Fluoride Action Network



Accumulating evidences have confirmed that liver is one of the more severely damaged organs during chronic fluorosis. However, the detail mechanism is unclear to data. At present, the objective of this study was to investigate the relationship between down-regulation of IKBKG gene expression and hepatocyte senescence induced by sodium fluoride (NaF).


Chronic fluorosis rats and NaF-exposure human liver L02 cells were reproduced the model of hepatocyte senescence in vivo and in vitro. The mRNA and protein levels of p16, p21 and IKBKG, the IL-8 level were determined. The role of IKBKG in fluoride-induced senescence of hepatocytes was explored by knock down in hepatocytes in vivo and in vitro.


The number of senescence-positive cells in rat liver tissues was increased as well as the level of IL-8 and the expression levels of p16, p21 and IKBKG in fluoride exposure to rat depending on the fluoride concentration. The similar results were obtained in NaF treated liver L02 cells, and the number of cells that stagnated in the G2 phase increased significantly. Further, our results confirmed that decreasing the expression of IKBKG in hepatocytes could reduce fluoride-induced hepatocyte senescence and the changes of senescence-related indicators both in vivo and in vitro.


These results indicated that the elevated expression of IKBKG was positive relation with the fluoride-induced senescence in hepatocytes, suggesting the hepatocyte senescence might have a special relationship with fluoride-caused liver damage. Because of the present results limitation, the mechanism of fluoride induced senescence in hepatocytes should be concentrated in the future in detail, especially the novel targets for fluoride induced liver injury.


cell senescence


4. Discussion

Cellular senescence is a pathophysiologica l phase in which the cell cycle is arrested and cells stop growing. In recent years, cellular senescence has been widely highlighted as one of the pathogenesis mechanisms that regulate various diseases such as tumors, degenerative diseases, and chronic diseases [20]. The reason and mechanism of cell senescence are not fully understood. Oxidative stress is the main external cause of cell senescence that is more generally accepted. Cells undergoing senescence have characteristic features, displaying a large and flat morphology: an increase in SA-?-gal, DNA damage, and a stable cell cycle arrest executed by interplaying between the RB and p53 tumor suppressor pathways (often accumulate the p16INK4a, p15INK4b, and p21Cip1/Waf1 cyclin-dependent kinase inhibitors that engage the RB pathway or mediate p53 effects accordingly) [21, 22]. Previous studies have shown that there are senescent cells in the liver of patients with non-alcoholic fatty liver (NAFLD) and cirrhosis. The increase in the number of senescent cells can promote the occurrence of NAFLD, but the underlying mechanism is not fully elucidated [23, 24].

It has been verified that the increased oxidative stress induced by fluoride was associated with DNA fragmentation in rat hepatocytes, which was one of the main mechanisms leading to liver damage. As oxidative stress is one of the main causes of cell senescence, these studies suggested that fluorosis may cause liver damage by inducing cell senescence. However, there are very few studies on the toxic effects of fluoride by inducing cell senescence. The present study investigated the effects of fluoride on hepatocyte senescence through in vitro and in vivo experiments.

The results showed that fluoride could induce cell senescence by ?-galactosidase staining on frozen sections of liver tissues of chronic fluorosis rats and L02 cells treated with different concentrations of NaF. With the increase of fluoride concentration, the SA-?-gal-positive cells were increased obviously, suggesting that the degree of senescence was positively correlated with the concentration of fluoride. Concerning the levels of p16 and p21, the hallmarks of cell senescence, it was found that the expression levels of p16 and p21 were increased in a concentration-dependent manner in fluorosis rats’ liver and NaF treated L02 cells, which further confirmed that fluoride could induce hepatocyte senescence. As p16 was the main tumor suppressor, which could induce cell senescence and cycle arrest. After exposure NaF to L02 cell, the cell cycle was arrested in the G2 phase by flow cytometry. At the same time, the level of IL-8 in the liver tissue of fluorosis rats and culture medium of fluoride-treated L02 cells were significantly increased. Taken together, our results confirmed that fluoride could induce hepatocyte senescence, which might play an important role in the fluoride induced liver damage.

It is clear now that SASP components actively participate in the senescence process. For example, key SASP factors (IL-6 and IL-8) act in an autocrine feedback loop to reinforce the senescence growth arrest. Nuclear factor-?B (NF-?B) is a master regulator of SASP and is used to further probe the impact of SASP biology on the senescence program [25]. A large number of studies suggested that NF-?B signaling was highly activated in cellular senescence [15]. NF-?B gene expression was also enhanced in primary rat hippocampal neurons by fluoride treatment in a dose-dependent manner, which meant fluoride might regulate cell senescence by activating NF-?B signaling [26]. The NF-?B system is an evolutionarily conserved signaling pathway, which can be triggered by not only immune activation but also diverse external/internal danger signals associated with senescence and even the aging process (oxidative and genotoxic stresses). IKBKG is generally called NEMO, important regulatory component of IKK complex linked upstream to genotoxic signals and IL-1 and TNF receptor mediated signaling [16]. Recent studies have shown that IKBKG protein plays a major role in the activation of NF-?B signals caused by DNA damage, which suggests that IKBKG may be involved in the process of cell senescence [17, 27]. Studies have found that IKBKG can also regulate liver diseases at different functional levels [28] [29]. This study further explored the role of IKBKG in fluoride-induced hepatocyte senescence, hoping to provide a theoretical basis for the pathogenesis of fluorosis.

The results showed that the expression of IKBKG increased in liver tissues of chronic fluorosis rats and liver L02 cells exposured with NaF in a concentration-dependent manner. The results suggested that fluoride might induce hepatocyte senescence via up-regulating the expression of IKBKG. To further verify the role of IKBKG in fluoride-induced hepatocyte senescence, tail vein injection of AAV-IKBKG and transfected sh-IKBKG to L02 cells were adopted to knock down the level of IKBKG. The results showed that compared with the fluorosis rat or L02 cells treated with fluoride, the SA-?-gal-positive cells were decreased, the expression levels of P16 and P21 decreased, the IL-8 level decreased in rat liver or L02 cells treated with NaF and down-regulation of IKBKG, and the number of L02 cells arrested in the G2 phase was also reduced.

In conclusion, the chronic fluorosis rats and human L02 cells exposed with NaF as to evaluate whether fluoride could induce hepatocyte senescence and its potential regulated mechanism. The results indicated that fluoride might cause liver damage by inducing cell senescence, and its mechanism might be related to the up-regulation of IKBKG expression in hepatocytes. This research might shed more light on the underlying pathogenesis of fluorosis, the senescence is complex pathophysiological process, IKBKG just as a pathological factors and further researches should be designed to fully elucidate the regulation signal pathway of IKBKG in the role of cell senescence in the toxicity of fluoride.


[1] A.P. Daiwile, S. Sivanesan, P. Tarale, P.K. Naoghare, A. Bafana, D. Parmar, K. Kannan, Role of fluoride induced histone trimethylation in development of skeletal fluorosis, Environ Toxicol Pharmacol 57 (2018) 159-165.

[2] R.W. Dharmaratne, Exploring the role of excess fluoride in chronic kidney disease: A review, Hum Exp Toxicol 38(3) (2019) 269-279.

[3] X.X. Zeng, J. Deng, J. Xiang, Y.T. Dong, K. Cao, X.H. Liu, D. Chen, L.Y. Ran, Y. Yang, Z.Z. Guan, Protections against toxicity in the brains of rat with chronic fluorosis and primary neurons Journal Pre-proof exposed to fluoride by resveratrol involves nicotinic acetylcholine receptors, J Trace Elem Med Biol 60 (2020) 126475.

[4] E. Perumal, V. Paul, V. Govindarajan, L. Panneerselvam, A brief review on experimental fluorosis, Toxicol Lett 223(2) (2013) 236-51.

[5] M.J. Regulski, Cellular Senescence: What, Why, and How, Wounds 29(6) (2017) 168-174.

[6] A. Bernadotte, V.M. Mikhelson, I.M. Spivak, Markers of cellular senescence. Telomere shortening as a marker of cellular senescence, Aging (Albany NY) 8(1) (2016) 3-11.

[7] A. Hernandez-Segura, J. Nehme, M. Demaria, Hallmarks of Cellular Senescence, Trends Cell Biol 28(6) (2018) 436-453.

[8] S. Lopes-Paciencia, E. Saint-Germain, M.C. Rowell, A.F. Ruiz, P. Kalegari, G. Ferbeyre, The senescence-associated secretory phenotype and its regulation, Cytokine 117 (2019) 15-22.

[9] J. Janikiewicz, J. Szymanski, D. Malinska, P. Patalas-Krawczyk, B. Michalska, J. Duszynski, C. Giorgi, M. Bonora, A. Dobrzyn, M.R. Wieckowski, Mitochondria-associated membranes in aging and senescence: structure, function, and dynamics, Cell Death Dis 9(3) (2018) 332.

[10] U. Efe, S. Dede, V. Yuksek, S. Cetin, Apoptotic and Oxidative Mechanisms in Liver and Kidney Tissues of Sheep with Fluorosis, Biol Trace Elem Res 199(1) (2021) 136-141.

[11] H.W. Wang, J. Liu, S.S. Wei, W.P. Zhao, S.Q. Zhu, B.H. Zhou, Mitochondrial respiratory chain damage and mitochondrial fusion disorder are involved in liver dysfunction of fluoride-induced mice, Chemosphere 241 (2020) 125099.

[12] Z. Zhang, B. Zhou, H. Wang, F. Wang, Y. Song, S. Liu, S. Xi, Maize purple plant pigment protects against fluoride-induced oxidative damage of liver and kidney in rats, Int J Environ Res Public Health 11(1) (2014) 1020-33.

[13] G.H. Song, F.B. Huang, J.P. Gao, M.L. Liu, W.B. Pang, W. Li, X.Y. Yan, M.J. Huo, X. Yang, Effects of Fluoride on DNA Damage and Caspase-Mediated Apoptosis in the Liver of Rats, Biol Trace Elem Res 166(2) (2015) 173-82.

[14] A.G. Wang, T. Xia, Q.L. Chu, M. Zhang, F. Liu, X.M. Chen, K.D. Yang, Effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocytes, Biomed Environ Sci 17(2) (2004) 217-22.

[15] J. Zhao, L. Zhang, A. Lu, Y. Han, D. Colangelo, C. Bukata, A. Scibetta, M.J. Yousefzadeh, X. Li, A.U. Gurkar, S.J. McGowan, L. Angelini, R. O’Kelly, H. Li, L. Corbo, T. Sano, H. Nick, E. Journal Pre-proof
Pola, S.P.S. Pilla, W.C. Ladiges, N. Vo, J. Huard, L.J. Niedernhofer, P.D. Robbins, ATM is a key driver of NF-kappaB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging, Aging (Albany NY) 12(6) (2020) 4688-4710.

[16] A.S. Shifera, Protein-protein interactions involving IKKgamma (NEMO) that promote the activation of NF-kappaB, J Cell Physiol 223(3) (2010) 558-61.

[17] A. Salminen, A. Kauppinen, K. Kaarniranta, Emerging role of NF-kappaB signaling in the induction of senescence-associated secretory phenotype (SASP), Cell Signal 24(4) (2012) 835-45.

[18] C.X. Wu, Y.H. Wang, Y. Li, Z.Z. Guan, X.L. Qi, Changes of DNA repair gene methylation in blood of chronic fluorosis patients and rats, J Trace Elem Med Biol 50 (2018) 223-228.

[19] X.L. Qi, K. Ou-Yang, J.M. Ren, C.X. Wu, Y. Xiao, Y. Li, Z.Z. Guan, Preventing expression of the nicotinic receptor subunit alpha7 in SH-SY5Y cells with interference RNA indicates that this receptor may protect against the neurotoxicity of Abeta, Neurochem Res 38(5) (2013) 943-50.

[20] S. He, N.E. Sharpless, Senescence in Health and Disease, Cell 169(6) (2017) 1000-1011.

[21] D.J. Baker, B.G. Childs, M. Durik, M.E. Wijers, C.J. Sieben, J. Zhong, R.A. Saltness, K.B. Jeganathan, G.C. Verzosa, A. Pezeshki, K. Khazaie, J.D. Miller, J.M. van Deursen, Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan, Nature 530(7589) (2016) 184-9.

[22] M. Pinto, A.M. Pickrell, X. Wang, S.R. Bacman, A. Yu, A. Hida, L.M. Dillon, P.D. Morton, T.R. Malek, S.L. Williams, C.T. Moraes, Transient mitochondrial DNA double strand breaks in mice cause accelerated aging phenotypes in a ROS-dependent but p53/p21-independent manner, Cell Death Differ 24(2) (2017) 288-299.

[23] M. Ogrodnik, S. Miwa, T. Tchkonia, D. Tiniakos, C.L. Wilson, A. Lahat, C.P. Day, A. Burt, A. Palmer, Q.M. Anstee, S.N. Grellscheid, J.H.J. Hoeijmakers, S. Barnhoorn, D.A. Mann, T.G. Bird, W.P. Vermeij, J.L. Kirkland, J.F. Passos, T. von Zglinicki, D. Jurk, Cellular senescence drives age-dependent hepatic steatosis, Nat Commun 8 (2017) 15691.

[24] A.M. Papatheodoridi, L. Chrysavgis, M. Koutsilieris, A. Chatzigeorgiou, The Role of Senescence in the Development of Nonalcoholic Fatty Liver Disease and Progression to Nonalcoholic Steatohepatitis, Hepatology 71(1) (2020) 363-374.

[25] Y. Chien, C. Scuoppo, X. Wang, X. Fang, B. Balgley, J.E. Bolden, P. Premsrirut, W. Luo, A. Chicas, C.S. Lee, S.C. Kogan, S.W. Lowe, Control of the senescence-associated secretory phenotype by NF-kappaB promotes senescence and enhances chemosensitivity, Genes Dev 25(20) Journal Pre-proof
(2011) 2125-36.

[26] M. Zhang, A. Wang, T. Xia, P. He, Effects of fluoride on DNA damage, S-phase cell-cycle arrest and the expression of NF-kappaB in primary cultured rat hippocampal neurons, Toxicol Lett 179(1) (2008) 1-5.

[27] J. Fu, D. Huang, F. Yuan, N. Xie, Q. Li, X. Sun, X. Zhou, G. Li, T. Tong, Y. Zhang, TRAF-interacting protein with forkhead-associated domain (TIFA) transduces DNA damage-induced activation of NF-kappaB, J Biol Chem 293(19) (2018) 7268-7280.

[28] T. Luedde, N. Beraza, V. Kotsikoris, G. van Loo, A. Nenci, R. De Vos, T. Roskams, C. Trautwein, M. Pasparakis, Deletion of NEMO/IKKgamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma, Cancer Cell 11(2) (2007) 119-32.

[29] H. Pereira, A.S. Dionizio, T.T. Araujo, M.D.S. Fernandes, F.G. Iano, M.A.R. Buzalaf, Proposed mechanism for understanding the dose- and time-dependency of the effects of fluoride in the liver, Toxicol Appl Pharmacol 358 (2018) 68-75.