Oxidative Damage To Mitochondria Induced By Occupational Hazards In Nerve Cells And Its Potential Protection

Mitochondrial dysfunction including oxidative phosphorylation efficiency,energy metabolism disturbance,mitochondrial oxidative stress and mitochondrial DNA (mtDNA) oxidative damage, is considered to make great contributions to various diseases in nervous system. The rapidly growing application of electromagnetic radiation (EMR) and nickel in modern technologies has raised considerable concerns about their potential hazardous effects on human health. It is reported that nervous system is one of the most important targets in the toxicity of EMR and nickel. The purpose of this study was to investigate whether the mitochondrial dysfunction was involved in the neurotoxicity of EMR radiation and nickel exposure. In addition, we would like to find some potential molecular or substances that could protect against the neurotoxicity induced by EMR radiation and nickel exposure. In order to address this issue, different kinds of nerve cells, including primary cultured cortical neurons, human neuroblasma SH-SY5Y cells and mouse neuroblasma Neruo2a cells, were exposed to 1800MHz radiofrequency radiation (RF) and various concentration of nickel. The main results and conclusions were as following:一,EMR radiation induced oxidative damage to mitochondria in nerve cells: the protective effects of Tfam(一) EMR radiation induced oxidative damage to mitochondria in cortical neuronsAt 6h,12h,24h and 48h after EMR radiation,ROS production obviously increased,the cell viability of cortical neurons significantly decreased,the content of ATP and mitochondrial membrane potential (△Ψm) markedly reduced. In addition, the contents of 8-hydroxy-2-deoxy-guanosine(8-OHdG) significantly elevated, while the mtDNA copy number and mtDNA transcripts (ND1,COX I and ND6) levels obviously decreased. It demonstrated that EMR radiation induced oxidative damage to mitochondria in cortical neurons.(二) EMR radiation effected the expression and import of Tfam, the ability of Tfam in binding mtDNA in cortical neuronsNumous studies indicate that mitochondrial dysfunction is largely attributable to mtDNA mutation. Tfam works as the key factor for the maintenance of mtDNA. In some pathophysiology situations, oxidative stress could cause the oxidative damage to mitochondria through disturbing the expression and activity of Tfam. Thus, in this part, we hypothesized that EMR radiation disturbed the expression and activity of Tfam,which might be related with the oxidative damage of mitochondria in radiated-nerve cells. As a result, we found that both the mRNA levels and protein levels of Tfam obviously elevated after EMR radiation. Additionally,import assay showed that EMR radiation decreased the import of S³⁵-Tfam from cytoplasm to mitochondria. Furthermore,electrophoretic mobility shift assay (EMSA) exhibited that the binding of Tfam to the mtDNA promoter significantly decreased after EMR radiation. It indicated that EMR radiation inhibited the import of Tfam and repressed the acticity of Tfam in binding mtDNA in mitochondria, which may lead to the oxidative damage to mitochondria in nerve cells.(三) Tfam overexpression protected mitochondria against oxidative damage induced by EMR radiation in SH-SY5Y cells1. We successfully constructed the plasmid expressing Tfam and transfected the recominated plasmid into SH-SY5Y cells. At 24h after EMR radiation,overexpression of Tfam could significantly reduce ROS production, reverse the decrease of cell viability, improve ATP content, effectively attenuate the oxidative damage of mtDNA induced by EMR radiaiton in SH-SY5Y cells. It suggested that Tfam overexpression protected mitochondria against oxidative damage induced by EMR radiation in SH-SY5Y cells.2. We disrupted the C-terminal tail of Tfam,which had no ability in promoting mtDNA transcripts but could still maintain the nucleoid structure of mtDNA and mtDNA copy number. As the same as the wild-type Tfam, disrupted Tfam also could significantly reduce ROS production, reverse the decrease of cell viability, improve ATP content, effectively attenuate the oxidative damage of mtDNA induced by EMR radiation in SH-SY5Y cells. It suggested that the protective effects of Tfam overexpression against EMR radiation-induced oxidative damage to mitochondria were largely attributed to the ability of Tfam in maintaining the nucleoid structure of mtDNA and mtDNA copy number.(å››)β-amyloid induced oxidative damage to mitochondria in nerve cells: the protective effects of Tfamβ-amyloid deposition has been proposed as the major pathogenic event in the development and progression of Alzheimer's disease (AD). Oxidative stress and mitochondrial dysfunction have been the provital mechanism involved in Aβ-mediated neurotoxicity. Evidence suggests that Aβenters mitochondria and interacts with the mitochondrial proteins, induces mitochondrial dysfunction and causes oxidative stress. In order to further verify the involvment of mitochondrial dysfunction in the neruotoxicity of EMR radiation and the protective effects of Tfam, we established another oxidative damage model of mitochondria by administration of Aβ_1-42 as a positive control. As expected, Aβ_1-42 caused oxidative damage to mitochondria in SH-SY5Y cells, which was similar with that in irradiated-cells. Both Tfam with or without C-terminal tail could effectively reduce oxidative stress induced byβ-amyloid, attenuate the oxidative damage of mtDNA and reverse mitochondrial dysfunction.二,Nickel exposure induced oxidative damage to mitochondria in nerve cells: the protective effects of melatonin(一) Nickel exposure induced oxidative damage to mitochondria in nerve cellsAfter primary cultured cortical neurons and mouse neuroblastoma Neuro2a cell lines were exposed to different concentrations of nickel chloride (NiCl₂) (0.125 mM, 0.25 mM, 0.5 mM and 1 mM) for 12 h or 0.5 mM NiCl₂ for various periods (0 h, 3 h, 6 h, 12 h and 24 h),we found that nickel significantly increased ROS production and caused the loss of cell viability both in cortical neurons and Neuro2a cells. In addition, nickel exposure obviously inhibited the mitochondrial function, disrupted the mitochondrial membrane potential, reduced ATP production and decreased mtDNA content. It indicated that nickel caused oxidative damage to mitochondria in nerve cells.(二) Melatonin pretreatment protected mitochondria against oxidative damage induced by nickel exposure in nerve cellsThe pretreatment of melatonin, a kind of efficient neuroprotective agents famous for its activity in reducing oxidative stress and maintaining mitochondrial function, efficiently attenuated the oxidative damages to mitochondria in nickel-treated nerve cells.In conclusion, our results demonstrated that EMR radiation induced oxidative stress and caused oxidative damage to mitochondria. It may be related to the inhibitory effects of EMR radiation on the import of Tfam in cortical neurons. In addition, Tfam overexpression effectively reduced oxidative stress and protected mitochondria against oxidative damage induced by EMR radiation in nerve cells. These protective effects were largely attributed to the ability of Tfam in maintaining the nucleoid structure of mtDNA and mtDNA copy number. Similarly, nickel exposure obviously induced oxidative damage to mitochondria, which were efficiently attenuated by melatonin pretreatment. All of our study indicated that EMR radiation and nickel exposure had deleterious effects on nerve cells through causing mitochondrial dysfunction. Importantly, it was potential to protect against the neurotoxicity of EMR radiation and nickel by reducing oxidative stress and maintaining mitochondrial function. It not only revealed the mechanism of neurotoxicity induced by EMR radiation and nickel exposure,but also provided the clues for protecting agaisnt EMR radiation and nickel exposure.