Characterization of a valine-lysine thiourea cross-link on rat globin produced by carbon disulfide or N,N-diethyldithiocarbamate in vivo.
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.
Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561, USA.
J C
JC
Erve
V
V
Amarnath
R C
RC
Sills
D L
DL
Morgan
W M
WM
Valentine
D000818:Animals; D002246:Carbon Disulfide / Q000633:toxicity*; D004050:Ditiocarb / Q000633:toxicity*; D005914:Globins / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D013890:Thiourea / Q000378:metabolism*; D014633:Valine / Q000378:metabolism
D005914:Globins; D004050:Ditiocarb; D013890:Thiourea; D014633:Valine; D008239:Lysine; D002246:Carbon Disulfide