Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol

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Abstract

Increased production of reactive oxygen species (ROS) by the mitochondrion has been implicated in the pathogenesis of numerous liver diseases. However, the exact sites of ROS production within liver mitochondria and the electron transport chain are still uncertain. To determine the sites of ROS generation in liver mitochondria we evaluated the ability of a variety of mitochondrial respiratory inhibitors to alter the steady state levels of ROS generated within the intact hepatocyte and in isolated mitochondria. Treatment with myxothiazol alone at concentrations that significantly inhibit respiration dramatically increased the steady-state levels of ROS in hepatocytes. Similar results were also observed in isolated mitochondria oxidizing succinate. Coincubation with antimycin or rotenone had no effect on myxothiazol-induced ROS levels. Myxothiazol stimulation of ROS was mitochondrial in origin as demonstrated by the colocalization of MitoTracker Red and dichlorofluorescein staining using confocal microscopy. Furthermore, diphenyliodonium, an inhibitor that blocks electron flow through the flavin mononucleotide of mitochondrial complex I and other flavoenzymes, significantly attenuated the myxothiazol-induced increase in hepatocyte ROS levels. Together, these data suggest that in addition to the ubiquinone–cytochrome bc1 complex of complex III, several of the flavin-containing enzymes or iron–sulfur centers within the mitochondrial electron transport chain should also be considered sites of superoxide generation in liver mitochondria.

Section snippets

Materials

Collagenase type I was obtained from Worthington Biochemical Corp. (Lakewood, NJ). 2,7-Dichlorofluorescin diacetate (DCFH-DA) and MitoTracker Red CM-H2XRos (MTR) were obtained from Molecular Probes, (Eugene, OR) and dichlorofluorescein (DCF) was obtained from PolySciences, (Warrington, PA). Antimycin, Hepes, 1-malic acid, 1-glutamic acid, EGTA, rotenone, succinate, and ADP were obtained from Sigma Chemical (St. Louis, MO) and diphenyliodonium chloride was from Aldrich Chemical (Milwaukee,

Inhibition of hepatocyte respiration by myxothiazol is concentration and time dependent

The efficacy of myxothiazol to inhibit hepatocyte respiration was examined over a 1-h period in cells incubated with different concentrations of myxothiazol (Fig. 1). In this set of experiments, hepatocyte respiration was well maintained in untreated hepatocytes (i.e., control) with only a 10% decrease over the course of the hour. A 5-min exposure to all concentrations of myxothiazol was sufficient to significantly inhibit hepatocyte respiration to about 20% of the level measured in control

Discussion

The effects of myxothiazol on mitochondrial respiration are well documented. It effectively inhibits mitochondrial respiration by preventing an electron being transferred from the ubiquinol form of ubiquinone to the Rieske iron–sulfur protein [28]. This property has been previously demonstrated with liver, skeletal muscle, [21] and heart mitochondria [29], [30] and was verified in the present study for liver mitochondria. Myxothiazol is also an effective inhibitor of cellular respiration, as

Acknowledgements

The authors thank Mr. Ken Grant for his technical assistance with the confocal microscopy studies. These studies were supported by USPHS Grants AA02887 and AA00279 (to C.C.C.) and AA12058 and AA13682 (to S.M.B.) from the National Institute on Alcohol Abuse and Alcoholism.

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