title: Identification of a carboxylesterase as the major protein bound by molinate.
abstract: Molinate, a herbicide widely used on rice, has been previously shown to cause testicular toxicity when a single dose is administered to Sprague-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 important in testicular toxicity. Both the sulfoxide and sulfone metabolites of molinate are electrophilic and molinate covalent binding to cellular macromolecules has been attributed to formation of these reactive metabolites. The present study has investigated the nature of the binding reaction of 14C-molinate as well as 14C-molinate sulfoxide and 14C-molinate sulfone in liver and testis microsomal preparations. All three compounds in preparations from both tissues bound extensively and tightly to only one protein of approximately 60 kDa molecular weight on SDS-PAGE. Isoelectric focusing PAGE revealed a pI of approximately 6.0 and native PAGE analysis revealed a native molecular weight of 180 kDa. These data, along with the ability of phenylmethylsulfonyl fluoride to block binding of the 14C-molinate, suggested the molinate-bound protein was an esterase. The protein was purified to homogeneity and MALDI-TOF mass spectral analysis was consistent with Hydrolase A, a carboxylesterase present in both liver and testis. N-terminal sequence analysis revealed 100% homology with Hydrolase A for the first 17 residues. The effect of molinate administration on in vivo esterase activity was assessed both by enzymatic measurement and by histochemical measurement. Molinate treatment caused a marked inhibition of nonspecific esterase activity in both liver and testis. In the testis, histochemical staining showed the esterase activity inhibited by molinate was localized primarily to the Leydig cell, consistent with the localization of Hydrolase A. From these data, it is proposed that molinate-induced inhibition of esterase activity in the Leydig cell could inhibit the mobilization of cholesterol esters required for testosterone biosynthesis.
authors.affiliation: Department of Environmental Toxicology, University of California, Davis 95616, USA.
authors.forename: W T
authors.initials: WT
authors.lastname: Jewell
authors.forename: M G
authors.initials: MG
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001381:Azepines / Q000378:metabolism*; D002219:Carbamates; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000032:analysis / Q000378:metabolism*; D006540:Herbicides / Q000378:metabolism*; D007985:Leydig Cells / Q000187:drug effects / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D010574:Pesticide Synergists / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012451:Safrole / Q000031:analogs & derivatives* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013450:Sulfones / Q000378:metabolism; D013737:Testis / Q000187:drug effects / Q000201:enzymology; D013859:Thiocarbamates
chemical_list: D001381:Azepines; D002219:Carbamates; D006540:Herbicides; D010574:Pesticide Synergists; D013450:Sulfones; D013859:Thiocarbamates; C009821:molinate; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D012451:Safrole; C005746:sulfoxide