Dog UDP-glucuronosyltransferase enzymes of subfamily 1A: cloning, expression, and activity.

Abstract

Understanding drug glucuronidation in the dog, a preclinical animal, is important but currently poorly characterized at the level of individual enzymes. We have constructed cDNAs for the 10 dog UDP-glucuronosyltransferases of subfamily 1A (dUGT1As), expressed them in insect cells, and assayed their activity as well as the activity of the nine human UGT1As, toward 14 compounds. The goal was to find out whether individual dUGT1As and individual human UGT1As have similar substrate specificities. The results revealed similarities but also many differences. For example, similarly to the human UGT1A10, dUGT1A11 exhibited high glucuronidation activity toward the 3-OH of 17-β-estradiol, 17-α-estradiol, and ethinylestradiol, and also conjugated the drug entacapone. Unlike the human UGT1A10, however, it failed to catalyze considerable rates of R-propranolol, diclofenac, and indomethacin glucuronidation. The estrogen glucuronidation assays revealed that dUGT1A8 and dUGT1A10 have a capacity to catalyze the formation of (linked) diglucuronides, an activity no human UGT1A exhibited. dUGT1A2-dUGT1A4 are homologs of the human UGT1A4, but none of them catalyzed N-glucuronidation of dexmedetomidine. Contrary to the human UGT1A4, however, dUGT1A2-dUGT1A4 catalyzed indomethacin and diclofenac glucuronidation. It may be concluded that, perhaps with the exception of UGT1A6, high similarities in substrate specificity between individual dog and human UGTs of subfamily 1A are rare or partial. Activity assays with liver and intestine microsomes of both dog and human further revealed interspecies differences, particularly in glucuronidation rates. In the dog, the microsomes assays also strongly suggested important roles for dUGTs of other subfamilies, mainly in the liver.