Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson's disease

Abstract

Parkinson's disease (PD) has been linked to mitochondrial dysfunction and pesticide exposure. The pesticide rotenone (ROT) inhibits complex I and reproduces features of PD in animal models, suggesting that environmental agents that inhibit complex I may contribute to PD. We have previously demonstrated that ROT toxicity is dependent upon complex I inhibition and that oxidative stress is the primary mechanism of toxicity. In this study, we examined the in vitro toxicity and mechanism of action of several putative complex I inhibitors that are commonly used as pesticides. The rank order of toxicity of pesticides to neuroblastoma cells was pyridaben > rotenone > fenpyroximate > fenazaquin > tebunfenpyrad. A similar order of potency was observed for reduction of ATP levels and competition for (3)H-dihydrorotenone (DHR) binding to complex I, with the exception of pyridaben (PYR). Neuroblastoma cells stably expressing the ROT-insensitive NADH dehydrogenase of Saccharomyces cerevisiae (NDI1) were resistant to these pesticides, demonstrating the requirement of complex I inhibition for toxicity. We further found that PYR was a more potent inhibitor of mitochondrial respiration and caused more oxidative damage than ROT. The oxidative damage could be attenuated by NDI1 or by the antioxidants alpha-tocopherol and coenzyme Q(10). PYR was also highly toxic to midbrain organotypic slices. These data demonstrate that, in addition to ROT, several commercially used pesticides directly inhibit complex I, cause oxidative damage, and suggest that further study is warranted into environmental agents that inhibit complex I for their potential role in PD.

Figure1.

Dose-dependent cell death in response to rotenone exposure. Cells were exposed to rotenone for 48 hr, and cell death was monitored between 24 and 48 hr using Sytox green fluorescence. Rotenone treatment at 1 μM, 100 nM, and 10 nM caused toxicity. Results show means ± SEM of six independent experiments. *p 0.05. A.U., Arbitrary units.

Figure 2.

Rotenone toxicity requires electron transfer through complex I. A, Cells infected with rAAV-NDI1 are resistant to rotenone inhibition of oxygen utilization. Where indicated, 5 mM glutamate (Glu), 5 mM malate (Mal), 5μM rotenone (Rot), 500μM flavone, 5 mM succinate (Succ), and 5 μM antimycin (AntA) were added. Note that oxygen utilization in NDI1 transfected cells was not sensitive to rotenone inhibition but that antimycin A still inhibited oxygen consumption. B, NDI1-transduced cells were resistant to rotenone (100 nM) toxicity but remained sensitive to hydrogen peroxide (300 μM) and azide (1 mM). Control and NDI1transduced cells were grown in 96 well plates, and cell death was analyzed 48hr after exposure to compounds. Results represent means ± SEM for three independent experiments.

Figure3.

Rotenone toxicity does not result solely from ATP depletion. A, ATP depletion after exposure to rotenone or 2-DG. Note that rotenone (100 nM) and 2-DG (1 mM) caused similar reductions in cellular ATP levels. Cells were treated for 6–8 hr before ATP measurements; similar results were obtained at 20 hr. Data represent means ± SEM for three to five independent experiments. *p 0.05 versus ATP levels in vehicle-treated wells. B, Although rotenone (100nM) and 2-DG(1mM) caused similar bioenergetic defects, only rotenone (100nM) was toxic. Cells were exposed to rotenone or 2-DG for 48 hr, and cell death was determined using Sytox green fluorescence. Results show means ± SEM of three independent experiments, six replicates per experiment. *p 0.05 versus death in vehicle-treated wells.

Figure4.

Rotenone-induced oxidative damage is dose-dependent and depends on electron transfer through complex I. Untransfected SK-N-MCandNDI1-transfected cells were exposed to rotenone for 24 hr, and then protein carbonyls were measured. Protein carbonyl levels are expressed as a percentage change from levels in solvent-treated cultures. NDI1-transfectedcells received 10 nM rotenone for 24 hr. Data shown are means ± SEM and represent five to six independent experiments. *p 0.05 versus solvent-treated controls.

Figure5.

Glutathione depletion potentiates rotenone toxicity. Cells were exposed to 10μM BSO for 24 hr to deplete cellular glutathione before treatment with 10 nM rotenone. Cell death was then analyzed using Sytox green fluorescence, and death at 36hr after rotenone treatment is shown. Results shown are means ± SEM for three independent experiments. *p < 0.05 versus solvent-treated controls; ^**p < 0.05 versus rotenone-treated wells.

Figure 6.

Antioxidants protect cells from rotenone toxicity. A, α-Tocopherol pretreatment [62.5 μM (gray column) or 125 μM (white column)] attenuated rotenone toxicity. Death was expressed as a percentage of maximal cell death after rotenone treatment. Results represent means ± SEM for four to five independent experiments. *p 0.05 versus rotenone-induced death (black column). B, α-Tocopherol pretreatment did not prevent ATP depletions after rotenone exposure. Cells were exposed to solvent or α-tocopherol (62.5 or 125 μM) before rotenone exposure, and ATP levels were measured. Column colors are as in A. Data shown are means ± SEM and represent three independent experiments. C, α-Tocopherol pretreatment prevented rotenone-induced oxidative damage. Cells were exposed to solvent or 125 μM α-tocopherol for 24 hr before rotenone (10 nM) treatment and before protein carbonyls were measured. Carbonyls are expressed as a percentage change from levels in solvent-treated cultures. Data shown are means ± SEM and represent five independent experiments. *p 0.05 versus solvent-treated controls. D, Coenzyme Q₁₀ (CoQ) pretreatment attenuated rotenone toxicity. Cells were pretreated for 24 hr with 12.5 μM coenzyme Q₁₀ before exposure to 10 nM rotenone. Cell death was followed for 48 hr after rotenone treatment, and the last 24 hr period is shown. ^**p < 0.05 versus rotenone-treated cells in the presence of CoQ and versus solventtreated cells; *p < 0.05 versus solvent-treated cells.

Figure 7.

α-Tocopherol protected dopaminergic neurons in midbrain slice cultures from rotenone oxidative damage and toxicity. A, Rotenone caused oxidative damage to midbrain slice cultures that was prevented by α-tocopherol treatment. Slice cultures were exposed to rotenone (50nM) for 7d in the presence of vehicle or-tocopherol (100M).Data are expressed as a percentage increase in protein carbonyl levels in rotenone-treated cultures. *p 0.05 versus solvent-treated cells. B–D, Rotenone toxicity in midbrains lice cultures was prevented by α-tocopherol. Midbrain slice cultures were exposed to vehicle (B) or 50 nM rotenone for 7 d in the presence of vehicle (C) or -tocopherol (100 M) (D). Rotenone exposure markedly reduced the number of TH processes in midbrain dopaminergic neurons (compare B and C). -Tocopherol treatment reduced the effects of rotenone on these degenerative changes (D).

Figure 8.

Rotenone-induced loss of TH immunoreactivity in the nigrostriatal pathway. TH immunocytochemistry in the striatum (A) and substantia nigra (B) of a vehicle-treated rat is shown. Rotenone treatment reduced TH immunore activity in both the striatum(C) and the substantia nigra (D).The rotenone-treated animal received 3.0mg/kg/d rotenone, subcutaneously, for 5d.

Figure 9.

Chronic rotenone infusion induced selective oxidative damage, with the greatest damage in the midbrain and the olfactory bulb. Chronic rotenone infusion increased protein carbonyl levels in soluble protein isolated from the striatum (n = 18), midbrain (n = 18), cortex (n 11), and olfactory bulb (n 12) but not the cerebellum (n = 10) or hippocampus (n = 10). Rotenone exposure increased insoluble protein carbonyl levels only in mid brain (n = 18) and olfactory bulb (n = 12). Data are expressed as a percentage of protein carbonyl levels in vehicle-treated animals. Results represent means ± SEM. *p < 0.05 compared with control.