Iron and paraquat as synergistic environmental risk factors in sporadic Parkinson's disease accelerate age-related neurodegeneration

Abstract

Extensive epidemiological data in humans and studies in animal models of Parkinson's disease (PD) suggest that sporadic forms of the disorder are not strictly genetic in nature but most likely because of combined environmental exposures over the period of the life-span coupled with increased genetic susceptibilities. Environmental paraquat and neonatal iron exposure have both been separately suggested as potential risk factors for sporadic forms of the disease. In this study, we demonstrate that combined environmental exposure to these two agents results in accelerated age-related degeneration of nigrostriatal dopaminergic neurons. Furthermore, pretreatment with the synthetic superoxide dismutase/catalase mimetic, EUK-189, significantly attenuated neuronal death mediated by combined paraquat and iron treatment. These findings support the notion that environmental PD risk factors may act synergistically to produce neurodegeneration associated with the disorder and that iron and paraquat may act via common oxidative stress-mediated mechanisms.

Figure 1.

Iron enhances paraquat-induced dopaminergic N27 cell apoptosis in vitro. A, N27 cells were treated with increasing concentrations of paraquat and 80 μm FeCl₂ for 24 h. Cell viability was measured via the MTT assay and expressed as a percentage of viability in control. B–F, Cells were treated with EUK-189 1 h before the addition of 80 μm FeCl₂ and 350 μm paraquat and cell viability at 24 h (B), cleaved caspase-3-positive cells at 18 h (C, E), and TUNEL-positive cells at 24 h (D, F) were measured. Error bars indicate mean ± SEM. n = 5. ^#p < 0.01, significantly from control; *p < 0.01, significantly from paraquat; **p < 0.01, significantly from paraquat alone; *** p < 0.01, significantly from paraquat plus FeCl₂ alone. White bar, Paraquat; black bar, paraquat plus FeCl₂.

Figure 2.

Inhibition of JNK kinase attenuates additional dopaminergic apoptosis induced by combined paraquat and iron in both N27 and primary mesencephalic cultures in vitro. N27 cells were treated with the indicated inhibitors 1 h before the addition of 350 μm paraquat or 350 μm paraquat plus 80 μm FeCl₂. A–C, Cleaved caspase-3-positive cells at 18 h (A), TUNEL-positive cells at 24 h (B), and cell viability at 24 h (C) were measured. Mesencephalic cultures were treated with the indicated inhibitors 1 h before the addition of paraquat or paraquat plus FeCl₂. D, E, Cleaved caspase-3-positive cells at 18 h (D) and cell viability at 24 h (E) were measured. Error bars indicate mean ± SEM. n = 4–5. ^#p < 0.05, significantly from paraquat alone; *p < 0.01, significantly from paraquat alone; **p < 0.001, significantly from paraquat plus FeCl₂ alone. Gray bars, Without inhibitors; white bars, SP600125; black bars, CEP-11004.

Figure 3.

Effects of combined neonatal iron and systemic paraquat administration on dopaminergic neurodegeneration in vivo with increasing age. A, Photomicrographs of SNpc TH-immunostained sections from 24-month-old mice. Original magnification, 10×. B, Quantitative stereological analysis of the number of TH-stained profiles from saline plus saline (purple bars), neonatal iron fed plus saline (green bars), saline plus paraquat (yellow bars), and neonatal iron plus paraquat (black bars). Error bars indicate mean ± SEM. n = 4–5. ^#p < 0.01, significantly from saline plus saline group;*p < 0.05, significantly from either neonatal iron fed plus saline group or saline plus paraquat group.

Figure 4.

Administration of EUK-189 attenuates combined neonatal iron and paraquat-induced dopaminergic neuronal death at 12 months of age. A, Photomicrographs of SNpc TH-immunostained sections. Original magnification, 10×. B, Quantitative stereological analysis of the number of TH-stained profiles from saline plus saline (gray bars), saline plus paraquat (white bars), and neonatal iron fed plus paraquat (black bars). Error bars indicate mean ± SEM. n = 4. ^#p < 0.01, significantly from saline plus saline plus vehicle group; *p < 0.05, significantly from saline plus paraquat plus vehicle group; ^##p < 0.01, significantly from saline plus paraquat plus vehicle group; **p < 0.01, significantly from neonatal iron fed plus paraquat plus vehicle group.

Figure 5.

3-Nitrotyrosine immunopositive cells count in the SNpc. A, An example of localization (arrows) of 3-nitrotyrosine-immunopositive staining (green) within dopaminergic neurons (red). 4′,6-Diamidino-2-phenylindole (blue) was used to counterstain nuclei. Scale bars, 20 μm. B, C, Quantitative analysis of double labeling for TH with 3-nitrotyrosine in the SNpc of 2-month-old (B) and 12-month-old (C) mice. White bars, Vehicle; black bars, EUK-189 treated. Error bars indicate mean ± SEM. n = 3. *p < 0.001, significantly from saline plus saline plus vehicle group; p̂ < 0.05, significantly from neonatal iron fed plus saline plus vehicle group; ^#p < 0.001, significantly from saline plus paraquat plus vehicle group; ^@p < 0.001, significantly from neonatal iron fed plus paraquat plus vehicle group.

Figure 6.

Administration of EUK-189 inhibits increased activation of the JNK signaling pathway elicited by combined paraquat and iron in SNpc versus paraquat alone. A, B, Phospho-JNK (A) and phospho-c-Jun (green; B) localized (yellow) within TH-positive SNpc neurons (red). 4′,6-Diamidino-2-phenylindole (blue) was used to counterstain nuclei. Original magnification, 10×. C, D, Representative higher-magnification images demonstrating localization of phospho-JNK (C) and phospho-c-Jun (green; D) within dopaminergic neurons (red; arrow). 4′,6-Diamidino-2-phenylindole (blue) was used to counterstain nuclei. Original magnification, 100×. E, F, Quantitative analysis of double labeling of TH with phospho-JNK (E) and phospho-c-Jun (F). Purple bars, Saline plus saline; green bars, neonatal iron fed plus saline; yellow bars, saline plus paraquat; black bars, neonatal iron fed plus paraquat. Error bars indicate mean ± SEM. n = 3. ^#p < 0.01, significantly from saline plus saline plus vehicle group; *p < 0.05, significantly from saline plus paraquat plus vehicle group; ^##p < 0.001, significantly from saline plus paraquat plus vehicle group; **p < 0.001, significantly from neonatal iron fed plus paraquat plus vehicle group.