CHARACTERIZATION OF A VALINE-LYSINE THIOUREA CROSS-LINK ON RAT GLOBINPRODUCED BY CARBON-DISULFIDE OR N,N-DIETHYLDITHIOCARBAMATE IN-VIVO

Previous in vivo studies have supported protein cross-linking by CS2 a s both a mechanism of neurotoxicity and a potential biomarker of effec t through the detection of a structure responsible for CS2-mediated pr otein cross-linking, namely, lysine-lysine thiourea. In this study, th e structure of a previously uncharacterized stable protein cross-link produced by CS2 in vivo involving lysine and the N-terminal valine of globin has been determined. Rats were exposed to 50, 500, and 800 ppm CS2 for 2, 4, 8, and 13 weeks by inhalation or to 3 mmol/kg N,N-diethy ldithiocarbamate administered orally on alternating days for 8 and 16 weeks. acid hydrolysis, using 6 N HCl, of globin from control and expo sed rats caused cyclization of the valine-lysine thiourea cross-link i n treated rats to isopropyl norleucyl thiohydantoin. The hydrolysate w as separated by size-exclusion chromatography, and the fraction that c oeluted with the synthetic deuterated isopropyl norleucyl thiohydantoi n internal standard was derivatized with lene-N,N,N-trimethylamino)phe nyl]-2-isothiocyanate and analyzed by liquid chromatography/tandem mas s spectrometry using selected reaction monitoring detection. Derivatiz ed isopropyl norleucyl thiohydantoin obtained from CS2-treated rats di splayed a cumulative dose response and was detectable at the lowest ex posure (50 ppm, 2 weeks) at levels of approximately 50 pmol/g of globi n. N,N-Diethyldithiocarbamate-treated rats, but not controls, also con tained a CS2-generated valine-lysine thiourea cross-link on globin. In vitro incubation of human hemoglobin with either CS2 or N,N-diethyldi thiocarbamate also resulted in the formation of CS2-generated valine-l ysine thiourea. These observations demonstrate the potential of thiour ea cross-linking involving a free amino terminus and epsilon-amino gro ups of lysine to accumulate in a long-lived globular protein and sugge st that cross-linking of globin may provide a specific dosimeter of in ternal exposure for CS2 capable of assessing exposure over subchronic periods.