Abstract
The stereo- and regioselective glucuronidation of 10 Δ4-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6β-, 7α-, 11β- or 17α-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6α- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6α-OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11α-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17β-glucuronidation and the high-affinity component of 16α-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16α- and 11α-OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6α- and 21-OHP may be used as selective “probes” for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.
Footnotes
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This work was supported by a grant from the National Health and Medical Research Council of Australia.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.013052.
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ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; HPLC, high performance liquid chromatography; 6α-OHP, 6α-hydroxyprogesterone; 6β-OHP, 6β-hydroxyprogesterone; 7α-OHAD, 4-androsten-7α-ol-3, 17-dione; 6β-OHAD, 4-androsten-6β-ol-3, 17-dione; TST, testosterone; 16α-OHP, 16α-hydroxyprogesterone; 17α-OHP, 17α-hydroxyprogesterone; 21-OHP, 21-hydroxyprogesterone; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferone-β-d-glucuronide; HLM, human liver microsome; HEK293, human embryo kidney 293 cell line; AZT, zidovudine; 11α-OHP, 11α-hydroxyprogesterone; 11β-OHP, 11β-hydroxyprogesterone.
- Received September 20, 2006.
- Accepted December 1, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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