CONTINUOUS MONITORING OF MITOCHONDRIAL-MEMBRANE POTENTIAL IN HEPATOCYTE CELL-SUSPENSIONS

We report a simple fluorometric method for the continuous monitoring o f mitochondrial membrane potential and cell viability in suspensions o f hepatocytes exposed in vitro to cytotoxic agents. Suspensions of fre shly isolated hepatocytes (10(6) cells/mL) preloaded with rhodamine 12 3 (Rh 123, 100 mu mol/L) are transferred to a thermostatically control led mixed cuvette to which the desired cytotoxic agent is added. Ph 12 3 is a cationic fluorophore that is actively accumulated by cells in d irect proportion to the mitochondrial membrane potential. Cell viabili ty was estimated by monitoring propidium iodide (PI) fluorescence. Exp osure of cell suspensions to the mitochondrial uncoupling agent FCCP c aused an immediate and titratable increase in Rh 123 fluorescence. Sub sequent treatment with digitonin did not change Rh 123 fluorescence, s uggeseting that Rh 123 equilibrates rapidly across the intact cell mem brane. Likewise, treatment of hepatocyte suspensions with inhibitors o f mitochondrial respiration (rotenone, cyanide, or menadione) caused a n immediate increase in Rh 123 fluorescence. This was accompanied by a progressive increase in PI fluorescence, suggesting a causal relation ship between mitochondrial depolarization and cell injury. In contrast , 1,4-benzoquinone caused a time-dependent and linear increase in PI f luorescence that paralleled changes in Rh 123 fluorescence. Comparing the time courses for changes in PI and Rh 123 fluorescence suggests th at for benzoquinone, the depolarization of the mitochondria is a conse quence rather than a cause of the cell injury. This modified procedure provides a simple and specific technique for continuously monitoring mitochondrial membrane potential and cell viability in suspensions of freshly isolated hepatocytes. The advantage is that there is no need t o separate cells from the incubation medium, making it possible to rec ord real-time changes in mitochondrial membrane potential and cell via bility throughout the in vitro exposure period.