Glucuronidation of Anabolic Androgenic Steroids by Recombinant Human Udp-Glucuronosyltransferases

Abstract

ABSTRACT: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 17␤-position. Most of the other enzymes glucuronidated hydroxyl groups at both the 3␣-and the 17␤-positions. Clear stereoselectivity was observed in glucuronidation of diastereomeric nandrolone metabolites (5␣-estran-3␣-ol-17-one and 5␤-estran-3␣-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 5␣-androstane-3␣,17␤-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.

Glucuronidation of Monohydroxylated Warfarin Metabolites by Human Liver Microsomes and Human Recombinant UDP-Glucuronosyltransferases

“…Unless otherwise specified, all other chemicals and reagents were purchased from Sigma-Aldrich. Recombinant human UGT 1A1, 1A3, 1A4, and 1A6 -1A10 were produced in baculovirus-infected insect cells as described previously Kuuranne et al, 2003). These preparations were shown to contain similar amounts of protein by Western blot analysis using an anti-His antibody directed at the His-tag modification present on each of these recombinant isoforms.…”

The Glucuronidation of Δ4-3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6α- and 21-Hydroxyprogesterone Are Selective Substrates for UGT2B7

“…A comparison of the glucuronidation of 13 anabolic androgenic steroids by recombinant human UGTs (Kuuranne et al, 2003) and data from activity-screening studies (Green et al, 1994;Beaulieu et al, 1996) suggests that UGT 2B15 and 2B17 preferentially glucuronidate 16␣-and 17␤-hydroxyl groups in ring D of androgens or pregnanes. Also consistent with data presented here, UGT2B7 has previously been shown in this and other laboratories to glucuronidate numerous hydroxylated C18-, C19-, and C21-steroids (Ritter et al, 1990;Jin et al, 1993Jin et al, , 1997Coffman et al, 1998;Turgeon et al, 2001;Kuuranne et al, 2003;Lepine et al, 2004). Apart from the substrates identified in the present study, UGT2B7 efficiently glucuronidates 3␣-hydroxyandrogens, 3␣-hydroxypregnanes and 3,4-catecholestrogens.…”

“…For example, it is well established that testosterone 6␤-hydroxylation is catalyzed selectively by CYP3A (Mei et al, 1999). Similarly, UGT1A and UGT2B enzymes appear to differentially contribute to the glucuronidation of hydroxylated derivatives of C18-(estrogens), C19-and C21-steroids (Jin et al, 1997;Turgeon et al, 2001;Kuuranne et al, 2003;Lepine et al, 2004). ⌬ 4 -3-Keto-hydroxylated C19-and C21-steroids provide a convenient series of compounds for investigating the regio-and stereoselectivity of hydroxysteroid glucuronidation.…”

Phenylalanine90 and phenylalanine93 are crucial amino acids within the estrogen binding site of the human UDP-glucuronosyltransferase 1A10

“…Details of the cloning and expression of UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, and 1A10 in baculovirus-infected Sf9 insect cells as His-tagged proteins and the preparation of enriched membrane fractions were reported previously [24,25].…”

“…A new derivative of the mammalian expression vector pcDNA 3.1 (+) (Invitrogen), called pcDNA 3.39, was prepared by replacing the Nhe1-Apa1 fragment of the multi-cloning site of pcDNA 3.1 (+) with the similarly-digested fragment from the multi cloning site of Litmus 39 (New England BioLabs, Ipswich, MA). The UGT1A10 gene was transferred from the pFastBac derivative pFB-XHC [24] into pcDNA 3.39 and site-directed mutagenesis was performed on this construct, pcDNA3.39-1A10, using the QuickChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). Single point mutations were introduced into pcDNA3.39-1A10 according to manufacturer instructions.…”