Vitamin A and its derivatives, especially retinoic acid, are involved in many key physiological processes, including regulation of growth and differentiation of many cell types. All-trans retinoic acid and 13-cis retinoic acid are interconvertible, naturally occurring isomers. However, when large amounts of retinoic acid (RA) are ingested, as a result of therapeutic treatment for skin disorders or for cancer, toxicity and teratogenicity can result. Glucuronidation of RA by UDP- glucuronosyltransferase (UDPGT, EC,2.4.17), a microsomal membrane enzyme, decreases the toxicity of retinoic acid. Retinoic acid beta-glucuronide (RAG) has been shown to express biological activity and antineoplastic action in a HL-60 cell line (Zile, et al, 1987). Similar results in the BA-HAN-1C cell line also demonstrated that RAG is as active as retinoic acid itself in inhibiting growth and inducing differentiation (Biesalski, et al., 1993). The UDPGT enzymes are a family of isoenzymes actively involved in the conjugation of UDP-glucuronic acid to a variety of structurally different compounds, including xenobiotics and endogenous substrates. Since the role of UDPGT in the metabolism of RA is essential to the regulation of its physiological function and detoxification, the proposed experiments are designed to characterize human hepatic RA UDPGT acidity and to establish which UDPGT isoenzyme(s) are specific for RA glucuronidation.
The specific aims of this proposal include; 1) To ascertain the kinetic and physical characteristics of human UDPGT isoenzyme(s) for retinoic acid; 2) To isolate the retinoic acid UDPGT isoenzyme(s) from human liver microsomes and to determine whether the human RA UDPGT isoenzyme(s) are the same or different from those isoenzymes characterized for p-nitrophenol (pNP), chloramphenicol (CAP) and bilirubin (BR);' and 3) To evaluate the role of cellular retinoic acid-binding protein (CRABP) as a coligand in retinoic acid conjugation and regulation. CRABP is the protein to which retinoic acid (RA) binds when it enters the cell and may reflect the biological requirement of these cells for RA. Holo-CRABP acts as a substrate for RA metabolism, providing direct transfer of RA to microsomal catabolizing enzymes (Napoli, et al., 1993). Experiments are proposed not only to evaluate holo-CRABP in glucuromidation, but also to evaluate differences between 13-cis and all- trans RA in these reactions. The results of these studies will contribute significantly to our understanding of the metabolic regulation of RAG.
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