Effects of titanium dioxide nanoparticles exposure on parkinsonism in zebrafish larvae and PC12
Introduction
As the world's population is steadily ageing, age related diseases such as neurological disorders have become a major public health concern. Parkinson's disease (PD) is ranked as the world's second prevailed neurodegenerative disease, which was characterized by severe dropping of dopaminergic neurons and formation of Lewy bodies (Mao et al., 2016). These years, environmental risk factors are of enormous interest in neurodegerative disease research. Nanomaterials have unique physicochemical properties such as small size and large surface area, and widely applied in cosmetics, optics, catalysis and medical fields (Zhang et al., 2016). Several nanoparticels, such as silica, silver and zinc oxide were reported could induce neurotoxicity in vitro and in vivo (Ansar et al., 2016, Begum et al., 2016). Actually, few studies evaluate the disease risks of nanomaterials and correlate the safety of nanomaterials with specific disease. Recently, Prof. Hu's group had firstly reported that graphene oxide (GO) GO could induced PD-like symptoms in zebrafish larvae (Ren et al., 2016). Therefore, it is an argent need for more detailed research to elucidate the potential neurodegerative disease risks of nanoparticles.
Titanium dioxide nanoparticles (TiO2 NPs) are one of the most highly manufactured nanomaterials and their usage has grown up to 10,000 tons annually worldwide between 2011 and 2014 (Miao et al., 2015). TiO2 NPs are widely used in cosmetics, paint and drug delivery system, which increases the possibility of their exposure to ecosystems (Chang et al., 2016). Therefore, it is urgent to elucidate their adverse effects on environment and organisms. Existing studies have shown that exposure to titanium dioxide can induce cytotoxic, genotoxic and oxidative effects in different cell lines (Han et al., 2013, Lopes et al., 2016). TiO2 NPs were also reported that they could disrupt the hemophilic interaction of VE–cadherin to cause endothelial cell leakiness (Setyawati et al., 2013). Moreover, it has been reported that TiO2 NPs were capable of accumulating in brain tissues and could stimulate oxidative stress and inflammatory responses to cause impairment on central nervous system in mice (Kumar et al., 2016). In zebrafish, low dose exposures of TiO2 NPs also may result in the brain damages and induce neurotoxicity (Palaniappan and Pramod, 2011, Purushothaman et al., 2014, Sheng et al., 2016). These results inspired us to explore the neurodisease risks of TiO2 NPs.
Neurons cells are one of the main cell types in brain tissue and play essential role in maintenance of brain function. The study of the neurotoxicity of nanoparticles is vital, but still in its infancy. Oxidative stress is considered an important pathway in promoting neuron damage. Nanoparticles were widely reported that they could induce the formation of reactive oxygen species (ROS) in vitro and in vivo (Duan et al., 2013, Zhao et al., 2013). In addition, TiO2 NPs could enter into primary neuronal cells to stimulate ROS in brain microglia and damage neurons (Long et al., 2007, Hong et al., 2015). Alpha-synuclein is the major component of Lewy bodies, and α-synuclein mutations contribute to early-onset PD. A number of studies have shown that α-synuclein, parkin, uch-l1 and pink1 were involved in dysfunction of the ubiquitin-proteasome system and Lewy body formation (Feng et al., 2014, Segura-Aguilar et al., 2016). Therefore, we hypothesized that TiO2 NPs may internalize in brain tissue and induce ROS generation, which influence the function of neurons, resulting in depletion of dopamine (DA) and interruption of ubiquitin-proteasome system related gene expression.
The main breakthrough of studies on neurotoxicity is the selection of desired model system. In this regard, zebrafish embryos were extensively investigated. Zebrafish (Danio rerio) embryos as the early life stages are sensitive and the signaling factors required for neurons development, which are same in fish and mammals (Paterson et al., 2011). Due to the transparent character, zebrafish embryos are facilitated on study of cellular uptake and toxic observation. Herein, zebrafish embryos and PC12 (a dopaminergic neuron-like cell line) were chosen as ideal PD model for studying neurotoxicity of nanoparticles. TiO2 NPs could accumulate in the brain of zebrafish larvae after co-incubation with zebrafish embryos and induce oxidative stress, which cause damage to the brain tissue and result in loss of DA secretion and dysregulation of ubiquitin-proteasome system related genes expression. Based on the neurotoxicity of TiO2 NPs in vivo, we conducted the in vitro research by investigating the effect of the TiO2 on dopaminergic neuron PC12. The decreased cell viability and mitochondria dysfunction of PC12 after exposure to TiO2 NPs further confirmed the relevance of TiO2 NPs and neurodegenerative disease.
Section snippets
Materials
TiO2 NPs from Sigma–Aldrich (#700347) were acquired from Sigma–Aldrich (#700347). DA ELISA kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dichlorodi hydrofluorescein (DCFH-DA) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). MitoRed was purchased from keyGEN BioTECH(Jiangsu, China). The anti-tyrosine hydroxylase (anti-TH) antibody was purchased from Proteintech. The ReverTra Ace® qPCR RT Kit and SYBR® Green Realtime PCR Master Mix were from
Results and discussion
As show in Fig. S1, TEM visualization of TiO2 NPs revealed its primary-particle spheroid morphology as those reported by the manufacturer and previous work (Li et al., 2014a, Li et al., 2014b). Generally, TiO2 NPs tended to aggregate when they were dispersed in suspensions and they exhibited average primary particle sizes of approximately 33.4 ± 1.9 nm. The hydrodynamic size in water is 149.4 ± 1.3 nm by DLS (Table S2). The DLS results showed agglomeration of the TiO2 NPs. The particle
Conclusions
In this study, we first showed that TiO2 NPs induced Parkinson's disease-like symptoms in zebrafish larvae and PC12. TiO2 NPs exposure induced zebrafish premature hatching and significantly disturbed locomotive activity, which suggested the potential risk of TiO2 NPs on zebrafish embryos development. The localization of TiO2 NPs in the brain tissue of zebrafish was found to induce ROS generation and induce cell death in hypothalamus region. More importantly, TiO2 NPs exposure affect the
Acknowledgments
The authors are grateful to National Natural Science Foundation of China (No. 31200890), and the Scientific Innovation Program for University Students in Zhejiang Province (No. 2014R403060).
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