CDNA Cloning and Functional Analysis of Minipig Uridine Diphosphate-Glucuronosyltransferase 1A1

Abstract

Uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies, because many of their physiological characteristics are similar to those of humans. In this study, the similarities and differences in enzymatic properties of UGT1A1 between humans and minipigs were precisely identified. Minipig UGT1A1 (mpUGT1A1) cDNA was firstly cloned by the rapid amplification of cDNA ends (RACE) method, and the corresponding protein as well as human UGT1A1 (hUGT1A1) enzyme was expressed in insect cells. Then the kinetics of estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation by recombinant UGT1A1s as well as human and minipig liver microsomes were analyzed. The homology between mpUGT1A1 and hUGT1A1 at the amino acid level was 80.9%. E-3OH and SN-38 glucuronidation by recombinant hUGT1A1 and mpUGT1A1 showed allosteric sigmoidal kinetics. The CL value (29.1 µL/min/mg protein) for E-3OH glucuronidation of mpUGT1A1 was significantly higher (1.4-fold) than that of hUGT1A1, whereas the CL value (0.83 µL/min/mg protein) for SN-38 glucuronidation was significantly lower (27%) than that of hUGT1A1; however, the kinetic models and parameter levels for E-3OH and SN-38 glucuronidation by human and minipig liver microsomes did not parallel those in the respective species. These findings suggest that the enzymatic properties of UGT1A1 are considerably different between humans and minipigs. The information on species differences in UGT1A1 function gained in this study should help with in vivo extrapolation of xenobiotic metabolism and toxicity.