diethyl-maleate and monobromobimane

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labeling.

Topics: Animals; Breast Neoplasms; Bridged Bicyclo Compounds; Buthionine Sulfoximine; Carcinoma; Carcinosarcoma; CHO Cells; Colonic Neoplasms; Cricetinae; Ethylmaleimide; Eukaryotic Cells; Evaluation Studies as Topic; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Glutathione; Humans; Maleates; Mammary Neoplasms, Experimental; Methionine Sulfoximine; Mice; o-Phthalaldehyde; Phenylmercury Compounds; Pyrazoles; Species Specificity; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury.

Topics: Animals; Bridged Bicyclo Compounds; Buthionine Sulfoximine; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Nucleus; Cells, Cultured; Cytosol; Ethylmaleimide; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Glutathione; Kinetics; Liver; Male; Maleates; Methionine Sulfoximine; Pyrazoles; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Subcellular Fractions; Vitamin K