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.
Lash
L H
LH
Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, NY 14642.
Elfarra
A A
AA
Rakiewicz-Nemeth
D
D
Anders
M W
MW
D000818:Animals; D001711:Biotransformation; D002470:Cell Survival / Q000187:drug effects; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D006882:Hydroxyapatites; D007668:Kidney / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D008190:Lyases / Q000302:isolation & purification / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D000637:Transaminases / Q000302:isolation & purification / Q000378:metabolism*
D006882:Hydroxyapatites; D006710:Homocysteine; D017886:Durapatite; D000637:Transaminases; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases