Bioactivation mechanism of cytotoxic homocysteine S-conjugates.

S-(1,2-Dichlorovinyl)-L-homocysteine is a much more potent nephrotoxin than the corresponding cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine (A. A. Elfarra, L. H. Lash, and M. W. Anders (1986) Proc. Natl. Acad. Sci. USA 83, 2667-2671). The objective of the present experiments was to test the hypothesis that the increased toxicity of homocysteine S-conjugates may be associated with the formation of the reactive metabolite 2-oxo-3-butenoic acid, which may arise via a nonenzymatic retro-Michael elimination reaction from the 2-oxo acid metabolites of homocysteine S-conjugates. S-(2-Benzothiazolyl)-L-homocysteine, which was a substrate for purified bovine kidney cysteine conjugate beta-lyase (glutamine transaminase K) and whose metabolism was dependent on the presence of a 2-oxo acid, was cytotoxic in isolated rat kidney cells and was toxic to rat renal mitochondria, whereas the cysteine S-conjugate S-(2-benzothiazolyl)-L-cysteine had little effect. L-Methionine sulfoximine, L-canavanine, and the Michael acceptor methyl vinyl ketone were cytotoxic. The 2-hydroxy acid analogs of S-(1,2-dichlorovinyl)-L-homocysteine and 2-oxo-3-butenoic acid, S-(1,2-dichlorovinyl)-2-hydroxy-4-mercaptobutanoic acid and 2-hydroxy-3-butenoic acid, respectively, which are expected to be metabolized by rat renal L-2-hydroxy (L-amino) acid oxidase to yield 2-oxo-3-butenoic acid, were also cytotoxic. To obtain evidence for the formation of 2-oxo-3-butenoic acid as a product of the metabolism of L-homocysteine S-conjugates and analogs, trapping experiments were conducted. S-(2-Benzothiazolyl)-L-homocysteine, S-(1,2-dichlorovinyl)-L-homocysteine, L-methionine sulfoximine, and L-canavanine were converted by snake venom L-amino acid oxidase to 2-oxo-3-butenoic acid, which was trapped by the nucleophile methanethiol to yield 4-methylthio-2-oxobutanoic acid; the trapped product was derivatized with 2,4-dinitrophenylhydrazine and was identified by its electronic absorption spectrum and by high-performance liquid chromatography. Similar trapping experiments conducted with kidney homogenates and purified beta-lyase were not successful. The data indicate that the bioactivation of homocysteine S-conjugates and analogs involves the enzymatic formation of the corresponding 2-oxo acids followed by a nonenzymatic retro-Michael elimination reaction to yield the Michael acceptor 2-oxo-3-butenoic acid, which may contribute to the observed cytotoxicity of homocysteine S-conjugates.