title: Carbon disulfide and N,N-diethyldithiocarbamate generate thiourea cross-links on erythrocyte spectrin in vivo.
abstract: CS2, a known neurotoxicant, is used in the viscose production of rayon and is also a decomposition product of N, N-diethyldithiocarbamate, a metabolic product of the drug disulfiram used in alcohol aversion therapy. Previous in vitro investigations have demonstrated the ability of CS2 to cross-link proteins through thiourea, dithiocarbamate ester, and disulfide structures. Although in vivo studies have supported protein cross-linking as both a mechanism of neurotoxicity and a potential biomarker of effect, the chemical structures responsible for CS2-mediated protein cross-linking in vivo have not been elucidated. In the present study, the structure of one type of stable protein cross-link produced on erythrocyte spectrin by CS2 in vivo is determined. Rats were exposed to 50, 500, and 800 ppm CS2 for 13 weeks by inhalation or to 3 mmol/kg N,N-diethyldithiocarbamate administered orally on alternating days for 8 weeks. Erythrocyte spectrin preparations from control and exposed rats were hydrolyzed using 6 N HCl and separated by size-exclusion chromatography. The fraction that coeluted with the synthetic deuterated lysine-lysine thiourea internal standard was derivatized with 3-[4'-[(N,N,N-trimethylamino)ethylene]phenyl] 2-isothiocyanate and analyzed by liquid chromatography tandem mass spectrometry using selected reaction monitoring detection. Lysine-lysine thiourea was detected in spectrin preparations obtained from CS2-treated rats at 500 and 800 ppm and N, N-diethyldithiocarbamate-treated rats, but not from controls. These results establish that CS2-mediated protein cross-linking occurs in vivo through the generation of Lys-Lys thiourea and that diethyldithiocarbamate can, through in vivo release of CS2, produce the same cross-linking structure. This observation supports the utility of cross-linking of peripheral proteins as a specific dosimeter of internal exposure for CS2 and provides a mechanistic explanation to account for the high-molecular-weight neurofilament protein species isolated from rats exposed to CS2 or N, N-diethyldithiocarbamate.
authors.affiliation: Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Erve
authors.forename: V
authors.initials: V
authors.lastname: Amarnath
authors.forename: D G
authors.initials: DG
authors.lastname: Graham
authors.forename: R C
authors.initials: RC
authors.lastname: Sills
authors.forename: A L
authors.initials: AL
authors.lastname: Morgan
authors.forename: W M
authors.initials: WM
authors.lastname: Valentine
mesh_terms: D000818:Animals; D002246:Carbon Disulfide / Q000494:pharmacology*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004050:Ditiocarb / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006868:Hydrolysis; D007202:Indicators and Reagents; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D011916:Rats, Inbred F344; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013049:Spectrin / Q000187:drug effects*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D013049:Spectrin; D012710:Serum Albumin, Bovine; D004050:Ditiocarb; D002246:Carbon Disulfide