Lumi Chikahisa (1,2,3), Yasuo Oyama (1,*), Eisuke Okazaki (1) and Katsuhiko Noda (1)
(1) Laboratory of Cell Signaling (Pharmacology), Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770, Japan (2) Anticancer and Antimicrobials Research Laboratory, Taiho Pharmaceutical Co., Ltd., Tokushima 771-01, Japan (3) Cancer Research Laboratory, Taiho Pharmaceutical Co., Ltd., Hanno 357, Japan (*) To whom correspondence should be addressed.
Abstract: We have developed a procedure to simultaneously estimate cell viability and the cellular level of nonprotein thiol (presumably glutathione) using two fluorescent dyes, 5-chloromethylfluorescein (5CMF) and ethidium, and rat thymocytes. Diethylmaleate and N-ethylmaleimide reduced, respectively, the intensity of 5CMF fluorescence to 0.23 and 0.1, relative to the control. Incubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, decreased the intensity of 5CMF fluorescence to 0.61. Results indicate that 5CMF fluorescence can be attenuated by agents that decrease the level of cellular nonprotein thiols, suggesting that 5CMF fluorescence is utilized for estimating the level of cellular glutathione. Hydrogen peroxide (10 microM to 3 mM) reduced the intensity of 5CMF fluorescence in a dose-dependent manner and increased the number of thymocytes stained with ethidium (presumably dead cells or cells with compromised membranes) at concentrations of 300 microM or greater. Reduction of cellular glutathione level seems to precede cell death in which oxidative stress is involved.
Keywords:
5-Chloromethylfluorescein, Cytotoxicity, Ethidium, Glutathione, Nonprotein thiol