ACID-LABILE ADDUCTS TO PROTEIN CAN BE USED AS INDICATORS OF THE CYSTEINE S-CONJUGATE PATHWAY OF TRICHLOROETHENE METABOLISM

Covalent binding of radiolabel to tissue proteins following [C-14]trichloroethene (TRI) exposure has been used as a measure of TRI activation. Cross binding of C-14 label does not differentiate between alternate routes of metabolism and can be confounded when there is significant metabolic incorporation of radiolabel. We examined the covalent association of C-14 label to hepatic and renal proteins in male F344 rats and B6C3F1 mice following oral treatment with [C-14]TRI and three metabolites of TRI: [C-14]trichloroacetate (TCA), [C-14]dichloroacetate (DCA) and [C-14]dichlorovinylcysteine (DCVC) in vivo. Association of radiolabel from [C-14]TRI with hepatic proteins reached a maximum at 2 and 4 h in moose and rat hepatic proteins, respectively. Association of radiolabel with renal proteins reached a maximum at 8 h in both species. An approach was developed based upon formation of protein adducts that release acetate and monochloroacetate (MCA) on acid hydrolysis. These adducts were found to be specifically associated with the activation of DCVC to reactive intermediates. Acetate and MCA were identified by using two different conditions of high-performance liquid chromatography (HPLC) separation with differing selectivity. Diethylmaleate and aminooxyacetic acid pretreatment inhibited the formation of these adducts from TRI, consistent with requirements for glutathione and P-lyase. No evidence of these adducts was detected following [C-14]TCA and [C-14]DCA treatment. Renal acid-labile adduct formation from 25 mg/kg DCVC was approximately 12-fold greater in male B6C3F1 mice than in male F344 rats. They accounted for 7.8 and 4.6% of the total adducts to renal protein in rats and mice, respectively. Acid-labile adducts formed from 1000 mg/kg TRI were approximately two times greater in mice than rats. In this case, they accounted for 1.4 and 3.3% of the total adduct formed in renal proteins from TRI (corrected for metabolic incorporation), respectively. This greater dilution of adducts associated with DCVC in renal proteins of the rat suggests that covalent binding of TRI has less specificity for the DCVC pathway in rats than in mice.