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Impact of fluoride exposure at different concentrations on preosteoblastic cells: Molecular, biochemical, and morphological insights.
| Tissue and Cell | Ferreira MKM, Sabino VG, Sousa JMM, Bittencourt LO, Ribeiro SB, Araújo AA, Barboza CAG, Souza-Rodrigues RD, Buzalaf MAR, Lima RR. | 96:103000.
Lipid peroxidation Bones/Joints Osteoblasts Oxidative Stress Cell Study

Abstract

Highlights

  • Fluoride in high concentrations modulates the viability of pre-osteoblastic cells.
  • Fluoride triggers oxidative stress, increasing levels of ROS and lipid peroxidation.
  • High levels of fluoride alter mitochondrial function and harm the phases of the cell cycle.
  • High fluoride triggers apoptosis/necrosis and irreversible changes in cell morphology.

Fluoride can harm various tissues depending on its concentration and exposure duration. While its effects on mineralized tissues like bones and teeth are known, few studies have explored its impact on preosteoblastic cells. This study examined the effects of fluoride on differentiating osteoblastic cells. M3CT3-E1 preosteoblastic cells were cultured for 24 h or 3, 5, or 7 days with fluoride concentrations of 1, 10, or 100 ug/mL. Cell viability, oxidative stress biomarkers, mitochondrial membrane potential, cell cycle distribution, apoptosis, and morphology were assessed. After statistical analysis, At 100 ug/mL, fluoride modulated cell viability in a dose- and time-dependent manner, increasing reactive oxygen species levels and lipid peroxidation (1.382 ± 0.163 vs. 0.826 ± 0.081; p = 0.0125). Oxidative stress markers such as superoxide dismutase (SOD) (4.008 ± 0.425 vs. 0.724 ± 0.474; p = 0.0025) and glutathione (GSH) (78.38 ± 4.506 vs. 45.65 ± 2.900; p = 0.0003) were also elevated. High fluoride concentrations impaired mitochondrial activity and disrupted the cell cycle, affecting the G0/G1, S, and G2/M phases. These changes caused irreversible damage, including apoptosis and alterations in cell morphology. High fluoride concentrations can significantly damage preosteoblastic cells, reducing viability, altering redox status, impairing mitochondrial function, and disrupting the cell cycle, leading to cell death. These findings underscore that fluoride toxicity is concentration-dependent and reinforce the safety of exposure to doses of fluoride such as those found in the optimally fluoridated water.

Introduction

It has already been established that excessive and prolonged exposure to high concentrations of fluoride can negatively impact health, especially mineralized tissues, causing skeletal and/or dental fluorosis (Yang et al., 2020). Thus, in addition to being an agent for promoting oral health and preventing caries (Yu and Yang, 2020), fluoride can also be an intoxicating agent (García and Borgnino, 2015). Because of this, guidelines and directives strongly debate the ideal form of exposure and the concentration that is sufficient for dentistry but does not cause damage to other tissues (Buzalaf, 2018, Toumba et al., 2019, Duffin et al., 2022).

In addition to naturally or artificially fluoridated water, other sources—such as food, tea, and some vegetables—can increase fluoride levels in the body. Fluoride is also released into the environment both naturally and through industrial activities (Singh et al., 2018, Zhu, 2016). This leads to its release into the atmosphere, which returns through rainfall (Walna et al., 2013), and is also capable of contaminating the soil (Gan et al., 2021), therefore affecting agricultural production (Singh et al., 2018). Previous studies have shown that fluoride is toxic to glial cells, modulating proteins and genes essential for maintaining cellular and energy metabolism, as well as MAP kinase (MAPK) signaling pathways (Puty et al., 2023). Furthermore, when investigating the potential interactions of fluoride with cells relevant to the oral cavity, such as ameloblast-like cells, fluoride has been shown to induce changes in gene expression, possibly associated with histone acetylation (Yamashita et al., 2024). Once ingested, fluoride has a tropism effect for mineralized tissues, with approximately 90 % of what is absorbed by the body being uptaken by bone (Buzalaf and Whitford, 2011, Mou et al., 2011). Considering its chemical properties, fluorine is part of the group of halogens, and is the most electronegative element of the periodic table. This means that its anion, fluoride, easily binds to cations, including Ca2+, one of the main components of hydroxyapatite [Ca10(PO4)6(OH)2], where it replaces hydroxyl ions, forming fluorapatite (Kimambo et al., 2019). Furthermore, at excessive doses, fluoride has the ability to modulate cellular metabolism and the redox system, reducing antioxidant capacity and promoting the establishment of oxidative stress, which is one of the main mechanisms of fluoride toxicity at high exposure levels (Sharma et al., 2017, Arab-Nozari et al., 2020, Nagendra et al., 2021).

Bone tissue homeostasis involves an active and constant remodeling mechanism, replacing old and damaged bone with new bone, in an uninterrupted cellular process, through an organized sequence of events, starting with the resorption of old bone by osteoclasts, followed by the deposition of new bone matrix by osteoblasts (Bolamperti et al., 2022, Wang et al., 2022). Specifically, skeletal fluorosis can be characterized as an imbalance in bone homeostasis caused by excessive exposure to fluoride that has been irremediably absorbed by bone tissue (Kabir et al., 2020). This pathology can be explained as a result of intracellular metabolic disorders of the main bone tissue model cells, osteoblasts and osteoclasts, through increased stimulation of the differentiation and activity of these cells (Jiang et al., 2020). On the other hand, imbalance in the redox system affects mitochondrial activity and membranes, inducing premature apoptotic processes in these cells (Gao et al., 2019, Tang et al., 2021, Avila-Rojas et al., 2022), which, depending on the exposure (concentration and time), can result in several changes, including osteoporosis, osteopenia, osteosclerosis, and calcification of ligaments (Qiao et al., 2021).

Therefore, given the crucial importance of preserving bone homeostasis and the relevance of fluoride in promoting oral health, it is essential to carry out a comprehensive investigation into the molecular, biochemical, and morphological effects of different concentrations of fluoride on bone cells that have not yet differentiated. In this way, the current study aims to clarify the effects of fluoride on this cell group, filling a gap in the literature.

Snippets:

Discussion

The current study investigated the effects of high concentrations of fluoride on one of the main bone cells, osteoblasts, without being fully differentiated, i.e., preosteoblasts, to verify the possible effects of fluoride on the activity, function, and morphology of these cells. Most of the damage occurred at the highest fluoride concentrations, affecting the viability, biochemical balance, proliferation, and even death of these cells via apoptosis. The damage was mostly found at the highest…

Conclusion

The current study demonstrated that exposure to high concentrations of fluoride (100 ug/mL) led to a decrease in the viability of preosteoblastic cells, disrupted redox balance, affecting mitochondrial functions, and induced cell cycle alterations, culminating in apoptosis and necrosis. However, such elevated fluoride levels are unlikely to be present in human plasma from regular consumption sources like water, beverages, and food…

ABSTRACT ONLINE AT
https://www.sciencedirect.com/science/article/abs/pii/S0040816625002800?via%3Dihub