1. Introduction

Fluoride is widespread and unevenly distributed in the environment, and it can be rapidly absorbed into the body via water, food, and air (Johnston and Strobel, 2020). Fluoride consumption at the prescribed level is essential for human health, whereas excessive fluoride exposure is harmful to health. Groundwater, minerals, soil, household chemical products such as varnishes, gels, mouthwashes, and toothpaste are significant sources of fluoride exposure. Others include industrial emissions and pesticide residues. (Lacson et al., 2020; Wang et al., 2019). Indeed, fluoride exposure in drinking water is the leading cause of fluorosis, threatening both skeletal and non-skeletal organs such as the liver, kidney, testes, thyroid, and brain (Johnston and Strobel, 2020; Yadav et al., 2018). The neurological system is the primary target of fluoride damage in humans (Dec et al., 2017). Neurotoxicants cause more damage to the developing brain than adult brain (Dobbing, 1971).

Fluorine can penetrate both the placental barrier and the blood-brain barrier (BBB) and enter the fetal brain (Atlanta. Agency for Toxic Substances and Disease Registry ATSDR, 2003). Noteworthy, infants and children retain more absorbed fluoride than adults (O’Mullane et al., 2016; Avvannavar, 2007). Prolonged exposure leads to fluoride accumulation in the brain, and consequent nervous system damage (Su et al., 2021). Epidemiological cross-sectional and prospective studies have found that fluoride exposure can negatively impact children (Valdez et al., 2017; Yu et al., 2018). In in vivo trials, embryonic and lactational exposure to fluoride alters the neurological functions of rats, resulting in decreased learning and memory capacity (Xin et al., 2021; Cao et al., 2019). In vitro studies have also documented biochemical changes induced by fluoride in brain cells, such as lipid peroxidation and inflammatory responses (Gao et al., 2008; Goschorska et al., 2018). Despite emerging evidence that fluoride exposure causes neurological development damage, the detailed mechanism underlying fluoride-induced developmental neurotoxicity is still largely unclear.

Due to their nonrenewable nature, neurons are unable to mitigate the load of harmful substances accumulated in the cells. Therefore, autophagy plays a dominant role in sustaining neuronal homeostasis by rapidly removing such substances (Shaikh et al., 2021). Autophagy is classified based on the pathways of degradation substrates into autolysosomes as macroautophagy, microautophagy, and chaperone-mediated autophagy (Mizushima and Levine, 2020). Macroautophagy, commonly referred to as autophagy, is a key intracellular degradation process. (Jimenez-Moreno and Lane, 2020). The dynamic process of autophagy is known as autophagic flux and involves several basic steps: autophagosome biogenesis and maturation, fusion of autophagosomes with lysosomes, and degradation of autophagic substrates within lysosome (Nie et al., 2021). Autophagic flux blockage results in the accumulation of pathogenic and misfolded proteins, as well as damaged organelles resulting in neuronal damage that underlies various neurological disorders (Blumenreich et al., 2020). Fluoride triggers defective autophagy and causes excessive apoptosis in human neuroblastoma SH-SY5Y cells, thus resulting in neurotoxicity (Zhou et al., 2019). We previously found that sodium fluoride (NaF) causes autophagic flux blockage, apoptosis, and reduced viability in SH-SY5Y cells and the Sprague-Dawley (SD) rat hippocampus (Niu et al., 2018). Chloroquine (inhibitor of autophagic degradation) exacerbated, whereas rapamycin (autophagy agonist) attenuated the reduced viability of SH-SY5Y cells caused by NaF (Niu et al., 2018). Although autophagic flux blockage is associated with fluoride-induced developmental neurotoxicity, the underlying mechanisms remain unknown.

Lysosomes are important subcellular organelles that receive and degrade macromolecules through endocytosis and autophagy (Zhang et al., 2021). The degraded products are transported out of lysosomes via membrane trafficking for reuse or energy production (Rudnik and Damme, 2021). Lysosomal degradation capacity is mainly dependent on more than 60 active hydrolases in their lumen, which catalyze the hydrolysis process to breakdown biomolecules (Trivedi et al., 2020). An acidic environment (pH 4.5–5.0) within the lysosomal lumen is vital for lysosomal degradation capacity by keeping the optimal environment for soluble hydrolases (Yamamoto et al., 2021). Ion channels and proton pumps are paramount in sustaining an acidic environment during lysosomal acidification. Vacuolar adenosine triphosphatase (V-ATPase) is a pH-sensitive multisubunit proton transporter that establishes and maintains an acidic environment within the lysosome by using the energy of ATP hydrolysis to pump H+ into the lysosome (Liu et al., 2021). Changes in V-ATPase expression and activity and abnormal lysosomal pH are associated with various neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases (Colacurcio and Nixon, 2016). However, the involvement of abnormal lysosomal pH in neurological damage induced by fluoride and the role and mechanism of V-ATPase in regulating lysosomal pH has not been clarified.

Therefore, we aimed to determine the role and mechanism of the lysosomal pH abnormalities that cause autophagic flux blockage in developmental fluoride neurotoxicity, particularly focusing on the involvement of V-ATPase in regulating lysosomal pH in neurons. Rats were given fluoridated drinking water ad libitum from the time of gestation, through delivery until the neonatal offspring reached the age of six months (simulating human exposure during a critical period of neurological development), and an in vitro model of NaF-treated SH-SY5Y cells, a cell line widely used to study developmental neurotoxicity. We explored the targets and molecular mechanism of fluoride-induced developmental neurotoxicity from the perspective of abnormal lysosomal pH leading to autophagic flux blockage. We also aimed to provide theoretical and scientific foundations for the prevention and treatment of fluorosis.