Jcl. Erve et al., CHARACTERIZATION OF A VALINE-LYSINE THIOUREA CROSS-LINK ON RAT GLOBINPRODUCED BY CARBON-DISULFIDE OR N,N-DIETHYLDITHIOCARBAMATE IN-VIVO, Chemical research in toxicology, 11(10), 1998, pp. 1128-1136
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.