Differential Susceptibility Of PC12 And BRL Cells And The Regulatory Role Of HIF-1? Signaling Pathway In Response To The Toxicity Of Methylmercury

Objective:MeHg is a widespread environmental pollutant,central nervous system is primary target of MeHg.MeHg also produces toxic damage to non-neural organs such as the liver.HIF-1?as a hypoxic regulator factor has been reported to play an important role in response to the toxicity of many chemicals and heavy metal,but the specific effect of HIF-1?has not been clearely reported.We used neuroal PC12 cells and hepatocyte BRL cells as the targets to explore the different sensitivities and study the effects of HIF-1?and its downstream proteins in the toxicity of MeHg.We also used different methods to upregulate the expression of HIF-1?to explain whether the role of HIF-1?in antagonizing is tissue-specific or not and explore more effective antagonists for different organ toxicity of MeHg.Methods:1.PC12 and BRL cells were exposed to different concentrations of MeHg?0,1,2.5,5,10?M?with different times?0,0.5,1,3,6 h?,the cell viability of PC12 and BRL was detected by MTT assay.The cell cytotoxicity of the two cell lines after exposed to MeHg was measured by LDH assay.The morphological changes were observed by optical microscope.The DCFH-DA method was used to detect the change of reactive oxygen species?ROS?.2.WB was used to detect the expression of HIF-1?and its downstream target proteins,GLUT-1,VEGF-A,EPO.The mRNA level of HIF-1?was detected by quantitative real-time PCR?qRT-PCR?.Pharmacology?2-MeOE2,10?M,0.5 h?was used to downregulating the expression of HIF-1?,WB was used to explore the expression of HIF-1?and its downstream proteins,MTT assay was used to detecte the cell viability to investigate the role of HIF-1?in MeHg toxicity.3.With the methods of upregulating of HIF-1?by genetics method?adenovirus overexpression?,WB was used to explore the expression of HIF-1?and its downstream proteins,MTT assay was used to detecte the cell viability to investigate the role of HIF-1?in MeHg toxicity.4.Prolinehydroxylase inhibitor?DHB,1 mM,6 h???CoCl₂,200?M,0.5 h?and proteasome inhibitor?MG132,1?M,24 h?were used to explore the possible mechanisms of MeHg downregulating the expression of HIF-1?and to explore the more effective antagonists for different organ toxicity of MeHg.Result:1.MeHg caused a time-and concentration-dependent decrease in cell viability in both cell types.The half-maximal inhibitory concentration values were determined for the MTT assay for cell-viability reduction were measured at varying intervals up to 6 h in the presence of MeHg at 5?M?PC12?or 10?M?BRL?.After 0.5 h incubation with 10?M MeHg,PC12 and BRL cell viability was decreased to 69.5±2.4%and 77.9±3.7%of controls,respectively?p<0.05?.Accordingly,0.5 h of treatment with10?M MeHg was used for subsequent experiments to explore the acute injury of MeHg.LDH release increased in a concentration-dependent manner and was significantly elevated relative to controls?p<0.05?in cells treated for 0.5 h with 2.5?M MeHg?PC12?or 10?M MeHg?BRL?.The levels of ROS in the two cells were also significantly increased after exposes to MeHg.2.WB results showed that MeHg treatment for 0.5 h significantly decreased the expression of HIF-1?in a concentration-dependent manner,at 2.5?M in PC12 cells and5?M in BRL cells.At 10?M MeHg,the protein level of HIF-1?decreased to¹2%and⁵4%of control cultures in PC12 and BRL cells,respectively.The protein levels of HIF-1?did not change significantly after MeHg treatment,whereas those of VEGF-A,GLUT-1 and EPO attenuated with increasing MeHg concentration.Measurement of HIF-1?mRNA using quantitative RT-PCR did not reveal significant alterations in mRNA levels in both MgHg-treated cells.2-MeOE2 inhibition of HIF-1?and target proteins enhanced MeHg-induced cytotoxicity in both PC12 and BRL cells further corroborating the cytoprotective role of HIF-1?.3.Genetic intervention was used to upregulating the expression HIF-1?.At a MOI of 100×10⁸ PFU/mL,HIF-1?levels increased compared with control values.Compared with MeHg alone,pretreatment with adenoviral Hif-1?before treatment with MeHg significantly increased the protein levels of HIF-1?and its downstream targets.Overexpression of HIF-1?also attenuated induced MeHg cytotoxicity in both cell lines.4.Pharmacological induction of HIF-1?and its targets by CoCl₂ protected BRL but not PC12 cells against MeHg-induced acute cytotoxicity.Pretreatment with the PHD inhibitor DHB could antagonize the toxicity of MeHg,especially in BRL cells.After pretreatment with the proteasome inhibitor?MG132,1?M,24 h?to upregulating the protein levels of HIF-1?could significantly antagonized the toxic effects of MeHg,especially in PC12 cells.Conclusion:Neuron-like PC12 cells are more sensitive to MeHg than non-neuronal BRL cells.MeHg decreased the protein levels of HIF-1?and its targets?GLUT-1,VEGF-A,EPO?in a concentration-dependent manner.HIF-1?was decreased at lower concentration of MeHg in PC12 cells could partly explaine the sensitively of PC12 cells towards the toxicity of MeHg.Hif-1?mRNA was detected with qRT-PCR,establishing the inability of MeHg to affect this gene at the transcription level and suggesting that its effects are secondary to the promotion of HIF-1?degradation via enhanced activity of PHD and proteasomal function.Pharmacological and genetic methods were used to upregulating the expression of HIF-1?which can antagonize the toxicity of MeHg in two cell lines,but the antagonism efficiency is different.The inhibitors of PHD could antagonize the toxicity of MeHg more significantly in BRL cells.MG132,as the proteasome inhibitor,also antagonize the toxicity of MeHg and more significantly in PC12 cells.On the one hand,it suggests that HIF-1?can serve as a general target to antagonize the toxicity of MeHg which provide a new direction for HIF-1?as a serological indicator for the diagnosis of MeHg poisoning.On the other hand,it provides a theoretical basis for selecting effective antagonists for MeHg induced toxicity of different systems.