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.     

Angiogenesis induction and regression in human surgical wounds

Angiogenesis in human wound healing is not well characterized, with only sparse information available regarding the maturation and fate of vessels formed as a consequence of human tissue repair. Therefore, this study aimed to establish the temporal profile of angiogenesis in human dermal wounds. Punch biopsies were obtained under local anesthesia from 45 patients following breast surgery. Scars were predominantly between 2 and 52 weeks after surgery but in five patients were > 52 weeks. Control samples were taken from breast skin peroperatively (n = 24). Quantification of vascular density was performed using the Chalkley grid, following antibody staining for platelet endothelial cell adhesion molecule. Vascular patterns, wound cellularity and morphology were also determined. Cumulative microvessel density was increased in all samples when compared to controls (p < 0.05). This was greatest 2 to 24 weeks following surgery 17 (15-21) median (range), decreased thereafter, but remained elevated compared to controls even in the mature scars > 52 weeks. Control tissue showed an ordered morphological arrangement of dermal structures, collagen, and elastic fibers. However, wounding resulted in marked structural distortion for up to 15 weeks. In conclusion, this study shows for the first time the prolonged persistence of both microvessels and cellularity (fibroblastic cells), in addition to structural distortion in human dermal wounds, which is in contrast to previous in vitro and in vivo studies.