Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells

Rotenone inhibits spontaneously and chemically induced hepatic tumorigenesi s in rodents through the induction of apoptosis. However, the mechanism for the induction of apoptosis by rotenone has not been defined. Mitochondrial dysfunction, in particular the induction of the mitochondrial membrane per meability transition (MPT), has been implicated in the cascade of events in volved in the induction of apoptosis. Inhibition of the mitochondrial elect ron-transport chain reduces the mitochondrial transmembrane potential (Delt a psi(m)), which may induce the formation of the mitochondrial permeability transition pore and the subsequent MPT. Fluorescent microscopy of Hoechst 33258-stained WB-F344 cells, a rat-liver cell line, was utilized to examine the effect of the mitochondrial respiratory chain inhibitor, rotenone (0.5 -5 mu M), atractyloside (5-10 mu M), and cyclosporin A (2.5-10 mu M) on apo ptosis. A time- and concentration-dependent increase in liver cell apoptosi s was observed following treatment with rotenone and atractyloside (11.7- a nd 7.7-fold, respectively, over solvent control). Cotreatment with 7.5- and 10 mu M-cyclosporin A for 12 h inhibited the apoptogenicity of 5-mu M rote none treatment. A similar effect was observed following cyclosporin A cotre atment with atractyloside. Rotenone induced a rapid increase in apoptosis ( within 20 min of treatment). By 2 h of treatment, the morphological appeara nce of apoptosis was similar to that observed in cultures treated continuou sly with rotenone for 12 h. Inhibition studies demonstrated that cyclospori n A prevented apoptosis if the exposure to it occurred prior to the 20-min threshold necessary to induce apoptosis by rotenone. Mitochondrial function was examined by staining with the mitochondrial membrane potential (Delta psi(m))-sensitive fluorochrome, MitoTracker Red (CMXRos) and confirmed util izing cytofluorometric analysis of DiOC(6)(3)-stained cells. Rotenone (5.0- mu M) and atractyloside (5.0-mu M) reduced the percent of CMXRos or DiOC(6) (3)-positive (Delta psi(m)-positive) liver cells within 15 min and througho ut the duration of the study (6 h) to approximately 65-80% and 50-80% of co ntrol. However, cotreatment with concentrations of cyclosporin A that inhib ited the apoptogenicity of rotenone and atractyloside prevented the rotenon e- and atractyloside-induced reduction of the Delta psi(m). Therefore, the apoptogenic effect of rotenone and atractyloside appears to occur rapidly ( within 20 min) and is irreversible once mitochondrial damage occurs. The in hibition of the rotenone- and atractyloside-induced apoptosis and mitochond rial dysfunction by cyclosporin A suggests the MPT may be involved in the i nduction of apoptosis by rotenone.