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 yield reactive sulfur-containing metabolites which bind covalently to cellular macromolecules. Mitochondria contain beta-lyase enzymes and are primary targets for binding and toxicity. Previously, mitochondrial protein and/or DNA have been considered as molecular targets for cysteine conjugate metabolite binding. We now report that metabolites of nephrotoxic cysteine conjugates form covalent adducts with rat kidney mitochondrial phospholipids. Rat kidney mitochondria were incubated with the 35S-labeled conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), S-(1,2-dichlorovinyl)-L-cysteine, and S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine. Quantitation of metabolite binding to whole mitochondria and to mitochondrial protein and lipid fractions revealed that as much as 42% of the 35S-label associated with the mitochondria was found in the lipid fraction. Total lipids were also extracted from 35S-treated mitochondria and separated by thin-layer chromatography. 35S-Containing metabolites were found in the lipid fractions from mitochondria treated with each of the conjugates. Lipids from both [35S]CTFC- and [35S]-TFEC-treated mitochondria contained major 35S-labeled lipid adducts which had similar mobility by thin-layer chromatography. Fatty acid analysis, 19F and 31P NMR spectroscopy, and mass spectrometric analyses confirmed that the major TFEC and CTFC adducts are thioamides of phosphatidylethanolamine.
Hayden
P J
PJ
W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
Welsh
C J
CJ
Yang
Y
Y
Schaefer
W H
WH
Ward
A J
AJ
Stevens
J L
JL
D000818:Animals; D002070:Butadienes / Q000378:metabolism* / Q000633:toxicity; D002855:Chromatography, Thin Layer; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D005227:Fatty Acids / Q000378:metabolism; D006845:Hydrocarbons, Fluorinated / Q000378:metabolism* / Q000633:toxicity; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D016339:Spectrometry, Mass, Fast Atom Bombardment; D005466:Fluorocarbons
D002070:Butadienes; D003545:Cysteine; D005227:Fatty Acids; D006845:Hydrocarbons, Fluorinated; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C039961:S-(1,2-dichlorovinyl)cysteine; C039330:S-pentachlorobuta-1,3-dien-yl-cysteine; C064116:S-(1,1,2,2-tetrafluoroethyl)cysteine; D005466:Fluorocarbons; C047601:S-(2-chloro-1,1,2-trifluoroethyl)cysteine