| Objective:Lead (Pb) neurotoxicity has received renewed interest with the growing evidencethat Pb contributes to Alzheimer’s disease (AD). However, the mechanism is not clear.In our previous study of long-term Pb exposure in vivo, a brain iron (Fe) overloadinduced by Pb was observed in elderly rats. It is well known that brain Fe overload isthe mechanism of AD. Therefore, we have reason to believe that Pb induced Feoverload and caused neurodegenerative disease. However, the mechanism or route ofPb-induced Fe overload is unknown. In the current study, the PC12rat adrenergicneural tumor pheochromocytoma cell line was selected as an in vitro Pb exposuremodel, the the effect of Pb exposure on cellular Fe homeostasis in PC12cells wasdetermined as well as the role of the iron exporter ferroportin1(FP1) and the ironimporter divalent metal transporter1(DMT1) were investigated.Methods:(1) The PC12cells were cultured at37°C in a humidified (5%CO2) incubator.After being exposed to0,0.01,0.1,1,10or100μM Pb for24,48or72h withdiffering Fe exposure (normal,10μM Fe2+or100μM DFO), the cellular Fe level wasdetected by the Perl’s straining and the cell viability was determined by the MTTassay.(2) After being exposed to0,0.01,0.1,1,10or100μM Pb for24,48or72h,the DMT1and FP1mRNA levels in PC12cells were examined by reversetranscriptase polymerase chain reaction assay (RT-PCR) and the correspondingprotein levels were analyzed by western blotting (WB).(3) The PC12cells were transfected with the pFP1-EGFP-N1plasmid andpEGFP-N1vector using the liposomal transient transfection assays, then evaluatedthe transfection effect of FP1by observing the expression of green fluorescent proteinGFP-positive cells and immunofluorescence (IF) detection.(4) After transfection, the PC12cells were exposed to10μΜ Pb for24h. UsingPerl’s staining and iron fluorescence dectection to determine the celluar Fe levels. Results:(1) Pb exposure induced celluar Fe increase of PC12cell: Perl’s staining showedthat Pb exposure could increase the intracellular Fe concentration in a time-andconcentration-dependent manner in PC12cells. Higher Fe supplement (10μM Fe2+)increased Pb induced Fe accumulation.(2) Pb exposure induced the viability decrease of PC12cell: MTT assay showedthat there were no significant changes in cell viability when PC12cells were exposedto varying concentrations of Pb in different Fe conditions for24h (P>0.05). From48to72h, the viability of PC12cells decreased in a concentration-dependent mannerwith the Pb concentration. Besides, higher Fe exposure aggravated the Pb inducedcytotoxicity.(3) Pb exposure influenced the DMT1expression: RT-PCR the DMT1(-IRE)mRNAlevels increased significantly compared with the control only when exposed to100μM Pb (P<0.05), and the DMT1(+IRE) mRNA levels increased in a time-andconcentration-dependent manner when the PC12cells were exposed to0.1-100μM Pbfor24h (P<0.05). The DMT1(+IRE) mRNA levels decreased in a time-andconcentration-dependent manner when exposed to Pb for48and72h.Western blottingfound that the DMT1protein levels increased in a time-and concentration-dependentmanner when the PC12cells were exposed to Pb for24h. However, the DMT1protein levels decreased in a time-and concentration-dependent manner whenexposed to Pb for72h.(4) Pb exposure decreased FP1expression: RT-PCR showed that the FP1mRNAlevels was significantly downregulated only when exposed to Pb for72h(P<0.05).Western blotting found that the FP1protein levels decreased in a time-andconcentration-dependent manner when the PC12cells were exposed to Pb for24h,48and72h.(5) FP1overexpression attenuated the Fe accumulation induced by Pb exposure:PC12cells were transfected with the pFP1-EGFP-N1plasmid and pEGFP-N1vectorusing liposomal transient transfection assays, and the FPN1protein levels detected byimmunofluorescent analysis increased3.3fold (P<0.05) in pFP1-EGFP-N1-transfected cells compared to the vector control. After being transfected, the PC12 cells were exposed to10μM Pb for24h. Perl’s staining found that the cellular Felevel decreased to0.4fold (P<0.05) in pFP1-EGFP-N1cells compared to pEGFP-N1vector control-transfected cells. And the fluorescence reverse quenching decreasedto0.33fold (P<0.05) in the FP1-transfected cells compared to the control cells.Conclusion:Pb exposure induce cellular Fe increase was due to downregulation of FP1expression, but not subiect to regulation of DMT1expression. Thus, FP1might be anovel target to prevent cellular Fe accumulation induced by Pb exposure andsubsequent neurotoxic consequences. |
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