Abstract
Paraquat (PQ), a herbicide used worldwide, causes fatal injury to organs upon high dose ingestion. Treatments for PQ poisoning are unreliable, and numerous deaths have been attributed inappropriate usage of the agent. It is generally speculated that a microsomal drug-metabolizing enzyme system is responsible for PQ toxicity. However, recent studies have demonstrated cytotoxicity via mitochondria, and therefore, the cytotoxic mechanism remains controversial. Here, we demonstrated that mitochondrial NADH-dependent PQ reductase containing a voltage-dependent anion channel 1 (VDAC1) is responsible for PQ cytotoxicity. When mitochondria were incubated with NADH and PQ, superoxide anion (O(2)(*)) was produced, and the mitochondria ruptured. Outer membrane extract oxidized NADH in a PQ dose-dependent manner, and oxidation was suppressed by VDAC inhibitors. Zymographic analysis revealed the presence of VDAC1 protein in the oxidoreductase, and the direct binding of PQ to VDAC1 was demonstrated using biotinylated PQ. VDAC1-overexpressing cells showed increased O(2)(*) production and cytotoxicity, both of which were suppressed in VDAC1 knockdown cells. These results indicated that a VDAC1-containing mitochondrial system is involved in PQ poisoning. These insights into the mechanism of PQ poisoning not only demonstrated novel physiological functions of VDAC protein, but they may facilitate the development of new therapeutic approaches.
Highlights
Paraquat (PQ2; methyl viologen, 1,1Ј-dimethyl-4,4Ј-bipyridinium dichloride) is an effective herbicide used in more than 120 countries [1]
These results indicated that PQ produced O2. in an NADH-dependent manner in mitochondria and damaged mitochondria followed by cell death
In this study we demonstrate that a mitochondrial system, not a microsomal system, is involved in PQ poisoning; PQ produces O2. by NADH-dependent oxidoreductase in the outer membrane of mitochondria and damages mitochondria, leading to cell death
Summary
Mitochondrial superoxide production in HeLa cells was detected using MitoSOX (Molecular Probes Inc., Eugene, OR), a red fluorescent mitochondrial superoxide indicator, according to the given protocol. Cells were pretreated with 1 mM PQ; Sigma-Aldrich) for 50 min at 37 °C and incubated with 5 M MitoSOX for 10 min in the dark. The medium was exchanged for fresh medium, and the cells were observed by a fluorescence microscope (Olympus IX70, Olympus Corp., Tokyo, Japan). Intracellular H2O2 production in HeLa cells by PQ was detected using 2Ј,7Ј-dichlorofluorescein-diacetate (DCFH; Molecular Probes) [17, 18]. Cells were pretreated with 1 mM PQ for 1 h at 37 °C, and the cells were incubated with 5 M DCFH for 20 min in the dark. The medium was exchanged for fresh medium; fluorescence images that appeared after the formation of 2Ј,7Ј-dichlorofluorescein (DCF) were observed by fluorescence microscopy
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