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. 2009 Oct 16;284(42):28642-9.
doi: 10.1074/jbc.M109.033431. Epub 2009 Aug 28.

Paraquat toxicity induced by voltage-dependent anion channel 1 acts as an NADH-dependent oxidoreductase

Hiroki Shimada  1 , Kei-Ichi Hirai, Eriko Simamura, Toshihisa Hatta, Hiroki Iwakiri, Keiji Mizuki, Taizo Hatta, Tatsuya Sawasaki, Satoko Matsunaga, Yaeta Endo, Shigeomi Shimizu
Affiliations

Paraquat toxicity induced by voltage-dependent anion channel 1 acts as an NADH-dependent oxidoreductase

Hiroki Shimada et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Damage to mitochondria caused by NADH-dependent O2˙̄ production induced by PQ. A, mitochondrial O2˙̄ production was visualized by MitoSOX in cells treated with PQ (1 mm) and BQ (0.2 mm). B, H2O2 production in isolated mitochondria was estimated by DCF florescence method. Mitochondria were incubated with 10 mm PQ, 2 mm NADH, and 0.3 mm BQ (*, p < 0.01, versus control). C, isolated mitochondria were incubated with 3 mm PQ, 2 mm NADH, and the 3000 IU/ml SOD. Upper panels, electron micrograph. Lower graph, percentages of intact mitochondria (*, p < 0.001, versus control). D, the survival rate of HeLa cells exposed to PQ (open circle) and PQ and 1 mm Trolox® (closed circle). Each point is the average of two to four experiments. Error bars represent S.E.
FIGURE 2.
FIGURE 2.
NADH-PQ oxidoreductase activity in the outer membrane extract. A, NADH (0.2 mm) was oxidized by the outer membrane extract in the presence of PQ (5 mm), but NADPH (0.2 mm) was not oxidized. B, PQ dose-dependent relationship to O2˙̄ production activity was observed by co-administration with NADH (0.1 mm) to the outer membrane extract (closed circle). In contrast, NADPH (0.1 mm) did not exert any such PQ effects (open circle). All error bars represent S.D. (n = 3).
FIGURE 3.
FIGURE 3.
Participation of VDAC1 in the NADH-PQ oxidoreductase activity and mitochondrial damage. A, O2˙̄ production in the outer membrane extract (closed circle) was inhibited by DIDS (100 μm; open circle, p < 0.001, n = 3) and anti-VDAC1 mAb (30 μg/ml; open triangle, p < 0.05, n = 3). Closed triangle, treated with normal IgG (30 μg/ml). Error bars represent S.D. (n = 3). B, the extract was immunoprecipitated with anti-VDAC1 mAb or normal IgG, and the NADH-oxidation activity of the supernatants was measured. Control, no treatment. *, p < 0.01, versus control. Error bars represent S.D. (n = 3). C, H2O2 production in isolated mitochondria by PQ (10 mm) co-administered with NADH (2 mm) was estimated by DCF florescence method. DIDS (100 μm) and anti-VDAC1 mAb (9 μg/ml) were inhibited H2O2 production. *, p < 0.001 with respect to the control. Each point is the mean of triplicate experiments. Error bars represent S.E. D, effects of anti-VDAC1 antibody on the NADH-PQ-dependent breakage of mitochondria were estimated. Isolated mitochondria were ruptured by the co-administration of PQ (3 mm) and NADH (2 mm), whereas the addition of anti-VDAC1 mAb (9 μg/ml) protected the mitochondria from such breakage. *, p < 0.01 versus the control. Each point is the mean of triplicate experiments. Error bars represent S.E.
FIGURE 4.
FIGURE 4.
VDAC1 is a component of NADH-PQ oxidoreductase. A, extracts obtained from the mitochondrial outer membrane by treatment with Triton X-100/deoxycholate (lane 1) or SDS/ Igepal CA-630 (lane 2) were run on SDS-PAGE, and the results were analyzed by Western blotting with anti-VDAC1 mAb. VDAC1 protein was detected by the mAb (arrowhead). B, DEAE fractions from the extracts containing oxidoreductase activity were examined by zymography with NADH and PQ in blue tetrazolium solution (lane 1). The active band was consistent with the anti-VDAC1 mAb-detected band (lane 2, arrowhead), and this band was excised and subjected to Western blot analysis using anti-VDAC1 mAb (lane 3; the arrowhead indicates VDAC1 protein). C, direct binding to VDAC1 was assayed using biotinylated (b-) PQ (closed circle) in competition with non-labeled PQ (open circle, 25 μm, open triangle, 250 μm). Error bars represent S.D. (n = 3). D, assay of NADH binding to the outer membrane extracts was performed. The extracts were trapped by immobilized anti-VDAC1 antibody and incubated with biotinylated NAD+. Bound biotinylated NAD+ was reduced by exposure to non-labeled NADH (p < 0.01, closed circles). When normal IgG was used for trapping, no NADH competition was detected (open circles). Broken lines represent 95% confidence interval of the control value. Error bars represent S.D. (n = 3).
FIGURE 5.
FIGURE 5.
Effects of VDAC1 overexpression on the PQ-dependent H2O2 production and the cytotoxicity in HeLa cells. A, lysates from VDAC1-overexpressing cells were subjected to Western blot analysis with anti-VDAC1 mAb. Control, cells transfected with empty vector; VDAC+, cells transfected with the vector bearing vdac1 cDNA. B, H2O2 production by PQ in VDAC1-overexpressing cells (VDAC1+) was higher than that of control cells (*, p < 0.001). Light bars, no treatment; dark bars, exposure to 1 mm PQ. Error bars represent S.D. (n = 3). C, the survival rates of VDAC1-overexpressing HeLa cells (closed circle) were lower than those of controls (open circle; p < 0.001). Error bars represent S.E. of triplicate experiments.
FIGURE 6.
FIGURE 6.
Effects of VDAC1 knockdown on the PQ-dependent H2O2 production and the cytotoxicity in HeLa cells. A, lysates from knockdown cells were subjected to Western blot analysis with anti-VDAC1 mAb. Control, cells transfected with the control siRNA; VDAC−, cells transfected with VDAC1 siRNA. The arrowheads indicate VDAC1 protein. B, NADH-PQ-dependent H2O2 production on mitochondria isolated from VDAC1-knockdown HeLa cells was estimated by DCF assay. Light bars, mitochondria incubated with 2 mm NADH only; dark bars, mitochondria were incubated with 10 mm PQ and 2 mm NADH. Error bars represent S.D. (n = 3). C, the survival rate of VDAC1-knockdown HeLa cells (VDA1-siRNA) after 24 h of exposure to 222 μm PQ was higher than that of control cells (p < 0.001). Error bars represent S.E. of triplicate experiments.
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