FORMATION OF MITOCHONDRIAL PHOSPHOLIPID ADDUCTS BY NEPHROTOXIC CYSTEINE CONJUGATE METABOLITES

Nephrotoxic cysteine conjugates derived from a variety of halogenated alkenes are enzymatically activated via the beta-lyase pathway to yiel d reactive sulfur-containing metabolites which bind covalently to cell ular macromolecules. Mitochondria contain beta-lyase enzymes and are p rimary targets for binding and toxicity. Previously, mitochondrial pro tein and/or DNA have been considered as molecular targets for cysteine conjugate metabolite binding. We now report that metabolites of nephr otoxic cysteine conjugates form covalent adducts with rat kidney mitoc hondrial phospholipids. Rat kidney mitochondria were incubated with th e S-35-labeled conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE C), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), S-(1,2-dichlo rovinyl)-L-cysteine, and S-(1,2,3,4,4-pentachlorobutadienyl)-L-cystein e. Quantitation of metabolite binding to whole mitochondria and to mit ochondrial protein and lipid fractions revealed that as much as 42% of the S-35-label associated with the mitochondria was found in the lipi d fraction. Total lipids were also extracted from S-35-treated mitocho ndria and separated by thin-layer chromatography. S-35-Containing meta bolites were found in the lipid fractions from mitochondria treated wi th each of the conjugates. Lipids from both [S-35]CTFC- and [S-35]-TFE C-treated mitochondria contained major S-35-labeled lipid adducts whic h had similar mobility by thin-layer chromatography. Fatty acid analys is, F-19 and P-31 NMR spectroscopy, and mass spectrometric analyses co nfirmed that the major TFEC and CTFC adducts are thioamides of phospha tidylethanolamine.