Effects Of Metal Ions On Neurocytotoxicity,DNA Methyltransferase,Metallothionein,and α-Synuclein

With the rapid economic development,heavy metal pollution has become one of the most serious environmental problems in China.Studies have shown that the pathogenesis of Parkinson’s disease is associated with heavy metals.In addition,heavy metals can affect the expression of DNA methytransferases（DNMTs）.DNMTs are widely expressed in the brain and dictate the transcriptional activities of genes through various epigenetic mechanisms.Their function alteration,activity change and abnormal expression can lead to various neurodevelopmental disorders,neuropsychiatric disorders and neurodegenerative diseases.Metallothioneins（MTs）can chelate metal ions,regulate physiological metal homeostasis,and effectively remove free radicals in the body.In addition,one pathological feature of PD is the abnormal aggregation ofα-synuclein（α-syn）,whose C-terminus can bind metal ions.The abnormal expression of this protein can damage the integrity of neurons,and cause irreversible effects on some normal physiological mechanisms.These negative effects may be important factors in the development of Parkinson’s disease.In this study,the cytotoxicity of heavy metal ions,including Zn²⁺,Fe³⁺,Cu²⁺and Cd²⁺,on SH-SY5Y cells was assessed.For each metal ion,two concentrations were selected to represent the chronic and acute toxic effects on SH-SY5Y cells.The effects of different metal ions on the expression of DNMTs,MTs andα-syn coding gene SNCA were detected by fluorescence quantitative PCR.Then,the intracellular reactive oxygen species（ROS）level was detected using the fluorescent probe dichlorofluorescein（DCFH-DA）to investigate the toxicity of metal ions.In addition,the prokaryotic expression and purification ofα-syn were performed,and the cytotoxicity ofα-syn monomers,oligomers,and fibers on SH-SY5Y cells was determined by cytotoxicity assay.The results showed that Zn²⁺almost did not affect the expression of DNMTs,while Fe³⁺and Cd²⁺mainly induced the expression of DNMT3A,DNMT3A was less affected by metal ions,and Cu²⁺induced the expression of DNMT1.The effects on MTs were as follows Metal ions upregulated the m RNA expression of MT1 and MT2 to various degrees,and the expression level of MT2 was higher than that of MT1.The expression of MT3 showed little change,but it was also induced by metal ions.The effects onα-syn are as follows:All four metal ions induced the expression of SNCA,with Fe³⁺and Cd²⁺exerting the greatest effect.ROS was significantly increased in cells treated with metal ions.For the aggregation and toxicity ofα-syn,it was found that oligomericα-syn formed after 30 h of incubation,and fibers formed after 96 h of incubation.The oligomers were the most toxic species to nerve cells at the same concentration.This study aimed at the toxicity of common metal ions to nerve cells and explored the effects of metal ions on the expression levels of DNMTs,MTs,and SNCA using quantitative Real-time PCR.In addition,the level of ROS produced by metal ions was assessed to reveal the effects of metal ions on nerve cells or their DNA in aspects of damage and epigenetic change.These findings will contribute to the basic research of heavy metal toxicology and provide experimental support for the assessment of environmental risks and the treatment of diseases caused by heavy metals.