Laser cytometric analysis of gossypol-induced cytotoxicity
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abstract
A number of vital fluorescence bioassays were employed in conjunction with laser cytometry to identify physiological endpoints altered by gossypol acetic acid and to evaluate the sensitivity of these assays for analysis of the mechanisms of gossypol-induced cytotoxicity. Endpoints chosen to explore cellular mechanisms of gossypol-induced cellular toxicity included mitochondrial and plasma membrane potential, intracellular Ca2+ and glutathione homeostasis, and cell-cell communication. Analyses of IC50 values for gossypol in 3 rodent cell lines (Clone 9, TM4, and SIGC) were comparable (2.5-3.3 M). Acute effects of gossypol on Clone 9 cells were detected in both steady-state and kinetic fluorescenee analyses, indicating a relatively narrow range between subtoxic and acutely toxic concentrations of gossypol at low micromolar concentrations rapidly after treatment. Steady-state fluorescence analyses of gossypol at doses (1.0 and 5.0 M gossypol) centered around the IC50 values of Clone 9, SIGC, and TM4 cells (2.5-3.3 M) revealed cytotoxicity at the 1.0 M dose with respect to mitochondrial membrane potential and gap junction-mediated intercellular communication (GJIC), whereas effects on plasma membrane potential, glutathione (GSH), and Ca2+ homeostasis were seen by 2 h at the 5.0 M gossypol dose. These studies support the utility of fluorescence assays to detect cytotoxicity in cells and suggest their potential for determination of early events in the chronology of gossypol-induced toxicity.
