Skip to main content

Roles of mitochondrial fission inhibition in developmental fluoride neurotoxicity: mechanisms of action in vitro and associations with cognition in rats and children

Abstract

Fluoride neurotoxicity is associated with mitochondrial disruption. Mitochondrial fission/fusion dynamics is crucial to maintain functional mitochondria, yet little is known about how fluoride perturbs this dynamics and whether such perturbation contributes to impaired neurodevelopment. Here in human neuroblastoma SH-SY5Y cells treated with sodium fluoride (NaF, 20, 40 and 60 mg/L), mitochondrial fission suppression exerted a central role in NaF-induced mitochondrial abnormalities and the resulting autophagy deficiency, apoptosis augmentation, and compromised neuronal survival. Mechanically, pharmacological inhibition of mitochondrial fission exacerbated NaF-induced mitochondrial defects and cell death through promoting apoptosis despite partial autophagy restoration. Conversely, genetic enhancement of mitochondrial fission alleviated NaF-produced detrimental mitochondrial and cellular outcomes by elevating autophagy and inhibiting apoptosis. Further suppressing autophagy was harmful, while blocking apoptosis was beneficial for cellular survival in this context. Consistently, using Sprague–Dawley rats developmentally exposed to NaF (10, 50, and 100 mg/L) from pre-pregnancy until 2 months of delivery to mimic human exposure, we showed that perinatal exposure to environmentally relevant levels of fluoride caused learning and memory impairments, accompanied by hippocampal mitochondrial morphological alterations manifested as fission suppression and fusion acceleration, along with defective autophagy, excessive apoptosis and neuronal loss. Intriguingly, the disturbed circulating levels of identified mitochondrial fission/fusion molecules were closely associated with intellectual loss in children under long-term environmental drinking water fluoride exposure. Collectively, our results suggest that mitochondrial fission inhibition induces mitochondrial abnormalities, triggering abnormal autophagy and apoptosis, thus contributing to neuronal death, and that the mitochondrial dynamics molecules may act as promising indicators for developmental fluoride neurotoxicity.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. Angmar-Mansson B, Whitford GM (1984) Enamel fluorosis related to plasma F levels in the rat. Caries Res 18(1):25–32

    CAS  PubMed  Article  Google Scholar 

  2. Barbier O, Arreola-Mendoza L, Del Razo LM (2010) Molecular mechanisms of fluoride toxicity. Chem Biol Interact 188(2):319–333

    CAS  PubMed  Article  Google Scholar 

  3. Bartos M, Gumilar F, Bras C, Gallegos CE, Giannuzzi L, Cancela LM, Minetti A (2015) Neurobehavioural effects of exposure to fluoride in the earliest stages of rat development. Physiol Behav 147:205–212

    CAS  PubMed  Article  Google Scholar 

  4. Bashash M, Thomas D, Hu H, Martinez-Mier EA, Sanchez BN, Basu N, Peterson KE, Ettinger AS, Wright R, Zhang Z, Liu Y, Schnaas L, Mercado-Garcia A, Tellez-Rojo MM, Hernandez-Avila M (2017) Prenatal fluoride exposure and cognitive outcomes in children at 4 and 6–12 years of age in Mexico. Environ Health Perspect 125(9):097017

    PubMed  PubMed Central  Article  Google Scholar 

  5. Choi AL, Sun G, Zhang Y, Grandjean P (2012) Developmental fluoride neurotoxicity: a systematic review and meta-analysis. Environ Health Perspect 120(10):1362–1368

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Choi AL, Zhang Y, Sun G, Bellinger DC, Wang K, Yang XJ, Li JS, Zheng Q, Fu Y, Grandjean P (2015) Association of lifetime exposure to fluoride and cognitive functions in Chinese children: a pilot study. Neurotoxicol Teratol 47:96–101

    CAS  PubMed  Article  Google Scholar 

  7. Corrado M, Scorrano L, Campello S (2012) Mitochondrial dynamics in cancer and neurodegenerative and neuroinflammatory diseases. Int J Cell Biol 2012(3):1–13

    Article  CAS  Google Scholar 

  8. Dec K, Lukomska A, Maciejewska D, Jakubczyk K, Baranowska-Bosiacka I, Chlubek D, Wasik A, Gutowska I (2017) The Influence of fluorine on the disturbances of homeostasis in the central nervous system. Biol Trace Elem Res 177(2):224–234

    CAS  PubMed  Article  Google Scholar 

  9. Du L, Wan C, Cao X, Liu J (2008) The effect of fluorine on the developing human brain. Fluoride 41:327–330

    CAS  Google Scholar 

  10. Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Cena V, Gallego C, Comella JX (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem 75(3):991–1003

    CAS  PubMed  Article  Google Scholar 

  11. Friedman JR, Nunnari J (2014) Mitochondrial form and function. Nature 505(7483):335–343

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Grandjean P, Landrigan PJ (2007) Developmental neurotoxicity of industrial chemicals. Lancet 369(9564):821–821

    Article  Google Scholar 

  13. Gui CZ, Ran LY, Li JP, Guan ZZ (2010) Changes of learning and memory ability and brain nicotinic receptors of rat offspring with coal burning fluorosis. Neurotoxicol Teratol 32(5):536–541

    CAS  PubMed  Article  Google Scholar 

  14. Guo X, Disatnik MH, Monbureau M, Shamloo M, Mochlyrosen D, Qi X (2013) Inhibition of mitochondrial fragmentation diminishes Huntington’s disease–associated neurodegeneration. J Clin Invest 123(12):5371–5388

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Hirzy JW, Connett P, Xiang Q, Spittle BJ, Kennedy DC (2016) Developmental neurotoxicity of fluoride: a quantitative risk analysis towards establishing a safe daily dose of fluoride for children. Fluoride 49(4):379–400

    CAS  Google Scholar 

  16. Ikeda Y, Shirakabe A, Maejima Y, Zhai P, Sciarretta S, Toli J, Nomura M, Mihara K, Egashira K, Ohishi M, Abdellatif M, Sadoshima J (2014) Endogenous Drp1 mediates mitochondrial autophagy and protects the heart against energy stress. Circ Res 116(2):264–278

    PubMed  Article  CAS  Google Scholar 

  17. Inoue-Yamauchi A, Oda H (2012) Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells. Biochem Biophys Res Commun 421(1):81–85

    CAS  PubMed  Article  Google Scholar 

  18. Izquierdo-Vega JA, Sanchez-Gutierrez M, Del Razo LM (2008) Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss. Toxicol Appl Pharmacol 230(3):352–357

    CAS  PubMed  Article  Google Scholar 

  19. Jiang C, Zhang S, Liu H, Guan Z, Zeng Q, Zhang C, Lei R, Xia T, Wang Z, Yang L, Chen Y, Wu X, Zhang X, Cui Y, Yu L, Wang A (2014) Low glucose utilization and neurodegenerative changes caused by sodium fluoride exposure in rat’s developmental brain. Neuromolecular Med 16(1):94–105

    CAS  PubMed  Article  Google Scholar 

  20. Kageyama Y, Zhang Z, Roda R, Fukaya M, Wakabayashi J, Wakabayashi N, Kensler TW, Reddy PH, Iijima M, Sesaki H (2012) Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage. J Cell Biol 197(4):535–551

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Kim J, Park BH, Lee JH, Park SK, Kim JH (2011) Cell type-specific alterations in the nucleus accumbens by repeated exposures to cocaine. Biol Psychiatry 69(11):1026–1034

    CAS  PubMed  Article  Google Scholar 

  22. Kim B, Kim JS, Yoon Y, Santiago MC, Brown MD, Park JY (2013) Inhibition of Drp1-dependent mitochondrial division impairs myogenic differentiation. Am J Physiol Regul Integr Comp Physiol 305(8):R927–R938

    CAS  PubMed  Article  Google Scholar 

  23. Liesa M, Shirihai OS (2013) Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell Metab 17(4):491–506

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Lin JR, Shen WL, Yan C, Gao PJ (2015) Downregulation of dynamin-related protein 1 contributes to impaired autophagic flux and angiogenic function in senescent endothelial cells. Arterioscler Thromb Vasc Biol 35(6):1413–1422

    CAS  PubMed  Article  Google Scholar 

  25. Liu HL, Lam LT, Zeng Q, Han SQ, Fu G, Hou CC (2009) Effects of drinking water with high iodine concentration on the intelligence of children in Tianjin, China. J Public Health (Oxf) 31(1):32–38

    Article  Google Scholar 

  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2– ∆∆CT Method. Methods 25(4):402–408

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Lou DD, Guan ZZ, Liu YJ, Liu YF, Zhang KL, Pan JG, Pei JJ (2013) The influence of chronic fluorosis on mitochondrial dynamics morphology and distribution in cortical neurons of the rat brain. Arch Toxicol 87(3):449–457

    CAS  PubMed  Article  Google Scholar 

  28. Manczak M, Calkins MJ, Reddy PH (2011) Impaired mitochondrial dynamics and abnormal interaction of amyloid beta with mitochondrial protein Drp1 in neurons from patients with Alzheimer’s disease: implications for neuronal damage. Hum Mol Genet 20(13):2495–2509

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Messer JS (2017) The cellular autophagy/apoptosis checkpoint during inflammation. Cell Mol Life Sci 74(7):1281–1296

    CAS  PubMed  Article  Google Scholar 

  30. Mishra P, Chan DC (2014) Mitochondrial dynamics and inheritance during cell division, development and disease. Nat Rev Mol Cell Biol 15(10):634–646

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11(1):47–60

    CAS  PubMed  Article  Google Scholar 

  32. Mumtaz N, Pandey G, Labhasetwar PK (2015) Global fluoride occurrence, available technologies for fluoride removal, and electrolytic defluoridation: a review. Crit Rev Env Sci Tec 45(21):2357–2389

    CAS  Article  Google Scholar 

  33. Niu Q, Chen J, Xia T, Li P, Zhou G, Xu C, Zhao Q, Dong L, Zhang S, Wang A (2018) Excessive ER stress and the resulting autophagic flux dysfunction contribute to fluoride-induced neurotoxicity. Environ Pollut 233:889–899

    CAS  PubMed  Article  Google Scholar 

  34. Nunnari J, Suomalainen A (2012) Mitochondria: in sickness and in health. Cell 148(6):1145–1159

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Parone PA, James DI, Da Cruz S, Mattenberger Y, Donze O, Barja F, Martinou JC (2006) Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis. Mol Cell Biol 26(20):7397–7408

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Peng K, Yang L, Wang J, Ye F, Dan G, Zhao Y, Cai Y, Cui Z, Ao L, Liu J, Zou Z, Sai Y, Cao J (2017) The interaction of mitochondrial biogenesis and fission/fusion mediated by PGC-1alpha regulates rotenone-induced dopaminergic neurotoxicity. Mol Neurobiol 54(5):3783–3797

    CAS  PubMed  Article  Google Scholar 

  37. Priault M, Salin B, Schaeffer J, Vallette FM, di Rago JP, Martinou JC (2005) Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast. Cell Death Differ 12(12):1613–1621

    CAS  PubMed  Article  Google Scholar 

  38. Rana A, Oliveira MP, Khamoui AV, Aparicio R, Rera M, Rossiter HB, Walker DW (2017) Promoting Drp1-mediated mitochondrial fission in midlife prolongs healthy lifespan of Drosophila melanogaster. Nat Commun 8(1):1–14

    CAS  Article  Google Scholar 

  39. Sarkar C, Pal S, Das N, Dinda B (2014) Ameliorative effects of oleanolic acid on fluoride induced metabolic and oxidative dysfunctions in rat brain: experimental and biochemical studies. Food Chem Toxicol 66(4):224–236

    CAS  PubMed  Article  Google Scholar 

  40. Sheridan C, Martin SJ (2010) Mitochondrial fission/fusion dynamics and apoptosis. Mitochondrion 10(6):640–648

    CAS  PubMed  Article  Google Scholar 

  41. Suzuki M, Bartlett JD (2014) Sirtuin1 and autophagy protect cells from fluoride-induced cell stress. Biochim Biophys Acta 1842(2):245–255

    CAS  PubMed  Article  Google Scholar 

  42. Teng Y, Zhang J, Zhang Z, Feng J (2017) The effect of chronic fluorosis on calcium ions and CaMKIIα, and c-fos expression in the Rat hippocampus. Biol Trace Elem Res 182(6):295–302

    PubMed  Google Scholar 

  43. Tu W, Zhang Q, Liu Y, Han L, Wang Q, Chen P, Zhang S, Wang A, Zhou X (2018) Fluoride induces apoptosis via inhibiting SIRT1 activity to activate mitochondrial p53 pathway in human neuroblastoma SH-SY5Y cells. Toxicol Appl Pharmacol 347:60–69

    CAS  PubMed  Article  Google Scholar 

  44. Westermann B (2010) Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 11(12):872–884

    CAS  PubMed  Article  Google Scholar 

  45. Yan N, Liu Y, Liu S, Cao S, Wang F, Wang Z, Xi S (2016) Fluoride-induced neuron apoptosis and expressions of inflammatory factors by activating microglia in Rat brain. Mol Neurobiol 53(7):4449–4460

    CAS  PubMed  Article  Google Scholar 

  46. Youle RJ, Karbowski M (2005) Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol 6(8):657–663

    CAS  PubMed  Article  Google Scholar 

  47. Youle RJ, van der Bliek AM (2012) Mitochondrial fission, fusion, and stress. Science 337(6098):1062–1065

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Yu X, Chen J, Li Y, Liu H, Hou C, Zeng Q, Cui Y, Zhao L, Li P, Zhou Z (2018) Threshold effects of moderately excessive fluoride exposure on children’s health: a potential association between dental fluorosis and loss of excellent intelligence. Environ Int 118:116–124

    CAS  PubMed  Article  Google Scholar 

  49. Zhang Z, Kageyama Y, Sesaki H (2012) Mitochondrial division prevents neurodegeneration. Autophagy 8(10):1531–1533

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Zhang BB, Wang DG, Guo FF, Xuan C (2015a) Mitochondrial membrane potential and reactive oxygen species in cancer stem cells. Fam Cancer 14(1):19–23

    CAS  PubMed  Article  Google Scholar 

  51. Zhang S, Zhang X, Liu H, Qu W, Guan Z, Zeng Q, Jiang C, Gao H, Zhang C, Lei R, Xia T, Wang Z, Yang L, Chen Y, Wu X, Cui Y, Yu L, Wang A (2015b) Modifying effect of COMT gene polymorphism and a predictive role for proteomics analysis in children’s intelligence in endemic fluorosis area in Tianjin, China. Toxicol Sci 144(2):238–245

    CAS  PubMed  Article  Google Scholar 

  52. Zhang S, Chen Y, Xue W, Hui G, Ma R, Jiang C, Gang K, Zhao G, Tao X, Zhang X (2016a) The pivotal role of Ca2+ homeostasis in PBDE-47-induced neuronal apoptosis. Mol Neurobiol 53(10):7078–7088

    CAS  PubMed  Article  Google Scholar 

  53. Zhang S, Niu Q, Gao H, Ma R, Lei R, Zhang C, Xia T, Li P, Xu C, Wang C, Chen J, Dong L, Zhao Q, Wang A (2016b) Excessive apoptosis and defective autophagy contribute to developmental testicular toxicity induced by fluoride. Environ Pollut 212:97–104

    CAS  PubMed  Article  Google Scholar 

  54. Zhang J, Zhu Y, Shi Y, Han Y, Liang C, Feng Z, Zheng H, Eng M, Wang J (2017) Fluoride-induced autophagy via the regulation of phosphorylation of mammalian targets of rapamycin in mice leydig cells. J Agric Food Chem 65(40):8966–8976

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

We would like to express our sincere thanks to all children donors who volunteered to participate in this study. We also sincerely thank the Tianjin Center for Disease Control and Prevention for its assistance for sample collection. This work was supported by grants from the State Key Program of National Natural Science of China (Grant No. 81430076), the National Natural Science Foundation of China (Grants No. 81502785 and No. 81773388) and the Fundamental Research Funds for the Central Universities (HUST 2016YXMS221 and HUST 2015ZDTD052).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Shun Zhang or Aiguo Wang.

Ethics declarations

Conflict of interest

Authors declared that there are no conflicts of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhao, Q., Niu, Q., Chen, J. et al. Roles of mitochondrial fission inhibition in developmental fluoride neurotoxicity: mechanisms of action in vitro and associations with cognition in rats and children. Arch Toxicol 93, 709–726 (2019). https://doi.org/10.1007/s00204-019-02390-0

Download citation

Keywords

  • Fluoride
  • Developmental neurotoxicity
  • Mitochondrial fission/fusion
  • Autophagy
  • Apoptosis