Subcellular localization of UGT1A10, the most active UGT against the dietary carcinogen PhIP and its major metabolite, N-OH-PhIP.

B62

The UDP glucuronosyltransferase (UGT), UGT1A10, plays an important role in the glucuronidation of a variety of endogenous and exogenous substances including many important carcinogens and is highly expressed in numerous target tissues for dietary cancers including the colon. Two classes of dietary carcinogens, heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs), are known to be detoxified by the UGT family of enzymes. Recently, our laboratory demonstrated that UGT1A10 has considerably more activity against PAHs in vitro than any other UGT family member. In this study we focused on the glucuronidation of the HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine (PhIP) and its bioactivated metabolite, N-OH-PhIP. We demonstrate that UGT1A10 is significantly more active against PhIP and N-OH-PhIP than any other UGT family member in vitro using whole cell homogenates of HEK293 cells specifically overexpressing individual UGTs. The order of glucuronidation activity against PhIP was 1A10>1A4>1A1>1A9>2B10. The other UGTs tested (1A3,1A6, 1A7, 1A8, 2B4, 2B7, 2B11, 2B15 and 2B17) were inactive. The order of glucuornidation activity against N-OH-PhIP was 1A10>1A1>1A4>1A8>1A9> 1A3>1A7. The other UGT family members examined (1A6, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17) showed no activity. Kinetic analysis revealed a 5-10-fold higher level of activity as compared to the next most active UGT against both PhiP and N-OH-PhiP as determined by K_m and V_max/K_m. Furthermore, we demonstrate that UGT1A10 glucuronidation activity can only be correctly assessed in vitro by using whole cell homogenates, as opposed to examining the microsomal fraction, since very low levels of UGT1A10 expression was observed in the microsomal fraction by Western blot analysis, indicating that the majority of UGT1A10 is not localized to the endoplasmic reticulum as is the case with other UGT family members. Experiments to determine the subcellular localization of UGT1A10 are currently underway.