IDENTIFICATION OF A CARBOXYLESTERASE AS THE MAJOR PROTEIN-BOUND BY MOLINATE

Molinate, a herbicide widely used on rice, has been previously shown t o cause testicular toxicity when a single dose is administered to Spra gue-Dawley rats. The sulfoxide metabolite of molinate also was capable of eliciting testicular damage but at lower dose levels than molinate , suggesting that metabolic activation via sulfoxidation could be impo rtant in testicular toxicity. Both the sulfoxide and sulfone metabolit es of molinate are electrophilic and molinate covalent binding to cell ular macromolecules has been attributed to formation of these reactive metabolites. The present study has investigated the nature of the bin ding reaction of C-14-molinate as well as C-14-molinate sulfoxide and C-14-molinate sulfone in liver and testis microsomal preparations. All three compounds in preparations from both tissues bound extensively a nd tightly to only one protein of approximately 60 kDa molecular weigh t on SDS-PAGE. Isoelectric focusing PAGE revealed a pi of approximatel y 6.0 and native PAGE analysis revealed a native molecular weight of 1 80 kDa. These data, along with the ability of phenylmethylsulfonyl flu oride to block binding of the C-14-molinate, suggested the molinate-bo und protein was an esterase. The protein was purified to homogeneity a nd MALDI-TOF mass spectral analysis was consistent with Hydrolase A, a carboxylesterase present in both liver and testis. N-terminal sequenc e analysis revealed 100% homology with Hydrolase A for the first 17 re sidues. The effect of molinate administration on in vivo esterase acti vity was assessed both by enzymatic measurement and by histochemical m easurement. Molinate treatment caused a marked inhibition of nonspecif ic esterase activity in both liver and testis. In the testis, histoche mical staining showed the esterase activity inhibited by molinate was localized primarily to the Leydig cell, consistent with the localizati on of Hydrolase A. From these data, it is proposed that molinate-induc ed inhibition of esterase activity in the Leydig cell could inhibit th e mobilization of cholesterol esters required for testosterone biosynt hesis. (C) 1998 Academic Press.