Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain

Abstract

Paraquat (PQ(2+)) is a prototypic toxin known to exert injurious effects through oxidative stress and bears a structural similarity to the Parkinson disease toxicant, 1-methyl-4-pheynlpyridinium. The cellular sources of PQ(2+)-induced reactive oxygen species (ROS) production, specifically in neuronal tissue, remain to be identified. The goal of this study was to determine the involvement of brain mitochondria in PQ(2+)-induced ROS production. Highly purified rat brain mitochondria were obtained using a Percoll density gradient method. PQ(2+)-induced hydrogen peroxide (H(2)O(2)) production was measured by fluorometric and polarographic methods. The production of H(2)O(2) was evaluated in the presence of inhibitors and modulators of the mitochondrial respiratory chain. The results presented here suggest that in the rat brain, (a) mitochondria are a principal cellular site of PQ(2+)-induced H(2)O(2) production, (b) PQ(2+)-induced H(2)O(2) production requires the presence of respiratory substrates, (c) complex III of the electron transport chain is centrally involved in H(2)O(2) production by PQ(2+), and (d) the mechanism by which PQ(2+) generates H(2)O(2) depends on the mitochondrial inner transmembrane potential. These observations were further confirmed by measuring PQ(2+)-induced H(2)O(2) production in primary neuronal cells derived from the midbrain. These findings shed light on the mechanism through which mitochondria may contribute to ROS production by other environmental and endogenous redox cycling agents implicated in Parkinson's disease.

FIGURE 1

Chemical reactions proposed to participate in mechanisms of H₂O₂ production by PQ²⁺.

FIGURE 2. PQ²⁺-induced H₂O₂ production in cellular fractions from the brain

Fluorometric (a) and polarographic (b) assays were used to measure H₂O₂ in the presence of malate and glutmate following the addition of 250 μM PQ²⁺ to equal amounts of protein from each rat brain fraction: mitochondria (solid line), cytosol (dotted line), and homogenate (dashed line).

FIGURE 3. Inhibition of PQ²⁺-induced H₂O₂ production in stimulated mitochondria

Fluorometric (a) measurement of H₂O₂ production from isolated mitochondria in the presence of succinate was measured in the presence of PQ²⁺(250 μM, □), antimycin A (◆), antimycin A and PQ²⁺(◇), PQ²⁺ and SOD (▲), and PQ²⁺and catalase (△). Polarographic (b) measurement was used to confirm results. Following the addition of 250 μM PQ²⁺, H₂O₂ production in mitochondria was monitored with inhibitors added as indicated: rote-none (solid line), catalase (dotted line), and antimycin A (dashed line). Rates of H₂O₂ production before and after the addition of inhibitor (steady state 1 and 2, respectively) were compared with obtain the percentage of H₂O₂ production under each condition (see Fig. 5).

FIGURE 4. Effect of ETC inhibitors on PQ²⁺-induced H₂O₂ production in isolated mitochondria

Mitochondria were stimulated with respiration substrates (malate plus glutamate (a) or succinate (b)), and H₂O₂ production was measured in the absence (closed bars) or presence (open bars) of 250 μM PQ²⁺. Inhibitors were added as described under “Experimental Procedures.” H₂O₂ production in mitochondrial fraction stimulated with malate plus glutamate as respiration substrates in the presence of PQ²⁺ was considered 100%, and all values are expressed as a percentage of this (mean ±S.E., n =3). *, p <0.05 compared with PQ²⁺-treated control mitochondria (one-way analysis of variance).

FIGURE 5. Effect of ETC inhibitors on PQ²⁺-induced H₂O₂ production in primary midbrain cultures

H₂O₂ production was measured in midbrain cultures incubated in buffer containing PQ²⁺ and inhibitors where indicated using a fluorometric method as described under “Experimental Procedures.” H₂O₂ production in the presence of PQ²⁺ was considered 100%, and all values are expressed as a percentage of this (mean ± S.E., n = 3). *, p < 0.05 compared with PQ²⁺-treated control mitochondria (one-way analysis of variance).

FIGURE 6. Proposed involvement of ETC in PQ²⁺-dependent H₂O₂ generation in brain mitochondria

Electrons feed into the ETC at the level of complex I or complex II and are transferred to ubiquinone (CoQ), complex cytochrome c (c), and complex IV sequentially. Complex III has the ability transfer these electrons to PQ²⁺, thus forming the PQ^+· radical, leading ROS formation and subsequent cell damage.