Karbowski M, Kurono C, Wozniak M, Ostrowski M, Teranishi M, Nishizawa Y, Usukura J, Soji T, Wakabayashi T
Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, Japan.
Free Radic Biol Med. 1999 Feb;26(3-4):396-409. doi: 10.1016/s0891-5849(98)00209-3.
Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-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 apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result 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 period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.
在生理和病理条件下诱导形成的巨型线粒体（MG）的病理生理意义及形成机制仍不清楚。我们现在提供证据表明，MG的形成可能是自由基介导的细胞凋亡的一个先决条件。使用荧光染料Mito Tracker Red CMXRos（CMXRos）通过共聚焦激光显微镜以及电子显微镜，在原代培养的大鼠肝细胞、大鼠肝细胞系RL - 34和IAR - 20以及肾细胞系Cos - 1中检测到MG，这些细胞用已知能产生自由基的各种化学物质处理22小时，这些化学物质包括肼、氯霉素、甲基乙二醛双脒腙、吲哚美辛、H2O2和红霉素。在用荧光染料羧基 - H2 - DCFDA对细胞进行染色监测时，在MG形成之前就检测到细胞内活性氧（ROS）水平显著升高。用上述各种化学物质孵育36小时或更长时间会诱导细胞出现明显的结构变化，这些变化是细胞凋亡的特征：细胞核浓缩、凋亡小体形成以及DNA梯状条带形成。用化学物质处理22小时的细胞停滞在G1期，然后凋亡的亚G1期细胞群体逐渐增加。通过CMXRos检测流式细胞术发现，氯霉素诱导的MG的膜电位与对照细胞中的线粒体相比有所降低。从用氯霉素或肼处理的大鼠肝脏中分离出的MG产生H2O2和O2-的速率低于对照动物肝脏线粒体的速率。基于目前的结果以及我们之前的发现，我们认为MG的形成可能是细胞在亚细胞水平对不利环境的一种适应性过程：当细胞暴露于过量自由基时，线粒体变大，氧气消耗速率降低。如本研究所示，MG氧气消耗的减少可能导致ROS产生速率降低。这同时会导致MG产生ATP的速率降低。如果细胞在一段时间内暴露于大量自由基，细胞内ATP水平降低可能会导致细胞发生凋亡变化。
