Activation Of Mitochondrial P53 Apoptotic Pathway Is Involved In Fluoride-induced Neurotoxicity

The work in this paper consists of three parts:Part I Impairment of learning and memory ability of offspring rats developmentally exposed to fluoride and its underlying mechanismsObjective: There has been great concern about the neurotoxicity of fluoride since it can cross through the blood-brain barrier to accumulate in the brain.It has been suggested that apoptosis plays a vital role in neurotoxicity of fluoride but whether p53-mediated apoptotic pathway is involved in is still unclear.This study aimed to investigate the effects of developmental exposure to sodium fluoride(NaF)on the learning and memory abilities of offspring rats and its underlying mechanisms.Methods: Thirty adult male and female Sprague-Dawley(SD)rats were grouped randomly and exposed to 10 and 50 mg/L of NaF via drinking water.Rats given tap water(fluoride < 1 mg/L)were taken as control.After 2 months of exposure,male and female within each group randomly mated(1:1),and then the pregnant rats were separately caged and continuously exposed to NaF throughout gestation and lactation.At the end of weaning,pups were re-caged in groups and treated with the same level of fluoride as their parents until 2 months after birth.At the end of exposure,female offspring rats were randomly selected and marked,and then Morris water maze(MWM)test was conducted to evaluate the learning and memory abilities of female pups,each group with six pups.After MWM test,the offspring were sacrificed to collect hippocampus by cervical dislocation to measure the protein levels of SIRT1,p53(acetyl K379),p53 and apoptosis-related proteins(including PUMA,cytochrome c(cyto c)and cleaved caspase-3)by Western blot.Results: In the place navigation test,compared with control at the same time,the escape latency of rats exposed to 50 mg/L of NaF was significantly longer at the third and fourth days.The escape latency of the control and fluoride-treated groups was all decreased with time,and the escape latency of rats exposed to 50 mg/L of NaF was significantly longer than that of control.In the spatial probe test,the numbers of platform crossing and the percentages of distance of target quadrant and time of target quadrant were significantly decreased after exposure to 50 mg/L of NaF compared with those of control.At the same time,exposure to 50 mg/L of NaF upregulated the protein levels of cleaved caspase-3,p53,PUMA,cyto c,p53(acetyl K379)and SIRT1 in the hippocampus of offspring rats.Conclusion: These results indicated that fluoride impaired the learning and memory abilities of offspring rats after developmental exposure to NaF and p53-mediated apoptotic pathway was activated during the process,suggesting that apoptosis mediated by p53 apoptotic pathway may be involved in the regulation of neurodevelopmental toxicity induced by fluoride in offspring rats,and SIRT1 may act as an upstream signal molecule of p53 pathway to regulate the process.Part II Fluoride induces apoptosis via activation of mitochondrial p53 pathway in human neuroblastoma SH-SY5 Y cellsObjective: The objectives of the present study were to investigate whether fluoride induces apoptosis in human neuroblastoma SH-SY5 Y cells and the role of p53-mediated apoptotic pathway in the process.Methods: CCK-8 assay was first carried out to determine the concentrations of NaF to treat SH-SY5 Y cells.After treatment with 20,40 and 60 mg/L of NaF for 24 h,the morphological changes of apoptotic cells were observed by Hoechst 33342 staining and the percentages of cells undergoing apoptosis were examined by Annexin V–FITC/propidium iodide assay.At the end of exposure,the expression levels of p53 and apoptosis-related proteins including Bax,Bcl-2,PUMA,cyto c,cleaved caspase-3 and cleaved PARP were measured by Western blot;mitochondrial translocation of p53 and Bax and mitochondrial release of cyto c were detected by immunofluorescence.In addition,SH-SY5 Y cells were pretreated with 10 ?M pifithrin-?,a specific inhibitor of p53 transcriptional activity,for 1 h before exposure to 60 mg/L of NaF for 24 h,and then the percentages of apoptotic cells were measured by flow cytometry and the levels of apoptosis-related proteins were measured by western blot.Results: When cells were exposed to 40,50,60 and 80 mg/L of NaF for 24 h,cell viabilities were significantly reduced(84.84%,70.76%,52.31% and 34.31% as compared with that of untreated cells),therefore,20,40 and 60 mg/L of NaF were used to treat SH-SY5 Y cells.The results of Hoechst 33342 staining and flow cytometry and upregulation of levels of cleaved caspase-3 and cleaved PARP showed that apoptosis was induced after treatment with 40 and 60 mg/L of NaF for 24 h in human neuroblastoma SH-SY5 Y cells.Exposure to 60 mg/L of NaF for 24 h significantly upregulated the levels of p53 and apoptosis-related proteins including PUMA and cyto c,whereas downregulated Bcl-2 in SH-SY5 Y cells.Meanwhile,40 and 60 mg/L of NaF increased p53 nuclear translocation,20,40 and 60 mg/L of NaF increased mitochondrial translocation of Bax,and 60 mg/L of NaF induced cyto c release from mitochondria to cytoplasm in SH-SY5 Y cells.Fluoride-induced increases of apoptotic rates and the levels of apoptosis-related proteins were significantly attenuated by inhibiting p53 transcriptional activity with pifithrin-?.Conclusion: Our study demonstrates that fluoride induces apoptosis via activation of mitochondrial p53 pathway which depends on p53 transcriptional activity in SHSY5 Y cells.Thus,SIRT1 may be a promising target to protect against neurotoxicity induced by fluoride.Part ? SIRT1 overexpression and resveratrol suppress p53 acetylation to alleviate fluoride-induced apoptosis in SH-SY5 Y cellsObjective: The objectives of this part were to investigate the effect of sodium fluoride(NaF)on p53 deacetylase SIRT1 and the role of SIRT1 in apoptosis induced by NaF in human neuroblastoma SH-SY5 Y cells.Methods: After exposure to 20,40 and 60 mg/L of NaF,SIRT1 deacetylase activity was determined with SIRT1 Assay Kit,and the expression levels of SIRT1 and p53(acetyl K382)were measured by Western blot in SH-SY5 Y cells.Cells were transfected with adenovirus encoding SIRT1 for 24 h before exposure to 60 mg/L of NaF for 24 h.Apoptosis was measured by flow cytometry and the levels of cleaved PARP,cleaved caspase-3,p53(acetyl K382)and p53 were measured by Western blot.Cells were pretreated with SIRT1 activator resveratrol for 2 h and then exposed to 60 mg/L of NaF for additional 24 h,and then the levels of cleaved PARP,cleaved caspase-3,p53(acetyl K382)and p53 were measured by Western blot..Results: Sixty mg/L of NaF significantly inhibited the deacetylase activity of SIRT1,and 40 and 60 mg/L of NaF significantly increased the level of SIRT1 and p53(acetyl K382)in SH-SY5 Y cells.Apoptosis and upregulation of cleaved caspase-3,cleaved PARP and p53(acetyl K382)induced by fluoride could be ameliorated by overexpressing SIRT1 in SH-SY5 Y cells;upregulation of cleaved caspase-3,cleaved PARP p53(acetyl K382)and p53 induced by fluoride could be ameliorated by pretreatment of resveratrol.Conclusion: Our study demonstrates that fluoride upregulates the levels of p53(acetyl K382)via inhibiting the activity of SIRT1 deacetylase,thereby activating the p53-mediated mitochondrial apoptotic pathway and inducing apoptosis in SHSY5 Y cells.Overexpression of SIRT1 or resveratrol pretreatment partially reversed fluorideinduced apoptosis,indicating that SIRT1 acts as the upstream signal regulator of p53 in the regulation of NaF-induced apoptosis in SH-SY5 Y cells.Therefore,SIRT1 could be a potential target and for the prevention and treatment of fluorine-induced neurotoxicity.