Effects of chronic fluorosis on the brain.

1.1. Overview

Endemic fluorosis (EF) is a chronic systemic disease characterized by skeletal fluorosis and dental fluorosis due to long-term excessive intake of fluoride, which affects residents living in fluoride-rich environments. A daily fluoride intake of more than 4 mg can cause chronic fluorosis. The disease is currently prevalent in more than 40 countries to varying degrees and poses a serious public health concern. Among Asian countries and areas, China and India are the two countries that are most widespread and severely struck by EF (Fluorides and oral health, 1994). In China, EF is mainly distributed in 14 provinces and municipalities including Guizhou, Shanxi, Gansu, Shandong, and Jiangsu. There are 275 endemic areas of fluorosis (composed of 2026 affected villages) in Jiangsu Province, and the prevalence of skeletal fluorosis is high in 9 counties (including Feng County, Peixian County, and Tongshan County), with Xuzhou City being the most EF-hit area in Jiangsu Province (Ren et al., 2021). There are three main types of EF in China: drinking water-related EF, coal-burning pollution-related EF, and tea-related EF, among which the drinking water-type is the most common. As stated in China’s Criteria for the Classification of Endemic Fluorosis-affected Areas (GB 17018–1997), the fluoride level in drinking water should not exceed 1 mg/L. According to the updated version of GB 17018–2011, the drinking water-type EF areas are defined as that with a fluoride level of > 1.2 mg/L in domestic water. In 1994, a World Health Organization expert committee recommended that the concentration of fluoride in drinking water should be 0.5–1.0 mg/L (Fluorides and oral health, 1994). The use of fluorinated water as a caries-preventing agent is considered to be an excellent representative of the active interventions for modern public health. After people fully elucidated the toxicokinetics of fluoride entering the brain, more and more studies found that fluoride has neurotoxicity. With the environmental pollution becoming more and more serious, the fluoride pollution of drinking water has become more and more common all over the world. In the train of the situation, the research on the mechanism of fluoride related nervous system damage and the development of prevention and treatment strategies have also become a hot research topic in many countries (especially developing countries). Since the immense and rapid progress of the effects of fluoride on the brain and its mechanisms, it is necessary to frequently review the literature to achieve an up-to-date overview of the subject. In addition, it is also the most noteworthy point that although the role of fluoride on dental and bone health is well known, its neuro-toxicological and suspected carcinogenic effects are debated (Grandjean, 2019, American Cancer Society, 2022). Based on above, we conducted this review.

1.2. Physicochemical properties and biological effects of fluorides

Fluorine is the 9th element in the periodic table and belongs to non-metals. As the most electronegative element, fluorine is extremely active and can form compounds with almost all other elements (including some rare gas elements), e.g., hydrogen fluoride, sodium fluoride, calcium fluoride, and ammonium fluoride, which are widely distributed in nature. Human body exposes to fluorides through the water, air, soil, and food. Drinking water is the most important source of fluorine exposure, followed by air and food. The main sources of fluorine in water are fluorine-containing minerals in rocks and sediments, such as fluorite, cryolite, fluoroapatite and hydroxyapatite, and clay minerals (Yadav et al., 2019). Fluorine is an essential trace element for metabolism and can enter all tissues. While being cumulative in the human body, it is mainly deposited in the teeth and bones. Meanwhile, fluoride in human body has a narrow safety range. A proper amount of fluoride can prevent dental caries and promote dental and skeletal health. However, inadequate or excess fluoride intake may undermine the normal physiological function of human body, causing damage not only to organs of the skeletal system but those of non-skeletal systems (such as the digestive system, endocrine system, nervous system, and reproductive system), resulting in acute or chronic fluorosis. Therefore, reducing fluoride in drinking water to the allowable level recommended by international or local organizations is of great significance for health (Baghal Asghari et al., 2017). Common methods include using alternative water sources, improving nutrition and defluoridating water (Lebrahimi et al., 2020, Ardekani et al., 2020). Among them, the research methods of water defluorination include precipitation/coagulation, reverse osmosis, electroflocculation, nanofiltration adsorption and ion exchange, etc (Yadav et al., 2019). Due to limited space and which go beyond the scope of this note, the detailed introduction of these methods will not be expanded in our review. Please refer to other relevant review for details (Solanki et al., 2022).

2.1. Effects of chronic fluorosis on fluoride concentration in brain tissue

In 1995, Mullenix et al. for the first time observed that fluoride concentration in some brain structures (e.g., hippocampus) elevated with the increase of fluoride content in the drinking water of rats and concluded that fluoride could cross the blood-brain barrier (BBB) and enter brain tissues (PJ et al., 1995). Niu et al. also found a 250-fold increase in fluoride levels in the brain of mice exposed to high-concentration fluoride (10 mg/L, 25 mg/L, and 50 mg/L) for 8 months comparing to the control mice (Niu et al., 2015a). Afterwards, numerous studies have shown that fluoride can cross the BBB either in ionic form or by binding to albumin. In addition, fluoride can be excreted through the drainage of cerebrospinal fluid to maintain the relative balance of fluoride concentration in the brain. However, long-term intake of high-concentration fluoride can cause fluoride accumulation in the brain, resulting in structural and functional damage to brain tissue. Animal experiments revealed that the fluoride concentration increased in the hippocampus, cerebral cortex, and midbrain of rats with chronic fluorosis, which was positively correlated with the dose of fluoride intake (CSReddy Nallagouni, 2017). Fluoride can penetrate the placental barrier and the BBB, and thus fluoride exposure during the fetal and neonatal periods is dangerous. Du et al. compared the fluoride levels in the brain tissues of 15 fetuses from an EF area that were aborted therapeutically at 5–8 months of gestational age with those of 16 fetuses at the same gestational age from a non-endemic area, and found that brain fluoride levels in fetuses from fluoridated areas (0.28 ± 0.140 ?g/g) were significantly higher than those from non-fluoridated areas (0.19 ± 0.06 ?g/g), confirming that fluoride can enter the fetus from maternal blood through the placental barrier during pregnancy and then accumulate in the fetal brain tissue after crossing the BBB and affect neurological development (Du, 1992).

2.2. Effects of chronic fluorosis on brain ultrastructure

The nervous system is sensitive to fluoride, and fluoride can accumulate in the brain after crossing the BBB, causing morphological and structural changes in animal and human brain tissues (Holland, 1979). The hippocampus is closely related to functions such as learning, memory and emotion. Niu et al. studied the ultrastructure of hippocampus in mice with long-term intake of high-fluoride drinking water and found significant pathological changes in the ultrastructure of neurons, synapses and myelin damage (Niu et al., 2018a). In addition, the mitochondria and endoplasmic reticulum (ER) in neurons were damaged, along with significantly increased intracellular lipofuscin. The neuronal synapses showed swelling and edema, and the mitochondria were severely degenerated. The electron density in synaptic gaps was increased, and the presynaptic and postsynaptic membranes were fused. Several studies have demonstrated that the accumulation of fluoride in the central nervous system (CNS) can cause morphological changes in neurons (especially changes in microtubule-associated protein expression and Nissl body concentration) as well as changes in neuronal activity and energy metabolism (Akinrinade et al., 2015, Lee et al., 2016, Niu et al., 2015b, Jiang et al., 2014a). Gui et al. investigated the hippocampal tissues of rats with coal-burning pollution-induced fluorosis and found that neuronal Nissl bodies in the hippocampal CA1 region were significantly reduced, indicating that the function of the neurons in synthesizing proteins was diminished (Gui et al., 2011). Significant neurodegenerative changes were found in the hippocampus, amygdala, cortex, and cerebellum of 10-week-old rats with chronic fluorosis, including a reduction in the size and amount of neurons in these regions, demyelination in the cerebral cortex and subcortical areas, and a decrease in the number of Purkinje cells in cerebellum. The altered cell morphologies included cell membrane folding, chromatin condensation, mitochondrial swelling, rough ER expansion, and a decrease in the number of synapses, seriously affecting synaptic function and plasticity (Bhatnagar et al., 2002).

2.3. Effects of chronic fluorosis on brain function

Brain tissues such as cerebral cortex, hippocampus, and cerebellum are closely related to learning, memory, emotion, and behavior, and their structural changes or damage can significantly affect the physiological functions of the brain (Fig. 1).

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Fig. 1. The damage caused by chronic fluorosis and the specific manifestation of central nervous system injury.

3.1. Fluorosis and oxidative stress

The oxidative stress theory has long been one of the hot spots in research on the pathogenesis of chronic fluorosis. Oxidative stress is caused by the disrupted balance between reactive oxygen species (ROS) synthesis and antioxidant enzyme activity. The term ROS encompasses superoxide anion radical (. O₂-), hydroxyl radical (·OH), and hydrogen peroxide (H₂O₂). Antioxidant enzymes basically include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). It has been observed from cellular experiments, in vivo animal experiments, and epidemiologic studies of chronic fluorosis that fluoride accumulation in the brain leads to increased ROS concentrations, reduced antioxidant enzyme activity, and increased lipid peroxidation. Increased oxidation of lipids and proteins was observed in the cerebral cortex, cerebellum, and medulla oblongata with access to 50 and 150?mg/L fluoride in drinking water (Dec et al., 2017).

In rats exposed to fluoride, the concentrations of fluoride were increased in the serum and brain, along with decreased SOD activity and increased lipid peroxidation (LPO) (Banala and Karnati, 2015). In addition, increased ROS synthesis, disturbed integrity, and altered mitochondrial membrane potential in neuronal mitochondria were observed in rats chronically drinking fluoride-contaminated water (13?mg/kg every 24?h). Antioxidants can protect cells from fluoride-induced lipid peroxidation. Fluoride can induce oxidative stress and regulate intracellular redox homeostasis (Hassan and Yousef, 2009, Mittal and Flora, 2006). Mitochondria are the main source of ROS. Excessive ROS production damages membrane phospholipids and induces lipid peroxidation, mitochondrial membrane depolarization, and apoptosis, which in turn leads to membrane damage, causing disruptions in cellular signaling pathways and triggering membrane lipid release and oxidation. Fluoride can directly interact with antioxidant enzymes and decrease their ability to scavenge free radicals. In hippocampal neuron cultures, after 48?h of incubation with fluoride ion (at concentrations of 40 and 80?mg/L), the ROS synthesis and malondialdehyde (MDA, a lipid peroxidation product and indicator in the organism) content were increased, along with the decrease in the activity of antioxidant enzymes SOD and GPx and in glutathione concentration (Zhang et al., 2007). The activity of CAT was increased in juvenile rats exposed to fluoride in drinking water (with concentrations of 30 and 100?mg/L), suggesting that ROS might have a protective effect against the deleterious activity of oxygen radicals in the organism (Guner et al., 2016). In adult rats exposed to fluoride at a dose of 20?mg/kg every 24?h, decreased glutathione concentration, antioxidant enzymes CAT, SOD, GPX, and glutathione reductase (GR) activities, as well as increased production of free radicals OH and NO were observed in brain tissues (Dec et al., 2017).

Therefore, the mechanism of brain injury caused by fluoride-induced oxidative stress can be summarized as follows: fluoride induces an increase in both ROS synthesis and lipid peroxidation as well as a decrease in antioxidant enzyme activities in neurons and glia, thus attenuating the body’s defense mechanisms (Zhang et al., 2015). In addition, oxidative stress can also activate different signaling pathways and initiate the apoptotic program. However, there are still many questions: is oxidative stress a causal factor or parallel phenomenon in the process of brain damage by fluorosis? Is it an initiating step or a triggering link? How do oxidative stress and other mechanisms interconnect and work together? Further research is warranted to answer these questions.

3.2. Apoptosis induced by fluorosis

Apoptosis, or the process of programmed cell death, is essential to maintain homeostasis. Our research showed increased level of apoptosis in PC-12 cells and BV2 microglia exposed to sodium fluoride (Zhang et al., 2017, Chen et al., 2017). The exact mechanism of fluoride-induced apoptosis remains unclear, however studies have shown that G protein-involved signaling pathways, calcium ions, p38 protein, MAP kinase, brain-derived neurotrophic factor (BDNF), and JNK pathway were involved in fluoride-induced apoptosis, and fluoride could induce the extrinsic, intrinsic, and ER-related apoptosis pathways (Dec et al., 2017, Ribeiro et al., 2017).

Extrinsic apoptotic pathway is also known as the death receptor pathway, in which death receptor such as Fas are stimulated by their ligands (e.g. Fas-L) to form a death-inducing signaling complex that activates caspase-8, which in turn triggers downstream caspase-3-mediated apoptosis. The Fas/Fas-L signaling may play a key role in fluoride-induced neural injury. Moreover, the expression of Fas, Fas-L, and caspases (?3 and ?8) were increased in human neuroblastoma (SH-SY5Y) cells treated with the high-concentration fluoride (Xu et al., 2011).

It is well known that TNF-? can activate the apoptosis pathway in neurons. One study found that the expression of TNF-? in the hippocampus of rats treated with fluoride (120?ppm) increased significantly, and microglia were activated to induce neuronal apoptosis and to increase the expression of inflammatory factors (Yan et al., 2016). NF-kB is a transcription factor involved in cell growth, cell cycle regulation, and cellular inflammatory processes. It showed that animals exposed to fluoride (30?mg/L), NF-kB expression was increased, which was correlated with elevated calcium and activated neuronal apoptosis through the extrinsic pathway (Zhang et al., 2011).

The intrinsic apoptotic pathway is also known as the mitochondrial/cytochrome C (Cyt c)-mediated pathway, in which the mitochondria are not only the center of the cellular respiratory chain and oxidative phosphorylation but also the apoptosis-regulation center. In animal experiments, Bcl-2 expression significantly decreased and caspase-12 (the initiator of the ER-related apoptotic pathway) level increased in brain cells of offspring rats exposed to fluoride during pregnancy. In addition, the number of apoptotic cells, the expression level of Cyt c, and the expression levels of caspase-9 and caspase-3 were significantly increased (Sun et al., 2017b). With increased fluoride concentrations, TUNEL assay detected an increase in apoptosis of rat neurons. Bax protein expression was increased and Bcl-2 protein expression was decreased in fluoride-treated rat brains (Yan et al., 2016). It is currently believed that excess intake of fluoride interferes with oxygen metabolism, causing an increase in ROS concentration in cells and alteration in intracellular mitochondrial outer membrane permeability, leading to increased Cyt c released from mitochondria into the cytoplasm, which further activates caspase-9 and caspase-3 and initiates neuronal apoptosis (Su et al., 2017). Another component of apoptotic signaling is the expression/regulation of pro- and anti-apoptotic genes, and abnormal Bcl-2/Bax expression has been shown to be involved in fluoride-induced apoptosis. Based on the close relationship between NF-?B and Bcl-2, Goschorska M et al. presented that fluoride ion in the brain increases apoptosis rate by activating the transcription factors (NF-?B, c-Jun) and proapoptotic proteins Bax, Fas, and P53. Simultaneously fluoride ion inhibits antiapoptotic proteins (Bcl-2 and Bcl-xl) synthesis (Goschorska et al., 2018).

ER plays a key role in protein folding and transport, and in maintaining calcium homeostasis in eukaryotic cells. When ER function is disturbed, the unfolded and misfolded proteins accumulate in the ER lumen, resulting in ER stress and activation of unfolded protein response (UPR), which is an evolutionarily conserved adaptive mechanism (Valenzuela et al., 2016). Prolonged or severe ER stress triggers apoptotic cell death through signaling cascades. ER stress is involved in neuronal disorders, leading to neurodegeneration and cognitive dysfunction (Hossain et al., 2015). ER stress plays an important role in fluoride-induced toxicity (Niu et al., 2018b), and both in vivo and in vitro experiments have shown that sodium fluoride exposure induced ER stress and activated UPR in the rat hippocampus. Excessive ER stress may lead to the failure of UPR-mediated adaptive mechanisms to compensate for the increased need for protein folding, thereby triggering the apoptotic program. Elevated levels of ER stress and associated apoptotic markers IRE1a, GRP78, caspase-12 and caspase-3 in SD rats exposed to sodium fluoride, suggesting the onset of neuronal apoptosis triggered by sodium fluoride. Park et al. found that 4-phenylbutyrate (4-PBA, a chemical chaperone that stabilizes unfolded proteins and promotes their correct folding) treatment decreased the expression of UPR markers in NaF-treated SH-SY5Y cells and also led to a significant decrease in ER stress-induced apoptosis in neurons (Park et al., 2012). Fluoride exposure increased the production of NO, which interferes with disulfide bond formation and leads to accumulation of misfolded proteins in ER, causing ER stress and ROS production (Bülbül and ?ireli, 2014). Therefore, the ER stress and associated apoptotic signaling are involved in fluoride-induced neurotoxicity, whereas fluoride induces apoptosis by interfering with signaling processes through multiple mechanisms.

3.3. Neurotransmitters and their receptors

Neurotransmitters are important regulators of brain function. Several studies have shown that fluoride accumulation in the nervous system affects the synthesis of neurotransmitters and the expression of their receptors. Acetylcholine is one of the neurotransmitters that are most closely related to cognition. It was found that the number of nAChR was significantly decreased in the brain of rats with chronic fluorosis and such decrease could be blocked by antioxidants, suggesting that fluoride-induced oxidative stress may be involved in nAChR deficiency in the brain (Spittle, 1994). Neuronal nicotinic receptors play a role in protecting nerves and modulating other transmitters, and are closely related to learning, memory, and cognitive functions. It has been found that chronic fluorosis can cause a decrease in neuronal nicotinic receptors, which occurs mainly at the post-transcriptional level. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are involved in glutamate synthesis, and their activities can be inhibited by excess fluoride (Niu et al., 2009, Yudkoff et al., 1991). Glutamate decarboxylase (GAD) catalyzes the conversion of the excitatory neurotransmitter glutamate to inhibitory neurotransmitter ?-aminobutyric acid (GABA). Fluoride increases GAD activity in the serum, hippocampus, and cerebral cortex of experimental animals, leading to a reduction in glutamate pools in the brain, which results in compromised synaptic transmission and cognitive impairment (Jiang et al., 2014b). Incubation of Bergmann glial cells isolated from rat cerebellum with fluoride caused a significant reduction in cellular lifespan, leading to impaired glutamate metabolism (Flores-Mendez et al., 2014). Glutamate is also involved in memory processes by stimulating specific ionotropic and metabotropic (mGluRs) receptors. Fluoride caused a significant decrease in mGluR5 expression in animal experiments (Jiang et al., 2014b). Increased levels of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) were detected in the striatum, hippocampus, and cerebral cortex of rats after 30 days of administration of drinking water containing NaF (100?mg/L) (Pereira et al., 2011). The concentration of 5-hydroxyindoleacetic acid, a metabolite of 5-HT, was found decreased in the brains of rats chronically exposed to fluoride, and it is speculated that fluoride increases the levels of 5-HT by reducing its metabolism (Cho et al., 1998). Reddy et al., however, had somehow different findings in animals given high-fluoride drinking water: increased levels of neurotransmitters including glutamate, epinephrine, and 5-HT and decreased levels of acetylcholine, norepinephrine, and dopamine in a dose-dependent manner (Reddy et al., 2021, Reddy et al., 2014). Therefore, further in-depth studies are needed to verify the effects of neurotransmitters on brain function in patients with chronic fluorosis and the mechanisms involved.

3.4. Interaction of fluoride and other elements

The main source of fluoride for human body is drinking water, in which fluoride can coexist with other elements, altering their kinetic and toxicological properties. It has been noted in the literature that aluminum-fluoride complexes can affect the nervous system and have neurotoxic properties, for instance, an animal experiment showed that long-term consumption of water containing aluminum fluoride and sodium fluoride can affect brain neurons and BBB in rats and cause morphological changes (Varner et al., 1998). Several studies have shown that concentrations of aluminum in drinking water 0.1–0.2?mg/L may increase the risk of AD (Kawahara, 2005). Fluoride may be a protective agent against AD in populations exposed to aluminum and fluoride ions (Colomina MTPeris-Sampedro F, 2017). In addition, both aluminum and fluoride ions are thought to exacerbate or cause neurodevelopmental disorders such as autism, attention deficit/hyperactivity disorder, dyslexia, and other cognitive impairments (Grandjean PLandrigan, 2014, Strunecka et al., 2018).

Arsenic is another element that have potential interaction with fluoride. Although the toxicity of arsenic and fluoride administered alone have been well known, the biological effects and mechanisms of their co-exposure remain controversial, and antagonistic, synergistic, and independent effects have been reported. Several epidemiological studies have indicated that co-exposure to fluoride and arsenic can increase the risk of lowered childhood IQ (Farooqi et al., 2017). Studies based on murine models identified that simultaneous administration of arsenic and fluoride cause less toxicity than given alone (Mittal and Flora, 2006) or exert antagonistic effects (Flora et al., 2009). Therefore, the toxic effects of co-exposure to arsenic and fluoride need to be clarified in future studies.