Determination of rat hepatocellular glutathione by reversed-phase liquid chromatography with fluorescence detection and cytotoxicity evaluation of environmental pollutants based on the concentration change.
Three methods for the determination of rat hepatocellular thiols by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection have been developed. The thiols in the cells were tagged with three fluorogenic reagents, SBD-F, ABD-F and DBD-F. These reagents could permeate into cells and effectively reacted with thiols to produce highly fluorescent derivatives. These derivatives fluoresced in the long wavelength region at around 530 nm (excitation at around 380 nm). The five biological thiols tagged were perfectly separated by reversed-phase liquid chromatography and were sensitively and selectively detected without any interference from endogenous substanaces. The main thiol in the cells was reduced GSH and the concentration was at the mM level. The proposed procedures were applied to the determination of hepatocellular GSH after treatment of environmental pollutants such as volatile organic compounds (VOC) and endocrine disrupting chemicals (EDC). From the comparison of intracellular GSH concentration, the test compounds were classified into four groups: compounds of strong depletion (eg triphenyltin chloride, hexachlorocyclohexene, nonylphenol, bromoacetic acid, 4-chlorobenzyl chloride and 1,3-dichloropropene), slight decrease (eg bisphenol A, benzo[a]pylene, carbon tetrachloride and benzene), slight increase (eg bromoform and toluene), and no effect (eg 1,1,1-trichloroethane, 1,1,2-trichloroethane and 1,2-dichloroethane). Although the decrease of GSH concentration does not reflect the cytotoxicity of chemicals, the proposed procedure utilizing isolated rat hepatpcytes seems to be useful for investigating the bioactivation of VOC, and EDC, etc.
Toyo'oka
T
T
Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. toyooka@ys2.u-shizuoka-ken.ac.jp
Tanabe
J
J
Jinno
H
H
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004785:Environmental Pollutants / Q000633:toxicity*; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione / Q000032:analysis*; D022781:Hepatocytes / Q000737:chemistry* / Q000187:drug effects; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence
D004785:Environmental Pollutants; D005456:Fluorescent Dyes; D005978:Glutathione