Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1‐O‐octadecyl‐2‐methyl‐rαc‐grycero‐3‐phosphocholine in p53‐defective hepatocytes

ABSTRACT The mechanism of induction of apoptosis by the novel anti‐cancer drug 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3) was investigated in p53‐defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 μM ET‐18‐OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET‐18‐OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET‐18‐OCH3 in the presence of the antioxidant α‐to‐copherol. Similar results were achieved when cells were switched acutely to choline‐deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET‐18‐OCH3 and CD decreased the phos‐phatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5′,6,6′‐tetramethylbenzimidazolcarbocyanine iodide (JC‐1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the in‐tracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET‐18‐OCH3 or CD. Taken together, these results suggest that the form of p53‐independent apoptosis induced by ET‐18‐OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.— Vrablic, A. S., Albright, C. D., Craciunescu, C. N., Salganik, R. I., Zeisel, S. H. Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine in p53‐defective hepatocytes. FASEB J. 15, 1739—1744 (2001)