDPGA). Its inhibition by UDP, on the other hand, was noncompetitive with respect to entacapone and competitive with respect to UDPGA. These inhibition patterns are compatible with a compulsory ordered bi bi mechanism in which UDPGA is the first-binding substrate. Despite the identical primary structure of the C-terminal halves of the UGT1A isoforms, there were marked differences in the respective K(m) values for UDPGA, ranging from 52 microM for UGT1A6 to 1256 microM for UGT1A8. Relative specificity constants were calculated for the eight UGT1A isoforms with 1-hydroxypyrene, 4-nitrophenol, scopoletin, 4-methylumbelliferone, and entacapone as aglycone substrates. The results demonstrated that seven of the UGT1A isoforms are capable of conjugating phenolic substrates with similar highest k(cat) values, and UGT1A4 has a lower relative turnover rate. The highest specificity constants were obtained for 1-hydroxypyrene, even with UGT1A6, which has been regarded as a specific isoform for small planar phenols. A k(cat) value of 1.9 s(-1) was calculated for the glucuronidation of scopoletin by purified UGT1A9.

Selected aspects of dobutamine glucuronidation were studied in detail. There are potentially four sites at which dobutamine can be conjugated to glucuronic acid. Three of the four dobutamine monoglucuronides that can be formed were enzymatically synthesized using pig liver microsomes, isolated, and characterized by tandem mass spectrometry, and (1)H and (13)C NMR spectroscopy. Analysis of dobutamine glucuronidation by liver microsomes from various sources revealed large variability in the ratios of the three regioisomers. Interestingly, catecholic dobutamine meta-O-glucuronide, by far the major product synthesized with human liver microsomes, was only a minor product for rat liver microsomes. Rabbit liver microsomes yielded diglucuronides, in addition to monoglucuronides. Activities of individual recombinant human UDP-glucuronosyltransferases (UGTs) were investigated, and the results suggested that dobutamine glucuronidation in the human liver is mainly carried out by UGTs 2B7 and 1A9. Among the extrahepatic UGTs, the formation of monoglucuronides, mainly catecholic meta-O-glucuronide, by UGTs 1A7 and 1A8 was similar to that by 1A9, whereas UGT1A10 also efficiently catalyzed the formation of catecholic dobutamine para-O-glucuronide.

A multidimensional study on the glucuronidation of anabolic androgenic steroids and their phase I metabolites by 11 recombinant human UDP-glucuronosyltransferases (UGTs) was carried out using liquid chromatographic-tandem mass spectrometric analyses. Large differences between the enzymes with respect to the conjugation profiles of the 11 tested aglycones were detected. Two UGTs, 1A6 and 1A7, did not exhibit measurable activity toward any of the aglycones that were examined in this study. Regioselectivity was demonstrated by UGTs 1A8, 1A9, and 2B15 that preferentially catalyzed hydroxyl glucuronidation at the 17beta-position. Most of the other enzymes glucuronidated hydroxyl groups at both the 3alpha- and the 17beta-positions. Clear stereoselectivity was observed in glucuronidation of diastereomeric nandrolone metabolites (5alpha-estran-3alpha-ol-17-one and 5beta-estran-3alpha-ol-17-one), whereas such specificity was not seen when analogous methyltestosterone metabolites were assayed. UGTs 1A1, 1A3, 1A4, 1A8, 1A9, 1A10, 2B4, 2B7, and 2B15 readily glucuronidated 5alpha-androstane-3alpha,17beta-diol, but none of them exhibited methyltestosterone glucuronidation activity. In agreement with the latter observations, we found that the methyltestosterone glucuronidation activity of human liver microsomes is extremely low, whereas in induced rat liver microsomes it was significantly higher. The homology among UGTs 1A7 to 1A10 at the level of amino acid sequence is very high, and it was thus surprising to find large differences in their activity toward this set of aglycones. Furthermore, the high activity of UGT1A8 and 1A10 toward some of the substrates indicates that extrahepatic enzymes might play a role in the metabolism of anabolic androgenic steroids.