Free radical-induced megamitochondria formation and apoptosis

Pathophysiological meaning and the mechanism of the formation of megamitoch ondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for Gee radical-mediated apoptosis. MG were detected in primar y cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidne y cell Line Cos-1 treated for 22 h with Various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydraz one, indomethacin, H2O2, and erythromycin using a fluorescent dye Mite Trac ker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive ox ygen species (ROS), monitored by staining of cells with a fluorescent dye c arboxy-H-2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize ap optosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G(1) phase, and apoptotic sub-G(1) populations then became gradually inc reased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mi tochondria in control cells. Rates of the generation of H2O2 and O-2(-) fro m MG isolated from the liver of rats treated with chloramphenicol or hydraz ine were found to be lower than those of mitochondria of the liver of contr ol animals. We suggest, based on the present results together with our prev ious findings, that the formation of MG may be an adaptive process at a sub cellular level to unfavorable environments: when cells are exposed to exces s amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may resul t in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain pe riod of time, lowered intracellular levels of ATP may result in apoptotic c hanges of the cell. (C) 1998 Elsevier Science Inc.