The mechanism for paraquat toxicity involves oxidative stress and inflammation: A model for Parkinson's disease.

摘要

Parkinson's disease (PD) is a neurodegenerative disorder known to affect the dopaminergic neurons in the substantia nigra. Epidemiological studies have shown an increased risk of developing PD with exposure to paraquat. Paraquat inhibits complex I of the mitochondrial respiration chain and generates of oxidative stress. In this study, we examined the source of reactive oxygen species (ROS) and the underlying signaling pathway for paraquat-induced cytotoxicity to BV-2 microglial cells. Paraquat-induced ROS production (including superoxide anion) in BV-2 cells was accompanied by translocation of p67phox, the cytosolic subunit of NADPH oxidase, to the membrane. Paraquat-induced ROS production was inhibited by NADPH oxidase inhibitors, apocynin and diphenylene iodonium (DPI), but not the xanthine/xanthine oxidase inhibitor, allopurinol. Apocynin and DPI also rescued cells from paraquat-induced toxicity. Inhibitors for either protein kinase C (PKC) or extracellular signal-regulated kinases (ERK1/2) could partially attenuate paraquat-induced ROS production and cell death; a combination of these two kinase inhibitors also partially attenuated paraquat-induced cytotoxicity. Rottlerin, a selective PKCδ inhibitor, also inhibited paraquat-induced translocation of p67phox.;Under inflammatory conditions, microglial cells respond to interferon-γ (IFNγ) and lipopolysaccharides (LPS) by induction of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). Because paraquat can cause cytotoxicity by increasing ROS production in microglial cells, we also tested whether paraquat may exacerbate cytotoxicity in the presence of NO produced by pro-inflammatory cytokines. Our results show that paraquat greatly inhibited cytokine-induced production of NO induced by both IFNγ and LPS but only moderately enhanced ROS production and cell cytotoxicity in microglial cells. Paraquat-induced attenuation of NO production was not due to either (1) inhibition of iNOS expression, (2) a direct interaction between paraquat with nitrite in the culture medium, or (3) interaction with NO produced by SNAP, an NO donor. Inhibition of NO production by L-Nil, a specific inhibitor for iNOS, did not decrease ROS production by paraquat. On the other hand, examination of nitrotyrosine, an indicator of protein adduct due to peroxynitrite formation, indicated increase due to IFNγ and IFNγ with paraquat. Addition of NADPH, cofactor for both NADPH oxidase and iNOS, did not increase paraquat-induced ROS production but inhibited IFNγ-induced NO production.;We wanted to determine the role of NADPH oxidase in neuronal cells. SH-SY5Y human neuroblastoma cells become dopaminergic neuronal-like after differentiation with retinoic acid. SH-SY5Y cells express the NADPH oxidase subunit p67 phox at lower amounts than microglial cells. Paraquat did induce the translocation of p67phox to the membrane. Paraquat also induced the upregulation of the p67phox subunit protein and NOX2 mRNA. It has been shown that increased gene expression of the cytosolic subunits can result in a dramatic increase in the generation of superoxide. Moreover, the low levels of paraquat that people are generally exposed to can upregulate gene expression of NADPH oxidase and increase superoxide production in neurons.;In conclusion, our data suggest that NADPH oxidase plays a significant role in the toxicity of paraquat, especially in microglia cells that may destroy the substantia nigra and lead to the development of PD. A better understanding of the underlying cause of this disease would help us to develop better strategies and pharmaceutical agents for prevention and treatment of PD.