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Fluoride Exposure Provokes Mitochondria-Mediated Apoptosis and Increases Mitophagy in Osteocytes via Increasing ROS Production

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Abstract

Fluoride is a persistent environmental pollutant, and its excessive intake causes skeletal and dental fluorosis. However, few studies focused on the effects of fluoride on osteocytes, making up over 95% of all bone cells. This study aimed to investigate the effect of fluoride on osteocytes in vitro, as well as explore the underlying mechanisms. CCK-8, LDH assay, fluorescent probes, flow cytometry, and western blotting were performed to examine cell viability, apoptosis, mitochondria changes, reactive oxygen species (ROS) and mitochondrial ROS (mtROS), and protein expressions. Results showed that sodium fluoride (NaF) exposure (4, 8 mmol/L) for 24 h inhibited the cell viability of osteocytes MLO-Y4 and promoted G0/G1 phase arrest and increased cell apoptosis. NaF treatment remarkably caused mitochondria damage, loss of MMP, ATP decrease, Cyto c release, and Bax/Bcl-2 ratio increase and elevated the activity of caspase-9 and caspase-3. Furthermore, NaF significantly upregulated the expressions of LC-3II, PINK1, and Parkin and increased autophagy flux and the accumulation of acidic vacuoles, while the p62 level was downregulated. In addition, NaF exposure triggered the production of intracellular ROS and mtROS and increased malondialdehyde (MDA); but superoxide dismutase (SOD) activity and glutathione (GSH) content were decreased. The scavenger N-acetyl-L-cysteine (NAC) significantly reversed NaF-induced apoptosis and mitophagy, suggesting that ROS is responsible for the mitochondrial-mediated apoptosis and mitophagy induced by NaF exposure. These findings provide in vitro evidence that apoptosis and mitophagy are cellular mechanisms for the toxic effect of fluoride on osteocytes, thereby suggesting the potential role of osteocytes in skeletal and dental fluorosis.

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The data of this study will be made available on reasonable request.

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Funding

This work was supported by the Natural Science Foundation of Zhejiang Province (LY21H060001), the National Natural Science Foundation of China (30900301), and the National Undergraduate Innovation and Entrepreneurship Training Program (202110349006).

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  1. Yun Zhang and Fanhe Dong contributed equally to this work.

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  1. College of Medicine, Shaoxing University, Huancheng West Road 508, Shaoxing, 312000, People’s Republic of China

    Yun Zhang, Fanhe Dong, Zihan Wang, Bingbing Xu, Tao Zhang, Qiqi Wang & Qiao Lin

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Contributions

Yun Zhang conceived and designed the experiments. Fanhe Dong, Zihan Wang, Bingbing Xu, and Qiqi Wang carried out the experiments. Tao Zhang and Qiao Lin processed the data. Yun Zhang and Fanhe Dong wrote the paper. The final manuscript was read and approved by all authors.

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Zhang, Y., Dong, F., Wang, Z. et al. Fluoride Exposure Provokes Mitochondria-Mediated Apoptosis and Increases Mitophagy in Osteocytes via Increasing ROS Production. Biol Trace Elem Res 201, 3994–4007 (2023). https://doi.org/10.1007/s12011-022-03450-w

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