Dose-dependent protein adduct formation in kidney, liver, and blood of rats and in human blood after perchloroethene inhalation

Perchloroethene (PER) was a widely used solvent and is an environmentalcontaminant. In bioassays for carcinogenicity, PER was found to increasethe incidence of liver tumors in mice and of renal tumors in male rats.Toxic effects of PER after repeated administration are likely caused bybioactivation. PER bioactivation occurs by two pathways. Oxidation bycytochrome P450 results in trichloroacetyl chloride, which binds to lipidsand proteins. Glutathione S-conjugate formation from PER and furtherprocessing of the formed S- (trichlorovinyl)glutathione toS-(trichlorovinyl)-L-cysteine, followed by cysteine conjugate beta-lyasecatalyzed cleavage, resulted in the reactive dichlorothioketene, whichbinds to proteins under formation of N epsilon-(dichloroacetyl)-L-lysine inproteins. The objective of this study was to comparatively quantify thedose-dependent formation of protein adducts from PER in rats and humansusing antibodies with high specificity for either Nepsilon-(trichloroacetyl)-L-lysine or N epsilon-(dichloroacetyl)-L-lysinein proteins. Male and female rats (n = 2, per concentration and time point)were exposed to 400, 40, and 10 ppm PER for 6 h and killed at various timepoints. Formation of N epsilon-(dichloroacetyl)-L-lysine and Nepsilon-(trichloroacetyl)-L- lysine in proteins was comparativelyquantified in subcellular fractions from liver and kidney and in blood. Inaddition, three male and three female human volunteers were exposed to 10and 40 ppm PER, and formation of protein adducts in blood was analyzedusing the antibodies and GC/MS after immunoaffinity enrichment of modifiedproteins. In liver and kidney subcellular fractions and blood of PER-exposed rats, dose-dependent formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was observed.Highest concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteinswere formed in kidney mitochondria, followed by kidney cytosol. Only lowconcentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins werepresent in liver proteins; blood concentrations of Nepsilon-(dichloroacetyl)-L-lysine in proteins were 5 to 10 fold lower thanin kidney mitochondria. Highest concentrations of N epsilon-(trichloroacetyl)-L-lysine were found in microsomal and cytosolic proteinsfrom the liver of rats exposed to PER. A higher protein adduct formationwas seen in PER-exposed-male than -female rats for N epsilon-(dichloroacetyl)-L-lysine in renal mitochondrial proteins, after exposureto 400 ppm PER. In human blood samples taken 0 and 24 h after the 6 hexposures to PER, N epsilon-(trichloroacetyl)-L-lysine- containing proteinswere present in low concentrations. N epsilon-(Dichloroacetyl)-L-lysine-containing proteins were not detected either byWestern blotting or GC/MS after immunoaffinity chromatography. The obtainedresults indicate a dose-dependent covalent binding of PER metabolites toproteins in rat liver, kidney, and blood and suggest that the concentrationof covalent protein adducts is much lower in blood of humans as compared tothe blood of rats exposed under identical conditions.