br Materials and methods br Results

Materials and methods
Results
Discussion Induction of neuronal cell death is the main neurotoxic mechanism of arsenite (Vahidnia et al., 2007). Previous studies have demonstrated that arsenite may trigger neuronal cell death via apoptosis, autophagy as well as necrosis (Yen et al., 2012; Lau et al., 2013; Selvaraj et al., 2013). However, the detailed mechanisms underlying how arsenite induces neuronal cell death remain unclear. Ferroptosis is an oxidative, iron-dependent form of cell death that is remarkably distinct from classic apoptosis, necrosis, autophagy and other forms of cell death (Cao and Dixon, 2016; Xie et al., 2016). In the present study, we revealed a novel mechanism that arsenite exposure may trigger ferroptotic cell death in the neuron, manifested by generation of ROS, LCL161 receptor of SOD activities, accumulation of MDA and Fe2+, imbalance of GSH and GSSG, ATP depletion and the alteration of ferroptosis-related signaling pathways. These findings further indicate that regulation of ferroptosis by specific agents may be a new strategy for the prevention of arsenite neurotoxicity. Arsenicosis is a serious biogeochemical disease in China mainly caused by drinking water from the wells contaminated by high concentrations of arsenite (Rodríguez-Lado et al., 2013). Although the maximum allowable concentration of arsenite in the drinking water is limited at 0.01 mg/L in China, the doses of arsenite are up to almost 4.44 mg/L in arsenite-contaminated area. Besides, in some industrial area, the level of arsenite may reach the extremely high level at about 10 mg/L (Wang et al., 2007; He and Charlet, 2013; Rodríguez-Lado et al., 2013). High levels of arsenite, ranging from 0.01 to 1.86 mg/L, in the ground water have been found in the 19 provinces in mainland China (He and Charlet, 2013). The applied concentration in the mice should multiple the interspecies uncertainty factor. A 10-fold uncertainty factor was usually applied each for intra- and inter-species variability, for a total uncertainty factor of 100. Thus, we used the environmental relevant doses, 0.5, 5 mg/L for arsenite chronic exposure. For reducing the potential inter-species variability between mice and human, we also chose the 50 mg/L as the high dose. Moreover, human may be exposed to arsenite via drinking water for a long time, even the whole life. The high risk of arsenite-related diseases, such as cancers, cardiovascular disease and neurodegenerative disease etc., may cause heavy health burden for the people who lived in the high arsenite areas (Naujokas et al., 2013). Therefore, in this study, the healthy C57BL/6 J mice were exposed to three doses of arsenite, 0.5 mg/L, 5 mg/L and 50 mg/L, for 6 months to simulate the environmental exposure of human as much as possible. In addition, these concentrations of arsenite have already been used in the previous studies to reveal the toxic effects of arsenite (Bardullas et al., 2009; Gao et al., 2013). In the previous study, condensed mitochondrial membrane density and reduction/vanishing of mitochondria crista have been observed in the ferroptotic cells (Cao and Dixon, 2016; Xie et al., 2016; Yang and Stockwell, 2016). We further confirmed these morphological changes of mitochondria by transmission electron microscopy. Given iron metabolism and lipid peroxidation signaling are increasingly recognized as central mediators of ferroptosis (Stockwell et al., 2017), we detected the following oxidative damage-related indicators, Fe2+, ROS, SOD, MDA and GSH/GSSG. Our results found that levels of total and mitochondrial ROS were significantly enhanced by arsenite exposure, and the contents of lipid peroxidation products, MDA, were sharply increased in a dose-dependent manner. These alterations also accompanied with the elevation of Fe2+ within both the cells and mitochondria, suggesting that accumulation of lipid peroxidation products and the production of ROS may mainly derive from disorders of Fe2+ metabolism in mitochondria. Interestingly, after chronic arsenite exposure, we observed that, although the reduced GSH contents were decreased and its oxidized form GSSG were increased in 5.0 and 50 mg/L arsenite group, GSH/GSSG ratio was significantly enhanced by 0.5 mg/L arsenite. Similar trends were observed in the determination of SOD activities. Our data found that, the low level of arsenite enhanced the activities of total SOD and Cu-Zn SOD, whereas medium and high doses of arsenite treatment decreased the activities of these two types of SOD. This phenomenon may be mainly caused by the hormesis effects of arsenite at relative low level. In other words, in response to low concentration of arsenite (<0.5 mg/L), cells may adaptably produce more reduced GSH and SOD to cope with the toxic effects of arsenite, but with increasing level of arsenite, the synthesized GSH and SOD cannot handle these adverse effects, the contents of GSH and activities of SOD will quickly exhaust. Thus, we observed that the GSH/GSSG ratio, total SOD and Cu-Zn SOD activities remarkably decreased in 5.0 and 50 mg/L arsenite-treated animals. Interestingly, we did not observe the alteration of Mn SOD activity in response to arsenite exposure, indicating that this enzyme may not be involved in this process.