Characterization of a valine-lysine thiourea cross-link on rat globin produced by carbon disulfide or N,N-diethyldithiocarbamate in vivo.

Erve JC, Amarnath V, Sills RC, Morgan DL, Valentine WM
Chem Res Toxicol. 1998 11 (10): 1128-36

PMID: 9778308 · DOI:10.1021/tx980077p

Previous in vivo studies have supported protein cross-linking by CS2 as both a mechanism of neurotoxicity and a potential biomarker of effect through the detection of a structure responsible for CS2-mediated protein cross-linking, namely, lysine-lysine thiourea. In this study, the 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-diethyldithiocarbamate administered orally on alternating days for 8 and 16 weeks. Acid hydrolysis, using 6 N HCl, of globin from control and exposed rats caused cyclization of the valine-lysine thiourea cross-link in treated rats to isopropyl norleucyl thiohydantoin. The hydrolysate was separated by size-exclusion chromatography, and the fraction that coeluted with the synthetic deuterated isopropyl norleucyl thiohydantoin internal standard was derivatized with 3-[4'-(ethylene-N,N, N-trimethylamino)phenyl]-2-isothiocyanate and analyzed by liquid chromatography/tandem mass spectrometry using selected reaction monitoring detection. Derivatized isopropyl norleucyl thiohydantoin obtained from CS2-treated rats displayed a cumulative dose response and was detectable at the lowest exposure (50 ppm, 2 weeks) at levels of approximately 50 pmol/g of globin. N, N-Diethyldithiocarbamate-treated rats, but not controls, also contained a CS2-generated valine-lysine thiourea cross-link on globin. In vitro incubation of human hemoglobin with either CS2 or N, N-diethyldithiocarbamate also resulted in the formation of CS2-generated valine-lysine thiourea. These observations demonstrate the potential of thiourea cross-linking involving a free amino terminus and epsilon-amino groups of lysine to accumulate in a long-lived globular protein and suggest that cross-linking of globin may provide a specific dosimeter of internal exposure for CS2 capable of assessing exposure over subchronic periods.

MeSH Terms (13)

Animals Carbon Disulfide Ditiocarb Globins Humans Lysine Male Mass Spectrometry Rats Rats, Inbred F344 Rats, Sprague-Dawley Thiourea Valine

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