No 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)

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and 3 H]atRA was light-and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA ␤-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-␤-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [ 3 H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.