Formation of mitochondrial phospholipid adducts by nephrotoxic cysteine conjugate metabolites

Abstract

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.

Characterization of a Phospholipid Adduct Formed in Sprague Dawley Rats by Chloroform Metabolism: NMR Studies

“…This PL adduct arose from nucleophilic attack by the amine of the PE polar head group on the reactive electrophilic carbonyl group of phosgene, in agreement with what demonstrated for other toxic compounds (31,32). Expected stable adducts of the bifunctional electrophile phosgene with nucleophilic molecules are either those in which two nucleophiles …”

“…Literature data indicate that 31 P resonances of lipid mixtures are dependent on solvent composition (17). In this work, spectra were acquired resuspending the dried isolated lipids in the same solvent system used for 13 C and 1 H studies, CDCl 3 -CD 3 OD (2:1 v/v), in which we found the 31 P resonances stable and reproducible.…”

“…Sine and squared sine functions were applied to t1 and t2 domains, respectively. 31 P spectra were acquired at 161.98 MHz under proton decoupling conditions using a Waltz sequence. 31 P frequencies were referred to phosphatidylcholine at 0.3 ppm.…”

“…31 P spectra were acquired at 161.98 MHz under proton decoupling conditions using a Waltz sequence. 31 P frequencies were referred to phosphatidylcholine at 0.3 ppm. The possible degradation of the samples during the NMR spectral acquisition was later checked by means of HPTLC separation.…”

Signalling the molecular stress response to nephrotoxic and mutagenic cysteine conjugates: Differential roles for protein synthesis and calcium in the induction of c‐fos and c‐myc mRNA in LLC‐PK1 cells

“…Therefore, we have been using this model to determine if these cytotoxic pathways also serve as signals for induction of stress-responsive genes. The first step in NCC-induced cytotoxicity or gene expression is cleavage of the conjugate by the enzyme cysteine conjugate P-lyase (Stevens et al, 1986), to yield reactive intermediates (Stevens et al, 1986;Chen et al, 1990) which acylate targets in the cell (Dekant et al, 1989;Commandeur and Vermeulen, 1990) including proteins (Hayden et al, 1991;Harris et al, 1992) and lipids (Hayden et al, 1992). An increase in cytosolic free calcium follows covalent binding of NCC metabolites in LLC-PK1 cells (Stevens et al, 1986;Vamvakas et al, 1990;Chen et al, 1994) and other renal epithelial cell models (Jones et al, 1986;Groves et al, 1990;van de Water et al, 1993van de Water et al, ,1994.…”