Activation of Fas Receptors, Caspase-8 and Caspase-3 by Fluoride Ions in Rat Erythrocytes in vitro

  • N. A. AgalakovaEmail author
  • T. I. Petrova
  • G. P. Gusev
Comparative and Ontogenic Biochemistry


The goal of the study was to demonstrate the ability of fluoride ions (F) to activate key components of the receptor-dependent apoptotic pathway, membrane Fas receptors, caspase-8 and caspase-3, in rat erythrocytes in vitro. Cells were incubated in the presence of increasing NaF concentrations (0.1–10 mM) for 1, 5 and 24 h. Caspase-8 and caspase-3 activities were assayed by flow cytometry, expression of Fas receptors by immunoblotting. It was found that the kinetics of stimulation of Fas receptors, caspases-8 and caspases-3 in rat erythrocytes by fluoride ions differs depending both on the fluoride concentration and exposure time. For instance, activation of caspases was observed as early as 1 h after incubation with fluoride, while treatment of erythrocytes with 5 mM NaF for 24 h increased the cell population with active caspases-8 and caspases-3 up to ca 15-16%. At the same time, expression of Fas receptors increased in a concentration-dependent manner only after 24 h of incubation with NaF. Thus, one of the mechanisms underlying premature death of rat erythrocytes induced by fluoride in vitro is the ability of the latter to stimulate messengers of the receptor-dependent apoptotic pathway. However, it is possible that caspase-8 and caspase-3 activation is, at least in part, independent of the membrane-associated mechanism of activation of Fas receptors.


rat erythrocytes fluoride ions caspases Fas receptors 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This study was carried out on the equipment of the Center for Collective Use at Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences.


  1. 1.
    Fluoride in drinking water, Bailey, K., Chilton, J., Dahi, E., Lennon, M., Jackson, P., and Fawell, J., Eds., WHO Press, Switzerland, 2006.Google Scholar
  2. 2.
    Pizzo, G., Piscopo, M.R., Pizzo, I., and Guiliana, G., Community water fluoridation and caries prevention: a critical review, Clin. Oral Invest., 2007, vol. 11, pp. 189–193.CrossRefGoogle Scholar
  3. 3.
    Peckham, S., Water fluoridation: a critical review of the physiological effects of ingestedfluoride as apub-lic health intervention, Sci. World J., 2014:293019. Scholar
  4. 4.
    Krishnamachari, K.A., Skeletal fluorosis in humans: a review of recent progress in the understanding of the disease, Prog. Food Nutr. Sci., 1986, vol. 10, pp. 279–314.Google Scholar
  5. 5.
    Bronckers, A.L., Lyaruu, D.M., and Den-Besten, P.K., The impact of fluoride on amelo-blasts and the mechanisms of enamel fluorosis, J. Dent. Res., 2009, vol. 88, pp. 877–893.CrossRefGoogle Scholar
  6. 6.
    Reddy, D.R., Neurology of endemic skeletal fluorosis, Neurol. India, 2009, vol. 57, pp. 7–12.CrossRefGoogle Scholar
  7. 7.
    Dec, K., Lukomska, A., Maciejewska, D., Jakubczyk, K., Baranowska-Bosiacka, I., Chlubek, D., Wasik, A., and Gutowska, I., The influence of fluorine on the disturbances of homeostasis in the central nervous system, Biol. Trace Elem. Res., 2017, vol. 177, pp. 224–234.CrossRefGoogle Scholar
  8. 8.
    Barbier, O., Arreola-Mendoza, L., and Del Razo, L.M., Molecular mechanisms of fluoride toxicity, Chem.-Biol. Interact, 2010, vol. 188, pp. 319–333.CrossRefGoogle Scholar
  9. 9.
    Agalakova, N.I. and Gusev, G.P., Molecular mechanisms of cytotoxicity and apoptosis induced by inorganic fluoride, ISRN Cell Biol, 2012. ID 403835. Scholar
  10. 10.
    Ribeiro, D.A., Cardoso, C.M., Yujra, V.Q., De Barros Viana, M., Aguiar, O. Jr., Pisani, L.P., and Oshima, C.T.F. induces apoptosis in mammalian cells: in vitro and in vivo studies, Anticancer Res., 2017, vol. 37, pp. 4767–4777.Google Scholar
  11. 11.
    Bratosin, D., Estaquier, J., Petit, F., Arnoult, D., Quantannens, B., Tissier, J.P., Slomianny, C., Sartiaux, C., Alonso, C., Huart, J. J., Montreuil, J., and Ameisen, J.C., Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria, Cell Death Differ., 2001, vol. 8, pp. 1143–1156.CrossRefGoogle Scholar
  12. 12.
    Berg, C.P., Engels, I.H., Rothbart, A., Lauber, K., Renz, A., Schlosser, S.F., Schulze-Osthoff, K., and Wesselborg, S., Human mature red blood cells express caspase-3 and caspase-8, but are devoid of mitochondrial regulators of apoptosis, Cell Death Differ., 2001, vol. 8, pp. 1197–1206.CrossRefGoogle Scholar
  13. 13.
    Mandal, D., Moira, P.K., and Basu, J., Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes, FEBS Lett., 2002, vol. 513, pp. 184–188.CrossRefGoogle Scholar
  14. 14.
    Mandal, D., Baudin-Creuza, V., Bhattacharyya, A., Pathak, S., Delaunay, J., Kundu, M., and Basu, J., Caspase 3-mediated proteolysis of the N-terminal cytoplasmic domain of the human ery-throid anion exchanger 1 (band 3), J. Biol. Chem., 2003, vol. 278, pp. 52551–52558.CrossRefGoogle Scholar
  15. 15.
    Mandal, D., Mazumder, A., Das, P., Kundu, M., and Basu, J., Fas, caspase 8-, and caspase- 3-de-pendent signaling regulates the activity of the ami-nophospholipid translocase and phosphatidylserine externalization in human erythrocytes, J. Biol. Chem., 2005, vol. 280, pp. 39460–39467.CrossRefGoogle Scholar
  16. 16.
    Bratosin, D., Tcacenco, L., Sidoroff, M., Cotoraci, C., Slomianny, C., Estaquier, J., and Montreuil, J., Active caspases-8 and -3 in circulating human erythrocytes purified on immobilized annexin-V: a cytometric demonstration, Cytometry, 2009, vol. 75A, pp. 236–244.CrossRefGoogle Scholar
  17. 17.
    Agalakova, N.I. and Gusev, G.P., Fluorideinduced death of rat erythrocytes in vitro Toxicol. In Vitro, 2011, vol. 25, pp. 1609–1618.CrossRefGoogle Scholar
  18. 18.
    Agalakova, N.I. and Gusev, G.P., Excessive fluoride consumption leads to accelerated death of erythrocytes and anemia in rats, Biol. Trace Elem. Res., 2013, vol. 153, pp. 340–349.CrossRefGoogle Scholar
  19. 19.
    Pietraforte, D., Matarrese, P., Straface, E., Gambardella, L., Metere, A., Scorza, G., Leto, T.L., Malorni, W., and Minetti, M., Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes, Free Radic. Biol. Med., 2007, vol. 42, pp. 202–214.CrossRefGoogle Scholar
  20. 20.
    Mukherjee, K., Chowdhury, S., Mondal, S., Mandal, C., Chandra, S., Bhadra, R.K., and Mandal, C., 9-O-Acetylated GD3 triggers programmed cell death in mature erythrocytes, Biochem. Bio-phys. Res. Commun., 2007, vol. 362, pp. 651–657.CrossRefGoogle Scholar
  21. 21.
    Kriebardis, A.G., Antonelou, M.H., Stamoulis, K.E., Economou-Petersen, E., Margaritis, L.H., and Papassideri, I.S., Storage-dependent remodeling of the red blood cell membrane is associated with increased immunoglobulin G binding, lipid raft rearrangement, and caspase activation, Transfusion, 2007, vol. 47, pp. 1212–1220.CrossRefGoogle Scholar
  22. 22.
    Agalakova, N.I. and Gusev, G.P., Fluoride induces oxidative stress and ATP depletion in the rat erythrocytes in vitro, Environ. Toxicol. Pharmacol., 2012, vol. 34, pp. 334–337.CrossRefGoogle Scholar
  23. 23.
    Burgstahler, A.W., Recent research on fluoride and oxidative stress, Fluoride, 2009, vol. 42, pp. 73–74.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  • N. A. Agalakova
    • 1
    Email author
  • T. I. Petrova
    • 1
  • G. P. Gusev
    • 1
  1. 1.Sechenov Institute of Evolutionary Physiology and BiochemistryRussian Academy of SciencesSt. PetersburgRussia

Personalised recommendations