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, 112 (2), 427-34

Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism

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Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism

Derek A Drechsel et al. Toxicol Sci.

Abstract

Exposure to environmental pesticides can cause significant brain damage and has been linked with an increased risk of developing neurodegenerative disorders, including Parkinson's disease. Bipyridyl herbicides, such as paraquat (PQ), diquat (DQ), and benzyl viologen (BV), are redox cycling agents known to exert cellular damage through the production of reactive oxygen species (ROS). We examined the involvement of the mitochondrial respiratory chain in ROS production by bipyridyl herbicides. In isolated rat brain mitochondria, H2O2 production occurred with the following order of potency: BV > DQ > PQ in accordance with their measured ability to redox cycle. H2O2 production was significantly attenuated in all cases by antimycin A, an inhibitor of complex III. Interestingly, at micromolar (< or = 300 microM) concentrations, PQ-induced H2O2 production was unaffected by complex I inhibition via rotenone, whereas DQ-induced H2O2 production was equally attenuated by inhibition of complex I or III. Moreover, complex I inhibition decreased BV-induced H2O2 production to a greater extent than with PQ or DQ. These data suggest that multiple sites within the respiratory chain contribute to H2O2 production by redox cycling bipyridyl herbicides. In primary midbrain cultures, H2O2 differed slightly with the following order of potency: DQ > BV > PQ. In this model, inhibition of complex III resulted in roughly equivalent inhibition of H2O2 production with all three compounds. These data identify a novel role for complex III dependence of mitochondrial ROS production by redox cycling herbicides, while emphasizing the importance of identifying mitochondrial mechanisms by which environmental agents generate oxidative stress contributing to parkinsonism.

Figures

FIG. 1.
FIG. 1.
Chemical structures of bipyridyl herbicides: PQ, DQ, and BV.
FIG. 2.
FIG. 2.
Dose-response curves of H2O2 production in isolated rat brain mitochondria stimulated by redox cycling bipyridyl herbicides. Mitochondria were stimulated with (A) malate + glutamate or (B) succinate + rotenone and treated with PQ (solid line), DQ (dashed line), or BV (dotted line) at increasing concentrations. EC50 values were calculated to determine the potency of each compound in terms of H2O2 production (see text).
FIG. 3.
FIG. 3.
Role of complexes I and III in redox cycling herbicide-induced mitochondrial H2O2 production. Isolated rat brain mitochondria were supplemented with malate + glutamate in the presence of (A) PQ, (B) DQ, or (C) BV. Inhibitors of the respiratory chain, rotenone (5 μg/ml; white bars) or antimycin A (10μM; gray bars), were added as indicated. Data expressed as H2O2 production rates in nanomolar per minute + SEM, n = 3–4. Bars with different letters indicate statistical significance from one another (p < 0.05, one-way ANOVA with Tukey’s posttest).
FIG. 4.
FIG. 4.
Role of complex III in bipyridyl herbicide–induced H2O2 production in isolated mitochondria. (A) Scheme of electron flow through complex III. The sites of inhibition by antimycin A, myxothiazol, and stigmatellin are indicated. Electron leak can occur at either of the ubiquinone-binding sites (Qo or Qi) to reduce molecular oxygen and form O2·−. (B) Isolated rat brain mitochondria were supplemented with succinate in the presence of rotenone to direct electron flow through complex III. PQ, DQ, and BV were used to stimulate H2O2 production where indicated in the presence of vehicle/dimethyl sulfoxide (DMSO) or complex III inhibitors. Data expressed as mean + SEM, n = 3. Bars with different letters indicate statistical significance from one another (p < 0.05, one-way ANOVA with Tukey's posttest).
FIG. 5.
FIG. 5.
H2O2 production by bipyridyl herbicides in primary midbrain cultures. Cultures were exposed to PQ, DQ, or BV for 8 h at 300μM. Data expressed as mean + SEM, n = 3. Bars with different letters indicate statistical significance from one another (p < 0.05, one-way ANOVA with Tukey's posttest).
FIG. 6.
FIG. 6.
Role of respiratory chain complexes in bipyridyl herbicide–induced H2O2 production in primary midbrain cultures. Cultures were exposed to PQ, DQ, or BV at 300μM for 8 h in the presence of respiratory chain inhibitors. Data expressed as percent control of vehicle for each bipyridyl herbicide group, mean + SEM, n =3. Bars with different letters indicate statistical significance from one another (p < 0.05, one-way ANOVA with Tukey's posttest).

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