4-Hydroxyretinoic Acid, a Novel Substrate for Human Liver Microsomal UDP-glucuronosyltransferase(s) and Recombinant UGT2B7

Abstract

It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OHRAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDPglucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OHRAc with 6-and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways. atRA 1 is a major metabolite of vitamin A (all-trans-retinol) that undergoes isomerization and metabolism in vivo, yielding 13-cis-retinoic acid (13-cis-RA), 9-cis-retinoic acid (9-cis-RA)

Expression of UGT2B7, a UDP-Glucuronosyltransferase Implicated in the Metabolism of 4-Hydroxyestrone and All-Trans Retinoic Acid, in Normal Human Breast Parenchyma and in Invasive and in Situ Breast Cancers

“…13,14,17 The marked reduction in expression of UGT2B7 in invasive breast cancers suggests an anticarcinogenic function for the enzyme. The proposition Figure 6.…”

“…30 -32 A second putative tumor suppressor function for UGT2B7, that of glucuronidation of atRA, is suggested by the recent identification of atRA as a second endogenous substrate of this isoenzyme. 17 Glucuronidation of atRA, like that of 4-OH-E 1 , can be viewed as a tumor-suppressor function: retinoyl-␤-glucuronide, the product of the reaction, has been shown in diverse studies to be a potent mediator of actions of RA, and glucuronidation may protect RA from oxidative metabolism. 3,18,19 UGT2B7 is the first and, thus far, the only member of the UGT family of isoenzymes known to have the potential to catalyze the glucuronidation of atRA with high catalytic efficiency.…”

“…While this study was in progress, UGT2B7 was shown to also catalyze with high efficiency the glucuronidation of all-trans retinoic acid (atRA). 17 This property of the enzyme may also offer protection against carcinogenesis, because glucuronidation preserves rather than abrogates actions of RA known to be important for maintaining epithelia in a differentiated state. 3,18,19 The finding reported here that UGT2B7 is expressed in the normal mammary epithelium, but that its expression is decreased or abrogated in invasive cancers, supports the notion that UGT2B7 serves a protective, tumor-suppressor function in human breast parenchyma.…”

Hepatic Cyp2d and Cyp26a1 mRNAs and Activities Are Increased During Mouse Pregnancy

“…All-trans-retinoic acid (atRA), dextromethorphan, and dextrorphan were purchased from Sigma-Aldrich (St. Louis, MO). 4-OH-RA and 4-oxo-RA were synthesized as previously described (Samokyszyn et al, 2000;Topletz et al, 2012). Optima-grade water, optima-grade acetonitrile, and ethyl acetate used for chemical analyses were purchased from Thermo Fisher Scientific.…”

Prediction of in vivo developmental toxicity of all-trans-retinoic acid based on in vitro toxicity data and in silico physiologically based kinetic modeling

“…Data on the in vitro kinetic conversion of ATRA by microsomes or specific biotransformation enzymes as reported in the literature (Ahmad et al 2000;Little et al 1997;Thatcher et al 2010;Lutz et al 2009;Samokyszyn et al 2000;supplementary Table 3) were used to determine the model parameter values for hepatic clearance. For the rat model, kinetic constants on ATRA metabolism obtained using liver, kidney, lung and brain fractions were used, whereas for the human model, kinetic constants on ATRA metabolism obtained using specific cytochrome P450 (CYP) and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes were used (supplementary Table 3).…”