Mammalian UDP-glucuronosyltransferase (UGT) isozymes are critical catalysts in the broad and critical function of detoxifying endogenous products and potentially injurious lipid-soluble phenols derived from the diet and environment. The isozymes detoxify by conjugation of glucuronic acid with metabolites, drugs, toxins, and environmental chemicals to water-soluble excretable products. The system prevents accumulation of lethal plasma levels of bilirubin, as well as inactivates many drugs and averts mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons--including benzo(a)pyrene-- found in cigarette smoke and automobile emissions. Moreover, UGT prevents accumulation of high concentrations of dietary-derived polyphenols that inhibit critical enzymes. The specific properties and enzymatic mechanism(s) allowing UGT to convert numerous structurally unrelated lipid-soluble phenols to innocuous glucuronides are unknown. An important research aim, therefore, is to understand the properties and mechanism(s) that enable this system to detoxify a vast number of agents maintaining chemical homeostasis. While analyzing UGTs to understand glucuronidation requirements in human colon cell lines, we discovered the isozymes require phosphorylation. As phosphorylation is mediated by at least 2 PKC isozymes, the action of classical PKC agonists and antagonists, as well as effects of PKC translocation-specific inhibitor peptides, confirmed the involvement of signaling events. Mutants for two predicted phosphorylation sites in UGT were dominant negative, while a third site indicated phospho-group(s) play a unique and novel role in catalysis by operationally controlling pH optima and substrate selection. Expression of UGT activity and its mutants in conjunction with over expression of PKCs demonstrated site-specific phosphorylation. Evidence from 10 different UGTs indicates phosphorylation is a broadly-based requirement. Hydrogen-peroxide stimulation of constitutive UGT and inhibition of hydrogen peroxide-enhanced activity by catalase and herbimycin suggest cellular oxidants are signal(s) for the PKC-UGT system. Results demonstrate a linkage between oxidative stress-stimulated phosphorylation of PKC and the critical cellular function of detoxification. As the promising immunosuppressant, mycophenolic acid (MPA), has been introduced into transplantation and autoimmune disease protocols, there are serious side-effects associated with high dosage requirements due to extensive glucuronidation. We found that the cellular distribution and biochemical properties of UDP-glucuronosyltransferase1A7 (UGT1A7) through UGT1A10--primary metabolizers of MPA-- contribute significantly to high oral-dose requirements. In-situ hybridization studies revealed UGT1A7-, UGT1A9-, and UGT1A10-mRNAs are located in GI mucosal epithelia; studies with microsomes isolated from adjacent specimens showed esophagus, ileum, duodenum, and colon have high to moderately high glucuronidating activities. Recombinant UGTs avidly glucuronidate MPA showing nearly linear increases in activity at concentrations as high as 800 ?M; only UGT1A9 showed typical saturation kinetics. Each isozyme generates ~80 % ether-linked and ~20% acylglucuronide. To establish in-vivo impact of MPA glucuronidation, we used general kinase inhibitor, curcumin, and highly specific protein kinase C inhibitor, calphostin-C, on LS180 colon cells to inhibit UGTs by targeting newly discovered phosphorylation requirement. Concentration dependent inhibition of radiolabeled orthophosphate labeling of UGTs by calphostin-C with parallel loss of activity confirmed their phosphorylation. Transient inhibition of glucuronidation by oral pretreatment with curcumin before MPA administration caused nearly 3-fold greater immunosuppression of antigen-stimulated spleen cytotoxic T-lymphocyte proliferation in mice. Hence, curcumin-pretreatment to inhibit GI-distributed UGT is a potential model for increasing bioavailability of highly glucuronidated drugs.
