Effects of cocaine and its oxidative metabolites on mitochondrial respiration and generation of reactive oxygen species.

Cocaine is capable of producing severe hepatocellular necrosis in laboratory animals and humans. The mechanism of cocaine hepatotoxicity is not well understood, but appears to result from the actions of one or more N-oxidative metabolites of cocaine. Mitochondria have been proposed as critical cellular targets for cocaine toxicity, and previous studies have found depressed mitochondrial respiration and increased mitochondrial generation of reactive oxygen species (ROS) in animals treated with cocaine. To examine the potential role of cocaine N-oxidative metabolites in these effects, mitochondrial respiration and ROS generation were examined in isolated mouse mitochondria treated with cocaine and its N-oxidative metabolites-norcocaine, N-hydroxynorcocaine, and norcocaine nitroxide. Cocaine, in concentrations of 0.25 or 0.5 mM, had no effect on state 3 respiration, state 4 respiration, respiratory control ratio (RCR), or ADP/O ratio. Norcocaine (0.5 mM) inhibited state 3 respiration, and N-hydroxynorcocaine (0.5 mM) inhibited both state 3 and state 4 respiration. Norcocaine nitroxide had the greatest effect on mitochondrial respiration; the lower concentration (0.25 mM) completely inhibited both state 3 and state 4 respiration. Preincubation of mitochondria with cocaine or metabolites increased the inhibitory effect of norcocaine and N-hydroxynorcocaine, but not cocaine. Cocaine, norcocaine, and N-hydroxynorcocaine (0.1 mM) had no effect on ROS generation during state 3 respiration, and cocaine and norcocaine decreased ROS generation under state 4 conditions. Norcocaine nitroxide interfered with the fluorescence ROS assay and could not be assessed. The results suggest that the effects of cocaine on mitochondrial respiration are due to its N-oxidative metabolites. Inhibition of mitochondrial respiration by the N-oxidative metabolites of cocaine may be the underlying cause for observed ATP depletion and subsequent cell death.