The pathogenesis of endemic fluorosis: Research progress in the last 5 years

Abstract Fluorine is one of the trace elements necessary for health. It has many physiological functions, and participates in normal metabolism. However, fluorine has paradoxical effects on the body. Many studies have shown that tissues and organs of humans and animals appear to suffer different degrees of damage after long‐term direct or indirect exposure to more fluoride than required to meet the physiological demand. Although the aetiology of endemic fluorosis is clear, its specific pathogenesis is inconclusive. In the past 5 years, many researchers have conducted in‐depth studies into the pathogenesis of endemic fluorosis. Research in the areas of fluoride‐induced stress pathways, signalling pathways and apoptosis has provided further extensive knowledge at the molecular and genetic level. In this article, we summarize the main results.

Research, held in India in 2016, focused on the pathogenesis of fluorosis at the molecular and genetic level. It not only explores the molecular mechanism of fluoride action in bone tissue damage, but also the toxic effects of fluoride on non-skeletal tissues, such as the nervous system, cardiovascular system, liver, kidney, reproductive system, thyroid and the progeny effect of fluoride. Therefore, in this article, we will review research work from the last five years from the perspective of the effects of fluoride on different tissues and organs of the body. The key words for pathogenesis of endemic fluorosis in the last 5 years are shown in Figure 1.

| The pathogenesis of dental fluorosis
Dental fluorosis is the earliest specific clinical manifestation of endemic fluorosis. The pathological changes mainly occur in enamel, but dentin and cementum are also involved. In recent years, research into the pathogenesis mainly focused on fluoride interference with protein secretion of ameloblasts, resulting in amelogenin hydrolysis and removal delay, and differences in susceptibility to fluoride due to individual genotypes.

| Effects of fluoride on the signalling pathway of ameloblasts
Development of the tooth germ involves the process of ameloblast and odontoblast differentiation, leading to tooth hard tissue formation. Fluoride may cause disordered protein synthesis by affecting the function of the endoplasmic reticulum in ameloblasts. Recent studies found that fluoride can cause glucoseregulated protein78 up-regulation in ameloblasts, 2 and activate inositol-requiring kinase 1α and transcription factor 6 pathway in unfolded protein reactions, 3 thereby interfering with the secretory function of ameloblasts, resulting in the development of dental fluorosis. Oxidative stress is also related to the occurrence of dental fluorosis. Excessive fluoride can induce oxidative F I G U R E 1 The key words for pathogenesis of endemic fluorosis in the last 5 years. From the perspective of the effects of fluoride on different tissues and organs of the body, research work from the last five years has mainly focused on effects at the molecular and genetic levels, such as fluoride-induced stress pathways, signalling pathways and apoptosis stress in ameloblasts, 4 and the fluoride-induced reactive oxygen species (ROS) production causes oxidative damage to mitochondria and DNA, 5 leading to activation of SIRT1/autophagy via ROS-mediated JNK signalling. In addition, excessive fluoride can induce apoptosis of ameloblasts. The expression of and CHOP 2 in ameloblasts increases with the increase of fluoride concentration. It is speculated that apoptosis induced by the endoplasmic reticulum stress pathway may play a role in the occurrence of dental fluorosis. High-fluoride partially activates the FasL, 6 p-ERK and p-JNK signalling pathways 7 of ameloblasts, leading to increased expression of apoptotic genes, indicating that oxidative stress is closely associated with apoptosis in dental fluorosis.
Fluoride can also induce apoptosis by increasing the phagocytic activity of mature ameloblasts, and the Bcl-2 signalling pathway is involved in this process. 8 Furthermore, there is evidence that autophagy is involved in dental fluorosis. 4 One study 9 observed that fluoride increased expression of Beclin1, which is required for autophagosome formation, and decreased the expression of mTOR, an autophagy-related complex, indicating that autophagy is involved in dental fluorosis.

| Relationship between dental fluorosis and genetic polymorphism
In recent years, different individuals with genotypes susceptible to fluoride began to attract the attention of researchers. In the same population with the same fluoride exposure levels, there is a large difference in the extent of dental fluorosis between individuals, which may be related to genetic background and individual susceptibility to fluoride. The site Alu I CT + TT of the calcitonin receptor (CTR) is a susceptible genotype in populations with coal-burning type fluorosis. The polymorphism of the CTR gene may affect ion metabolism during tooth mineralization, resulting in differences in the occurrence of dental fluorosis at the same fluoride level. 10 Some studies have also shown a relationship between different loci of the same gene and dental fluorosis. Ten years ago, a case-control study showed a possible association between polymorphisms (Pvu II and Rsa I) in the COL1A2 gene and dental fluorosis in high fluoride-exposed populations. 11 However, recently, a cross-sectional study showed that the presence of an A/C polymorphism in the COL1A2 gene was not associated with the severity of dental fluorosis in drinking water-type fluorosis. 12 Another study showed that the polymorphisms in the enamel matrix genes AMBN, TFIP11, and TUFT1 were associated with dental fluorosis. 13   pathway is associated with fluoride-induced bone formation and bone resorption. 18 Another study shows that fluoride-induced osteoblast apoptosis may be regulated through ROS levels and mitochondrial membrane potentials. 19 In addition, fluoride can affect hormone levels, thereby contributing to active bone turnover. Studies have shown that increased secretion of parathyroid hormone (PTH) plays an important role in the pathogenesis of fluoride-induced osteogenesis and accelerated bone turnover, 20 and that PTH participates in the process of fluoride modulation of SOST/Sclerostin and RANKL expression. 21 Another study confirmed that insulin not only stimulates the activity of osteoblasts, but also enhances the role of fluoride in stimulating osteoblast activity. 22

| Effect of fluoride on osteoclasts
One of the pathological changes of skeletal fluorosis is the development of osteoporosis and osteopetrosis. In the development of skeletal fluorosis, the active function and absorption of osteoclasts plays an important role in the pathogenesis of osteoporosis. One report elucidated that the transforming growth factor (TGF) beta receptor 1/Smad3 pathway participated in the mechanism of biphasic modulation of osteoclast mode, regulated by fluoride. 23 RANKL is necessary for osteoclast formation.

| Effect of fluoride on chondrocytes and cellular matrix in bone and cartilage
Excessive fluoride interferes with bone metabolism partly by affecting the extracellular matrix of bone tissue. Osteoblasts are active but form immature woven bone in fluorosis. The structure and arrangement of collagen obviously differ from those of mature lamellar bone. Collagen is one of the important components of bone and cartilage tissue, with type I collagen being the main type of collagen in bone tissue, while in cartilage the main type is type II collagen. Excessive fluoride can cause metabolic abnormalities of bone and cartilage collagen. Studies have shown that excess fluoride can cause type I collagen disorder, leading to changes in the ultrastructure of bone tissue. 26 Another study showed that

| Effect of fluoride on the nervous system
Fluoride, like other halogen elements, can penetrate into the brain through the blood-brain barrier. In recent years, the action of fluoride on the nervous system has attracted attention. Exposure to high levels of fluoride in water was found to be significantly associated with reduced levels of intelligence in children. 33 Catechol-O-methyltransferase (COMT) gene polymorphisms may be related to the IQ of children exposed to fluoride in drinking water. 34 Another study showed that high fluoride exposure may be a risk factor for cognitive dysfunction of the elderly over the age of 60 in drinking water-type fluorosis areas where drinking water-type fluorosis is endemic. 35 Based on the results of the population survey, animal experiments in recent years have also focused on the study of the hippocampus which is responsible for learning and memory functions, since it is known that fluoride can damage the morphology of the hippocampus. 36,37 Fluoride also significantly changed hippocampal structural parameters of offspring after mothers were exposed to water fluorosis. 38 Excessive fluoride can lead to increased production of nitric oxide and JNK signalling pathway activation, 39

| Effects of fluoride on the cardiovascular system
The vascular wall is rich in collagen fibres, and collagen fibres are one of the major sites of action of fluoride. An epidemiological study in an endemic fluorosis area showed that excess fluoride intake has a certain connection with the incidence of adult hypertension, carotid atherosclerosis and the degree of disease lesions. The mechanism may be related to elevated levels of endothelin 1 in plasma caused by fluoride 46 and leads to inflammation of the oxidative stress system and endothelial activation. 47 The PI3K/AKT/eNOS pathway also plays a crucial role in the reduced expression of NO caused by excessive fluoride exposure. 48 Based on these results, we speculated that fluoride may be involved in cardiovascular disease by causing damage to the vascular wall and myocardial injury, but its specific mechanism needs further study. Excessive intake of fluoride has been shown to alter the renal function parameters, and oxidative stress 50,58 and the NF-κB signalling F I G U R E 2 Common alterations in fluorosis-apoptosis. Caspases, a family of cysteine proteases, are the central regulators of apoptosis. FasL can activate initiator caspases (Pro-caspase 8 and 10), then cleave and activate the effector caspases 3, 6 and 7, leading to apoptosis. Fluoride exposure can activate these signalling pathways and induce apoptosis. In addition, excessive fluoride induces stress pathways such as oxidative stress and endoplasmic reticulum stress, thus promoting apoptosis. Many signalling pathways such as Erk1/2 and PI3K/Akt induce anti-apoptotic Bcl-2 family members. These Bcl-2 family members protect the integrity of mitochondria, preventing Cytochrome C release and the subsequent activation of caspase-9. TNF-α may activate both pro-apoptotic and anti-apoptotic pathways. TNF-α can induce apoptosis by activating caspase 8 and 10, but can also inhibit apoptosis via NF-κB. Fluoride exposure can inhibit these survival signalling pathways can significantly increase sperm mitochondrial DNA copy number, and reduce nuclear DNA integrity. 63 A study indicated that fluoride exposure aggravates the degree of reproductive toxicity, such as sperm density, motility, viability and morphology as well as the testicular biochemical parameters in diabetic mice. 64 Fluorosis-induced spermatogenic cell apoptosis induced through the oxidative stress-mediated JNK and ERK signalling pathways, and antioxidants such as VE or lycopene can reduce these reactive oxygen species-induced toxic effects. 65 Another study provided

| Relationship between fluoride and diabetes
One report 75

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (81502762).

Wei Wei and Sun Dianjun analysed the references, Wei Wei
wrote the paper, Pang Shujuan classified the references.

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.