Toxicity Of Zinc On MES23.5 Dopaminergic Cells And The Possible Mechanisms Involved In This Process

Parkinson's disease(PD) is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons originating in the substantia nigra pars compacta(SNpc) and terminating in the striatum(Str).This disorder is characterized symptomatically by bradykinesia,resting tremor and rigidity.The exact pathogenesis of PD has not been revealed yet.However,lots of researches suggest that multiple factors might be involved,such as heredity,environment,oxidative stress, inflammation and decline in growth factors levels.With the development of our life, people began to pay more attention to the role of environment which might be involved in the pathogenesis in PD.Among the multiple environmental factors,metal ions have been suggested to play an important role in the process of dopaminergic neurons death.Neurochemical,clinical and epidemiological studies have indicated that occupational exposure to zinc appears to be a risk factor for PD.Evidence demonstrated that zinc level increased in the substantial nigra(SN) of Parkinson's patients by 50-54%and in the SN of 6-hydroxydopamine (6-OHDA)-induced PD rats,indicating that there might be some relationship between zinc and the pathogenesis of PD.Moreover,intranigral infusion of zinc in rats induced apoptosis on dopaminergic neurons.Thus it is supposed that zinc may be involved in the pathogenesis of PD by apoptotic pathway.However,up to now,the ion mechanisms underlying zinc-induced apoptosis for dopaminergic neurons still remain unclear.In the present study,we selected MES23.5 dopaminergic cells as our model to investigate the toxicity of zinc and the possible mechanisms of zinc-induced apoptosis. First we observed whether zinc could cause cell apoptosis.MTT assay and Hoechst 33258 staining were used to detect cell viability and cell apoptosis.Then we investigated whether zinc could decrease the cell function.Western blots, semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR) and high performance liquid chromatography-electrochemical detection(HPLC-ECD) were used to detect tyrosine hydroxlase(TH) protein level,mRNA expression and DA contents in MES23.5 cells.Furthermore,to explore the possible mechanisms of cell apoptosis caused by zinc,we investigated the changes of potassium current density by Whole-cell patch clamp recording.Using RT-PCR and flow cytometry,we also detected the changes of Bcl-2,Bax mRNA expressions,the changes of mitochondrial transmembrane potential(△Ψm) and caspase-3 activity.Finally,TEA,the blocker of potassium channels was added to the culture medium to investigate whether it could inhibit the cell apoptosis caused by zinc.The results were as follows:1.Incubating MES23.5 cells with various concentration of zinc for 24 h could influence the cell viability.Higher concentration of zinc(70,80,and 90μmol/L) markedly decreased the cell viability(P＜0.01);whereas 30,40 and 50μmol/L zinc had no effect on cell viability(P＞0.05).Moreover,after 60μmol/L zinc treatment for 24 h,the cell viability slightly decreased but not to a statistical level.2.Incubating MES23.5 cells with various concentration of zinc for 24 h could induce cell apoptosis.The nuclei of 60 and 90μmol/L zinc treated cells appeared hypercondensed(brightly stained) and fragmentation of chromatin.The percentage of cell apoptosis after exposure to 60 and 90μmol/L zinc for 24 h was 13.38±0.6%and 22.67±1.14%,respectively(P＜0.01).There were no differences between 30μmol/L zinc and control groups(P＞0.05).3.After treatment with 60μmol/L zinc for 24 h,the TH protein levels was decreased significantly compared to the control(P＜0.01);similarly,the TH mRNA expression in MES23.5 cells were down-regulated and the DA content also decreased.4.MES23.5 cells expressed a well developed voltage-gated outward potassium current with delayed rectifier characteristics and TEA sensitive.The enhancement of potassium current density by zinc was time-dependent.It began to be increased at 4h after exposure to zinc,reached to the peak at 8h,but slowly decreased after that(16h)(P＜0.01).Add TEA to the medium could fully block the enhancement of potassium current density.5.After treatment with 60μmol/L zinc for 24 h,the Bcl-2 mRNA expression was decreased significantly compared to the control(P＜0.05).No changes were observed in Bax mRNA expression.The ratio of Bcl-2/Bax was decreased compared to the control(P＜0.05). 6.After treatment with 60μmol/L zinc for 24 h,the△Ψm in MES23.5 cells was decreased markedly,compared to the control(P＜0.01).7.The caspase-3 activity was not changed after treatment with zinc for 1-8 h(P＞0.05),increased at 16 h(P＜0.05),and markedly increased at 24 h(P＜0.01).8.The percentage of apoptotic cells was not changed after treatment with zinc for 1-8 h(P＞0.05);Remarkable increases were observed after treatment with zinc for 16 and 24 h(P＜0.01).9.TEA could partly inhibit the apoptosis.Incubating MES23.5 cells with 60μmol/L zinc for 24 h could markedly increase the caspase-3 activity,whereas co-incubation with zinc and TEA decreased the caspase-3 activity.By Hoechst 33258 staining assay,we also observed that co-incubation with zinc and TEA partly inhibited the percentage of apoptotic cells.In conclusion,zinc decreased the viability of MES23.5 cells,reduced the TH protein and mRNA expression,and down-regulated the DA contents.The probable mechanism underlying zinc-induced damage might be pro-apoptosis by enhancing TEA-sensitive potassium channel activity,further decreasing the Bcl-2 mRNA expression and the ratio of Bcl-2/Bax,increasing the caspase-3 activity,and ultimately leading to cell apoptosis.Potassium channel blocker,TEA,could partly inhibit the toxicity of zinc.