Mechanisms participating in oxidative damage of isolated rat hepatocytes.

The aim of the study was to evaluate time course and dose dependence of peroxidative damage induced by tert-butyl hydroperoxide (tBHP) in rat hepatocytes cultured in suspension and in monolayer. At the lowest (0.1 mM) concentration, decrease of cytosolic glutathione and discharge of mitochondrial membrane potential (MMP) could be detected. Significant increases in leakage of lactate dehydrogenase and in malondialdehyde concentrations together with decrease of pyruvate-dependent respiration were detected at higher tBHP concentrations (above 0.5 mM) and after longer periods of incubation. Changes in plasma membrane integrity were observed at 1 mM concentration of tBHP. Succinate-dependent oxidation was most resistant to peroxidative damages. Opening of the mitochondrial permeability transition pore was responsible for the discharge of mitochondria membrane potential. In the presence of cyclosporine A and succinate, the membrane potential could be restored. Our data showed that the most sensitive indicators of the peroxidative damage are changes of cytosolic glutathione concentration and MMP.

Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode.

Mitochondrial membrane potential (Deltapsi(m)) plays important roles in the normal function of cells and in pathobiochemical situations. The application of ion-selective electrodes for the measurement of Deltapsi(m) is important for studying normal biological reactions and pathways and mitochondrial diseases. We constructed and optimized a computerized device for real-time monitoring of the Deltapsi(m), which included modification of tetraphenylphosphonium (TPP(+))-selective membrane that improved reproducibility of the TPP(+)-selective electrode. Application of MATLAB software increased the sensitivity of the system. We tested our improved device for membrane potential measurements of isolated mitochondria (in absolute scale of millivolts). In addition, we assessed relative changes of Deltapsi(m) (as changes in TPP(+) concentration) of digitonin-permeabilized cells (hepatocytes, control transmitochondrial cybrids, HeLa G and BSC-40) after addition of substrates, inhibitors, and uncoupler of respiratory chain. Our system can be successfully used for studies of many aspects of the regulation of mitochondrial bioenergetics and as a diagnostic tool for mitochondrial oxidative phosphorylation disorders.