METABOLISM OF TETRACHLOROETHENE IN RATS - IDENTIFICATION OF N-EPSILON-(DICHLOROACETYL)-L-LYSINE AND N-EPSILON-(TRICHLOROACETYL)-L-LYSINE AS PROTEIN ADDUCTS

Tetrachloroethene causes renal tumors in male rats after inhalation exposure. Tetrachloroethene is metabolized by cytochrome P-450 and by glutathione conjugation. Cytochrome P-450-dependent oxidation results in the formation of trichloroacetyl chloride, which may acylate cellular nucleophiles; glutathione conjugation results in the formation of S-(1,2,2-trichlorovinyl)glutathione, which is metabolized to the corresponding cysteine S-conjugate. S-(1,2,2-Trichlorovinyl)-L-cysteine is activated by renal cysteine conjugate beta-lyase to give dichlorothioketene. Covalent binding of this electrophile is presumably responsible for the renal toxicity of tetrachloroethene. In this report, we demonstrate the formation of protein adducts formed from tetrachloroethene using SDS-PAGE and immunochemical detection with rabbit anti-trifluoroacetyl serum. This serum recognizes dichloroacetylated rabbit serum albumin prepared by chemical modification of rabbit serum albumin with S-ethyl dichlorothioacetate and exhibited a high specificity for N-epsilon-(dichloroacetyl)-L-lysine residues in proteins as shown by competitive ELISA. In the liver of [C-14]tetrachloroethene-treated rats, the antibody recognized several modified proteins in microsomes. A protein adduct in rat liver identified by GC/MS after hydrolysis was N-epsilon-(trichloroacetyl)-L-lysine. Western blots of renal fractions from rats treated with [C-14]tetrachloroethene (200 mg/kg) or S-(1,2,2-trichlorovinyl)-L-cysteine (40 mu mol/kg, iv) suggested the presence of modified mitochondrial and cytosolic proteins; no modified proteins were detected in microsomes. Proteins of identical molecular weight were modified by tetrachloroethene and by S-(1,2,2-trichlorovinyl)-L-cysteine in vivo. Radioactivity derived from [C-14]tetrachloroethene comigrated in SDS-PAGE with the proteins in kidney mitochondria and cytosol recognized by the antibody. Incubation of renal mitochondria and cytosol with S-(1,2,2-trichlorovinyl)-L-cysteine in vitro also gave modified proteins; no modified proteins were found in incubations containing (aminooxy)acetic acid, an inhibitor of beta-lyase. In renal mitochondria incubated with S-(1,2,2-trichlorovinyl)-L-cysteine, N-epsilon-(dichloroacetyl)L-lysine was identified as a modified amino acid by GC/MS/SIM after protein hydrolysis. N-epsilon-(Dichloroacetyl)-L-lysine was predominantly present in renal proteins after administration of tetrachloroethene to rats. The detection of dichloroacetylated proteins in kidney after tetrachloroethene exposure further supports a role for beta-lyase in the chronic renal toxicity of tetrachloroethene.