Neurotoxic Effects of Fluoride in Endemic Skeletal Fluorosis and in Experimental Chronic Fluoride Toxicity

1. Professor of Biochemistry, KVG Medical College, Sullia. 2. Associate Professor of Biochemistry, Father Muller Medical College, Mangalore, Karnataka, India.

Abstract

Fluoride has a significant impact on the human health. Traces of fluoride are beneficial in preventing dental caries and osteoporosis, but the long-term intake of high levels of fluoride, mainly via drinking water, causes detrimental effects. High levels of fluoride cause dental and skeletal fluorosis and even the soft tissues are not spared from fluoride toxicity. Fluoride can cross the blood brain barrier and it can cause adverse effects on the brain cell architecture, metabolism, enzymes, the oxidantantioxidant status and on neurotransmitters and overall adverse effects on the mental functions. Fluoride induces the generation of free radicals, it increases lipid peroxidation, it impairs the antioxidants, it inhibits the key enzymes of the metabolic pathways, it impairs energy generation, and it inhibits protein synthesis. Excitoxicity, which has been proposed as the major mechanism in the neurotoxic manifestations of fluoride, needs a detailed and critical evaluation.

Brain, Excitotoxicity, Fluoride, Fluorosis, Neurotoxicity

Introduction
Fluorine is a highly electronegative trace element which is the 13th most abundant element in the earth’s crust (1). It is highly reactive and it occurs ubiquitously as fluorides in nature. Extensive research has been carried out on the chemistry and the biology of fluoride, and on its impact on the human health. Fluoride, in trace concentrations, is required to prevent dental caries, while the longterm consumption of excess fluoride, mainly via drinking water, leads to a spectrum of toxic manifestations which are referred to as fluorosis (1). Fluorosis, as a global public health problem, has been receiving wide and deserving attention in recent years. Fluoride has been shown to be toxic, not only to the skeletal tissues, but also to the non-skeletal tissues such as the brain, liver, pancreas, endocrines and the kidney (1),(2).

Fluoride can cross the blood brain barrier (3), and it can cause adverse effects on the brain cell architecture, metabolism, enzymes, the oxidant-antioxidant status and on neurotransmitters and overall adverse effects on the mental functions (4),(5),(6),(7). In this review, we have focussed on the effects of chronic fluoride toxicity on the structural and functional aspects of the brain in experimental animals and on the neurotoxicity of fluoride in endemic skeletal fluorosis.

The Environmenta l Sources and the Levels of Fluoride
Human beings are exposed to fluoride from various sources such as ground water, food ,air, drugs, cosmetics, tooth paste and dental applications (1),(8). The effects of fluorides on the human health stem largely from dissolved fluorides which are present in ground water and drinking water contributes to more than 60% of the total fluoride intake (1),(9). In rocks and soil, fluoride occurs in a wide variety of minerals, which include fluorspar (CaF2), cryolite (3 NaF AlF2), apatites ( 3 Ca2 [PO4] 2 Ca[F] 2), mica and hornblende (1). Volcanic rocks and salt deposits of marine origin also contain significant amounts of fluoride. The fluoride levels are found to be high in soft, alkaline, calcium-deficient waters, and in interior arid areas. A water fluoride level of 0.5-1.0 ppm is considered to be optimum, which is required to prevent dental caries, above which (>1.0 ppm ) clinical manifestations of dental and skeletal fluorosis are seen (1),(9).

The fluoride content of the top soil is increased by the addition of phosphate fertilizers, pesticides and irrigation water and by the deposition of gaseous, particulate emissions. The fluoride content of foods is significantly affected by the fluoride content in the water which is used for growing crops and for processing foods (1). Tea, marine fish, animal tissues and certain vegetations are found to contain highly significant amounts of fluoride. The fluorides in air originate from the dusts of fluoride containing soils, gaseous industrial wastes, the domestic burning of coal fires and from gases which are emitted in areas of volcanic activity. Many of the lakes of the Rift Valley system with high volcanic activity are found to have fluoride concentrations upto 2800 ppm (1),(10).

Fluoride and the Nervous System
1. The Neurological Manifestations of Skeletal Fluorosis
The most obvious and the earliest clinical manifestation of fluoride toxicity is dental fluorosis, which can develop in children, due to the intake of high levels of fluoride during the period of tooth development, and clinically, it is the marker of fluoride toxicity in the first six years of life (1). The fluoride accumulation in bones over a long time results in skeletal fluorosis and its early symptoms include stiffness and pain in the joints. Crippling skeletal fluorosis is associated with osteosclerosis, calcification of the tendons and the ligaments, and bone deformities (1),(11),(12),(13). Fluorosis exhibits neurological problems such as a tingling sensation in the fingers and toes, nervousness and depression. In the advanced stages of fluorosis, neurological manifestations such as paralysis of the limbs, vertigo, spasticity in the extremities, and impaired mental acuity, are observed in human beings (1),(12),(13).

2. Fluoride Accumulation in the Brain
Fluoride is known to enter the brain and the blood brain barrier fails to exclude fluoride from the nervous tissue (3). The transport of fluoride through the blood brain barrier is an active transport system which is similar to that of other halogens and ionic substances, and the normal CSF/blood fluoride ratio is less than 1.0 (14).

Mullenix et al., observed an accumulation of fluoride in the important regions of the brain, especially in the hippocampus (mean 0.993 ppm F at 125 ppm water fluoride during weanling), which was found to increase as the fluoride levels in the drinking water increased (4). The administration of NaF (20mg/kg, 14 days) caused an increased accumulation of fluoride in the brain of fluoride-treated rats (0.293 μg/g dry tissue; + 8%) , as compared to that in the control rats (7). Maternal exposure to 100 ppm fluoride in drinking water, resulted in fluoride accumulation (upto 2.14 μg/ g tissue) in the brain of young rats when compared to controls (0.27 – 0.64 μg/ g tissue) (15).

3. Fluoride and the Brain Cell Health
Fluoride is toxic to the brain and chronic fluoride intoxication causes abnormalities in the brain cell architecture. There are many reports of histological abnormalities in the brain tissue of animals which were exposed to high levels of fluoride directly or during the foetal and weanling stages via the mother (4),(5),(16),(17),(18). The passage of fluoride thorough the placenta of mothers with chronic fluorosis and its accumulation within the brain of the foetus is shown to have an adverse impact on brain development. Du et al. studied the brains of foetuses from endemic fluorosis areas, that were aborted therapeutically at the 5th–8th month of gestation, in comparison to the foetuses from non-endemic areas (16). They observed reductions in the mean volume of the neurons, the numerical density of the volume, volume density, and in the surface density of the mitochondria in the foetuses from endemic fluorosis areas (16). Offspring rats which were exposed to high fluoride (45 ppm) and low iodine for 90 days exhibited neurotoxic changes in brain, which were indicated by neurons with pycknosed nuclei, decreased Nissl substance, and elongated dendrites (19). The rats which were exposed to high fluoride and low iodine in water (100 ppm F, from one month to 20 months), showed considerable DNA damage of up to 92%, in the brain cells (20).

There have been attempts to assess the morphological changes in the various subregions of the brain in fluoride-treated animals. In a study which was done by our group, rats were exposed to 30 or 100 ppm fluoride (as NaF) in drinking water during their foetal (maternal exposure), weanling (maternal exposure), and post weaning stages of life until the age of ten weeks. Young rats which were exposed to 30 ppm fluoride did not show any notable alterations in the brain histology, whereas the rats which were exposed to 100 ppm fluoride showed significant neurodegenerative changes in the hippocampus, amygdala, motor cortex and the cerebellum. The changes included a decrease in the size and the number of the neurons in all the regions of brain, a decrease in the number of the Purkinje cells in the cerebellum, and signs of chromatolysis and gliosis in the motor cortex (5). Subcutaneous injections of sodium fluoride ( 5-50 mg/ml/kg/day for 15 weeks) in rabbits caused loss of the molecular layer and the glial cell layer in the brain tissues; chromatolysis and a ballooned appearance of the Purkinje neurons; vacuolization in the perikaryon; and the presence of spheroid bodies in the neuroplasm (18).

The chronic administration of fluoride as NaF or AlF3 in the drinking water (1 ppm F, 52 weeks) resulted in distinct morphological alterations in the brain of rats ; in the reduction of the neuronal density,chromatin clumping, enhanced protein staining, pyknosis and vacuolation. The presence of ghost-like cells in the left hemisphere was more prominent in the AlF3 –treated group than in the NaFtreated group (21).The fluoride toxicity also resulted in abnormal alterations in the cerebrovasculature (21).

4. Effect of Fluoride on the Metabolism in the Brain
Fluoride is an inhibitor of many enzymes which require divalent cations as cofactors. The enzymes which are inhibited by fluoride are concerned with energy metabolism, the metabolism of proteins and amino acids, and the scavenging of free radicals. Hence, fluoride is considered as a metabolic poison, and it is known to alter the metabolic pathways in tissues such as the liver, muscle and the brain (22),(23),(24),(25),(26). With regards to the effect of fluoride on the metabolism in the brain, studies have shown that fluoride (as NaF) impairs the activities of the enzymes which are concerned with the metabolism of lipids, proteins and nucleic acids, and transmission of the nerve impulse (7), (27), (28), (29), (30), (31). The exposure to fluoride (30 or 100 ppm for 3–7 months) caused changes in the membrane lipids in the brain (30). Sodium fluoride administration decreased the contents of phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl serine, and it increased the ubiquinone levels in the brain cell membrane (30),(31). Subcutaneous injections of sodium fluoride (5-50 mg/kg/day, 100 days) increased the contents of the total lipids, phospholipids and the triglycerides in the brain of rabbits (27). With regards to the effect of chronic fluoride intoxication on the enzymes which were concerned with energy metabolism, the fluoride administration (NaF at a dose of 20 mg/kg/day, 14 days) in mice reduced the activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) in the brain (7).

Fluoride inhibits the activities of the enzymes which are concerned with membrane function and nerve impulse transmission. Sodium fluoride reduced the activities of sodium-potassium ATPase, magnesium ATPase, calcium ATPase and acetyl cholinesterase in the brain (7). The administration of 5 or 50 ppm fluoride for 6 months to rats resulted in the decreased activities of acetylcholinesterase and butyrylcholinesterase in the brain tissue, and the effect was more pronounced with 5 ppm F (32). Contrary to this observation, maternal exposure to high levels of fluoride (5,15,50 ppm F) and continuation of the same treatment after birth till 80 days, resulted in an elevated activity of acetylcholinesterase in the cerebral synaptic membranes (33). Long et al., observed a significant reduction in the number of nicotinic acetylcholine receptors in the brain of rats which were exposed to sodium fluoride (34).

The inhibitory effect of fluoride on the synthesis of nucleic acids and protein in the brain, has been reported by few authors . The oral administration of sodium fluoride (NaF, 6 and 12 mg/kg body weight/day, for 30 days) caused a significant, dose-dependent reduction in the DNA, RNA, and the protein contents in the cerebral hemisphere, cerebellum, and in the medulla oblongata of the brain in mice. After the withdrawal of the treatment for 30 days, a partial but significant amelioration occurred (35). The fluoride intoxication in rabbits resulted in decreased contents of the total, soluble and the basic proteins, with an increase in the free amino acids in the brain (28).

5. Fluoride and the Antioxidant System of the Brain
Fluoride is known to induce oxidative stress and to impair the functioning of antioxidants in the brain. Various studies on experimental animals have observed an increased lipid peroxidation and decreased or increased levels of antioxidants in the brain tissue, on exposure to sodium fluoride. The fluoride administration (100 ppm, in drinking water for 3 months) in adult rats resulted in increased malondialdehyde (MDA), glutathione (GSH), glutathione S-transferase (GST), glutathione peroxidase (GSH-Px) and ascorbic acid in the brain (36). The administration of 5 or 50 ppm fluoride for 6 months to rats resulted in a decreased total antioxidant capacity and in increased MDA, in the brain (32). Krechniak and Inkielewicz observed a strong positive correlation of the brain fluoride content with the degree of oxidative stress; the brain fluoride content showed a positive correlation with MDA and the protein carbonyls,and a negative correlation with GSH and GSH-Px in the brain of rats which were subjected to chronic fluoride toxicity 37). Fluoride (at a dose of 20 mg/kg/day, 14 days) increased the activity of the prooxidant enzyme, xanthine oxidase and it reduced the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and GST in the brain of mice (7).

The maternal exposure to fluoride and fluoride intoxication at the early stages of life is shown to cause more pronounced effects on the oxidant-antioxidant status in the brain than the fluoride exposure at a later stage (adolescent/adult) of life (6),(15), (36), (38).Rats which were exposed to 100 ppm fluoride (as NaF) in drinking water during the last one week of intrauterine life (through maternal exposure) and which were then exposed up to ten weeks after birth, showed elevated MDA and GSH-Px levels, and decreased levels of total glutathione, GSH and ascorbic acid in the brain (6). The maternal exposure to 100 ppm fluoride in drinking water, resulted in increased lipid peroxidation, and in decreased levels of SOD, CAT, GSH-Px, GST and GSH in the brain of offspring rats (15). Basha et al., carried out a study to assess the effect of fluoride (100, 200 ppm in drinking water) on the oxidative stress in the brain, for three generations of rats ; they observed that the fluorideinduced increase in the lipid peroxidation and the decreases in the antioxidants were more pronounced with the second and third generations as compared to the first generation of the rats (38).

Fluoride, in combination with arsenic, is shown to have immense effect on the oxidant–antioxidant status in brain. Fluoride and arsenic singly or in combination caused increased levels of dehydroascorbic acid and lipid peroxides, and a decrease in the SOD, CAT, GSH-Px, GSH and the ascorbic acid levels in the brain of rats; the effect was more pronounced with a combination of fluoride and arsenic than with these compounds independently (39).

6. Fluoride and Mental Functions
There have been reports of decreased mental acuity and impaired mental functions, both in experimental animals which were subjected to fluoride toxicity, and in fluorotic children. In 1937, Kaj Roholm , the pioneer of fluoride research, published his findings on skeletal fluorosis with excessive tiredness, sleepiness, indisposition, headache, and giddiness in cryolite workers. The first case of skeletal fluorosis was reported in Andhra Pradesh in India in 1937 (40),(41). After this, many researchers have reported neurological manifestations such as tingling sensations, loss of the sensations of pain, temperature and touch, altered reflexes, and loss of the sphincter control in skeletal fluorosis patients (12),(40),(41). Many studies have shown that the children in fluorosis endemic areas were prone to mental retardation and that their IQ was low. Researchers, mainly from China, have reported that the IQ of children from high-fluoride, endemic fluorosis areas was significantly lower as compared to that of children from areas with less than 1 ppm fluoride in drinking water (42), (43), (44), (45), (46). Higher drinking water fluoride levels were found to be associated with higher rates of mental retardation (IQ <70) and borderline intelligence (IQ 70-79). The incidence of Down’s syndrome was shown to be higher among births to younger mothers in high-fluoride areas (46). High levels of fluoride in drinking water depressed the learning and memory abilities of the brain, and caused behavioural deficits even in rats and mice (4),(17),(32), (47),(48).

7. Mechanisms of the Neurotoxicity of Fluoride
7.1. Fluoride Induces Oxidative Stress
Fluoride exerts its toxic effects on the brain by multiple mechanisms; the primary phenomenon which is involved in the neurotoxicity of fluoride appears to be oxidative stress. Fluoride is known to induce the generation of free radicals and to result in the consequent oxidative stress (49),(50). Because of its high electronegativity, F–forms strong hydrogen bonds, especially with the –OH and –NH moieties in biomolecules, and it has a potent ability to form stable complexes with polyvalent metal cations like Al3+, Fe3+, and Mg2+ (49),(50).

We observed elevated MDA, GSH-Px and vitamin C levels, and reduced levels of GSH, uric acid and SOD in the blood of children with endemic skeletal fluorosis (51). While the elevated MDA levels which indicate increased lipid peroxidation, is a universal finding in the animal studies on oxidative stress in fluorosis, varied observations have been made on the changes in the antioxidants in blood and tissues, including the brain. The elevations in the antioxidants have been attributed to an adaptive response and the protective mechanism of the tissue to fluoride-induced oxidative stress, while the decreased levels of the antioxidants have been attributed to their depletion on combating the reactive oxygen species which are generated in chronic fluoride toxicity (15), (32), (36), (38), (49), (52). An in vitro study has shown that fluoride inhibits the antioxidant enzyme, SOD (53).

7.2. Fluoride Inhibits Enzymes and Alters Metabolic Pathways The biotoxicity of the fluoride ions results mainly from their inhibitory effect on the activity of many enzymes, mostly of ATP production and those which synthesize protein and DNA (22), (23), (24), (25), (26). This is associated with the high chemical activity of the F ion and its affinity to Ca2+ and Mg2+, which catalyze a number of enzymatic reactions. Few such enzymes include enolase, ATPases, cholinesterases, arginase, ACP, SDH, esterases, isocitrate dehydrogenase, phosphatases and aconitase (22), (23), (24), (25), (26). Inhibitions of the enzymes of glycolysis, the citric acid cycle and the electron transport chain have an adverse impact on the energy generation in the mitochondria. Studies have shown that the treatment with sodium fluoride decreases the activities of LDH, SDH, ALT, AST, CK, ATPases and actetyl cholinesterase in the brain of fluorideintoxicated animals (7),(32). These findings indicate that fluoride impairs the energy generation, the membrane function, the amino acid metabolism and the nerve impulse transmission. Fluoride is shown to alter the metabolism of glucose, lipids and amino acids in various tissues (27),(28),(54).

7.3. Fluoride Inhibits the Synthesis Of DNA, RNA And Protein, And Induces DNA Damage
In vitro studies have revealed that NaF affects the cellular protein synthesis by impairing the peptide chain initiation (55). The incubation of He La cells with NaF resulted in the inhibition of protein synthesis, disaggregation of the polyribosomes, accumulation of the 80 S ribosomes and in the decrease of the free ribosomal subunits. After the removal of NaF, the normal level of the free ribosomal subunits was restored at the expense of a random dissociation of the ribosomes (55). In vitro studies have also shown that fluoride inhibits the incorporation of amino acids into a polypeptide chain (55). The decreased levels of proteins, DNA and RNA, and the increased content of the free amino acids in the brain of animals which were exposed to chronic fluoride toxicity substantiated the findings of the in vitro studies (28),(35). Fluoride is also known to induce DNA damage and apoptosis in the brain (20).

7.4. Fluoride acts synergistically with aluminium and mediates the effects through the G-protein–coupled receptors
Aluminium and fluoride have a close association in drinking water sources, the environment and in cooking utensils, where they form aluminofluoride complexes. Aluminium reinforces fluoride’s stimulation of the G protein-coupled receptor-mediated signal transduction (56). Brain cells have their second messenger systems mediated through the activation of the G-protein coupled receptors, which are known to be influenced by fluoride alone or in combination with aluminium. These mechanisms include, activation of adenylate cyclase, increase in the levels of cyclic AMP; activation of protein kinase C and the subsequent activation of mitogenactivated protein kinase C(MAPK) and the phosphoinositide second messenger system (56), (57).

7.5. Excitotoxicity as the central mechanism in the neurotoxicity of fluoride
Excitotoxicity by microglial activation is proposed to play a central role in the neurotoxicity of fluoride (58). It is a universal mechanism which is known to be involved in many neurological disoders, and in the neurotoxic manifestations of many compounds and heavy metals. Excitoxicity is caused due to the overstimulation of the glutamate receptors in the neurons, leading to activation of the microglia (the immune cells of the brain) which release cytokines and other immune factors, finally causing brain cell death. The glutamate receptors get overstimulated because of the accumulated glutamate as a result of the impaired glutamate transporters of the brain. The cascade of excitoxicity is mediated through the reactive oxygen species (ROS) and the reactive nitrogen species (RNS), which may also be the causative factors in excitoxicity (58).

The theory of excitotoxicity as a central mechanism in the neurotoxicity of fluoride has been proposed by Blaylock , and circumstantial evidences are available to propose the excitotoxicity in fluorosis (58). Many animal studies have shown that fluoride accumulates in the areas of the hippocampus, dentate gyrus, and in the superficial areas of the amygdala, cortex and the cerebellum, leading to histopathological abnormalities (4),(5),(18),(19). These areas have an abundance of glutamate receptors. Fluoride is known to induce the generation of ROS and RNS in the brain (7), (36), which are the principal mediators of the excitoxicity. As has been explained under the mechanisms, 1 and 2, fluoride impairs the energy metabolism and the mitochondrial functions. Fluoride alone, or in combination with aluminium, triggers a cascade of molecular events through the G protein-coupled receptors, the second messenger systems, –c AMP, c GMP and the phosphoinositides and through the activation of protein kinase C, thereby stimulating the processes which are involved in the microglial activation (58). However, there is a lack of direct experimental evidences which shows that fluoride specifically activates the microglia, and that fluoride stimulates the glutamate receptors.

Summary
Fluoride is a neurotoxin and it makes a serious adverse impact on the developing brain. Impaired mental functions are observed among children in endemic fluorosis areas and in experimental animals with fluoride-induced neurotoxicity. Fluoride induces the generation of free radicals, it increases lipid peroxidation, it impairs antioxidants, it inhibits the key enzymes of the metabolic pathways, it impairs energy generation, and it inhibits protein synthesis. The animal experiments which were done on chronic fluoride toxicity have reported varied findings, which might be due to the differences in the dose, duration and the mode of the fluoride administration, the animal species which was used, and the organspecific metabolic responses. Excitotoxicity, which is proposed as the central mechanism in the neurotoxic effects of fluoride, needs critical evaluation by mechanistic studies and there is a need for extensive research on the amelioration of the fluoride-induced pathology of the brain.

Acknowledgement

The authors wish to dedicate this review paper to their mentors, Professor P.Gopalakrishna Bhat (Professor of Biochemistry, KMC Manipal) and Professor S. Hanumanth Rao (Former Professor of Biochemistry, MR Medical College, Gulbarga), two inspiring teachers. We gratefully remember the fluorosis patients from Kheru Nayak Thanda, Gulbarga, on whom our first research paper was published.

DOI and Others

ID: JCDR/2012/3590:0055

Date of Submission: Nov 10, 2011
Date of peer review: Feb 06, 2012
Date of acceptance: Apr 01, 2012
Date of Publishing: May 31, 2012