Mammalian UDP-glucuronosyltransferase (UGT) isozymes are critical catalysts in the broad and vital function of detoxifying endogenous products and potentially injurious lipid-soluble phenols derived from the diet and the environment. The isozymes maintain chemical homeostasis and detoxify by conjugating metabolites, drugs, toxins, and environmental chemicals with glucuronic acid, yielding water-soluble excretable products. The system prevents accumulation of lethal plasma levels of bilirubin, inactivates many drugs, and averts mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons, including benzo(a)pyrene, which is found in cigarette smoke and automobile emissions. Moreover, UGT prevents accumulation of high concentrations of dietary-derived polyphenols that inhibit critical enzymes. An important research aim is to understand the specific, still unknown properties and enzymatic mechanism(s) that allow UGT to convert numerous structurally unrelated lipid-soluble phenols to innocuous glucuronides.

Phosphorylation of UGT Proteins with Signalling
Basu, Kole, Kubota, Mitra, Pennington
While attempting to understand the glucuronida-tion requirements of UGTS by using human colon cell lines, we discovered that the isozymes require phosphorylation. Phosphorylation is mediated by at least two protein kinase C (PKC) isozymes while the action of classical PKC agonists and antagonists, as well as the effects of PKC translocation-specific inhibitor peptides, confirmed the involvement of signalling events. Mutants for two predicted phosphorylation sites in UGT were dominant negative, and a third site indicated that the phospho-group(s) plays a unique and novel role in catalysis by 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 that phosphorylation is a broadly based requirement.

Figure 12

Cellular localization of mRNA encoding UGT1A1 and UGT1A7-10. In situ hybridization with antisense probes (A) and sense probes (B). The esophageal images were magnified 50x; all others were magnified 200x. Numbers below each panel represent glucuronidation by microsomes isolated from adjacent tissue in a two-hour incubation (pmol/hr/gm tissue) for comparison with the RNA levels depicted by in situ analysis. The substrates were bilirubin, thymol, phloretin, apigenin, and epigallocatechin 3-0-gallate for UGT1A1 (pH 6.4), UGT1A7 (pH 7.6), UGT1A8 (pH 7.0), UGT1A9 (pH 6.4), and UGT1A10 (pH 6.4), respectively. Substrates are 95 percent specific, except thymol and phloretin, which were 60 and 33 percent specific for UGT1A7 and UGT1A8, respectively. ND: not detectable.

Hydrogen peroxide stimulation of constitutive UGT and inhibition of H202-enhanced activity by catalase and herbimycin suggest that cellular oxidants are signal(s) for the PKC-UGT system. Our results demonstrate a linkage between oxidative stress–stimulated phosphorylation of PKC and the critical cellular function of detoxification.

Effect of Location and Properties of UGT in the Control of Gastrointestinal Chemical Absorption
Basu, Kole; in collaboration with McDonagh
Using Northern blot, in-situ, and biochemical analyses, we determined distribution and function of the bilirubin-metabolizing isozyme UGT1A1 and of the UGT1A7-10 isozymes. We found, for the first time, that the isozymes are segmentally distributed throughout the gastrointestinal (GI) mucosal epithelia (Fig. 12A and B). Recombinant isozymes exhibited pH optima of 6.4 or 7.6 and/or broad ranges; increasing substrate concen-trations either did not effect or progressively inhibited activity. Under different optimal conditions, all isozymes exhibited wide substrate specificity for diet- and environmentally associated chemicals. We demonstrated the impact of glucuronidation on drug and chemical absorption at the GI level by exploiting our recent finding that UGTs require phosphorylation. [33P]Ortho-phosphate incorporation into immunoprecipitable UGTs demonstrated phosphorylation. The simultaneous loss of UGT radiolabeling and activity in cell lines by the general kinase inhibitor curcumin and the PKC-specific inhibitor calphostin-C confirmed the requirement for phosphorylation. Using curcumin-treated mouse duodenal loops, we demonstrated that free bilirubin (a marker) uptake dramatically increased while the levels of bilirubin-glucuronides in portal blood, lumen, and tissue declined. Our results represent the first direct evidence that UGTs control GI absorption of polyphenols and that maintenance of their phosphorylation is required to limit chemical absorption.

Immunosuppressant Activity of Mycophenolic Acid Improved by Transient Inhibition of Gastrointestinal UGT
Basu, Kole
In view of extensive glucuronidation and resulting high dosage requirements, the promising immunosuppressant mycophenolic acid (MPA), used now in transplantation and autoimmune disease protocols, exhibits serious side effects. We found that the cellular distribution and biochemical properties of UGT1A7-10, primary metabolizers of MPA, contribute significantly to high oral-dose requirements. In situ hybridization studies revealed that UGT1A7-, UGT1A9-, and UGT1A10-mRNAs are located in GI mucosal epithelia; studies with microsomes isolated from adjacent specimens showed that 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 mM; only UGT1A9 showed typical saturation kinetics. Each isozyme generates about 80 percent ether-linked and about 20 percent acylglucuronide. To establish the in vivo impact of MPA glucuronidation, we treated LS189 colon cells with the general kinase inhibitor curcumin and the highly specific protein kinase C inhibitor calphostin-C to inhibit UGTs, targeting the newly discovered phosphorylation requirement. Concentration-dependent inhibition of [33P]ortho-phosphate labeling of UGTs by calphostin-C, with parallel loss of activity, confirmed the UGT’s phosphorylation. Transient inhibition of glucuronidation by oral pretreatment with curcumin before MPA administration caused nearly a three-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 the bioavailability of highly glucuronidated drugs.