It is well established that NKA activity is inhibited by fluoride [ 117 138 ]. Furthermore, the results from human studies provide a biological gradient by which serum fluoride levels inhibit NKA activity in adults [ 137 138 ]. Considering the exclusion criteria and number of participants in these latter studies, these findings are significant. Notably, the inhibitory effect was found to occur in adults at serum fluoride levels < 5.0 µM and the inhibitory effects increased significantly as serum F levels increased in a dose dependent manner. [ 137 138 ]. For example, Arulkumar et al. observed that at serum ionic fluoride levels of 14.75 µM the activity of NKA declined by approximately 60 per cent compared to controls [ 137 ]. Considering that NKA activity is inhibited by hypothyroidism, the potential role of water fluoridation or fluoride intake with respect to increased prevalence of hypothyroidism must not be overlooked [ 139 140 ]. Interestingly, Kheradpisheh et al. in a case control study recently found that fluoride levels in drinking water has impacts on thyroid hormones even in the standard concentration of less than 0.5 mg/L. Among healthy participants without thyroid disease median TSH levels were found to increase when drinking water fluoride levels increased from 0–0.29 mg/L to 0.3–0.5 mg/L. Moreover, among subjects with clinically diagnosed hypothyroidism the effects of fluoride intake from drinking water on TSH were highly significant [ 140 ]. This finding is consistent with the Peckham study in England, which reported an increased prevalence of hypothyroidism, in communities where drinking water was artificially fluoridated [ 139 ]. As previously described, loss of NKA activity has been implicated in the pathogenesis of neurodevelopmental, neuropsychiatric and neurodegenerative disorders, as well as increased risk of cancer, metabolic, pulmonary and cardiovascular disease. Therefore, it is plausible that fluoride inhibition of NKA may have previously unforeseen consequences on public health and health inequalities, particularly in countries where drinking water is artificially fluoridated. However, studies examining the molecular mechanisms by which fluoride may contribute to such health inequalities are lacking. This relationship is particularly important given that exposure to fluoride can occur through water, food and other common sources such as dental products, in addition to occupational and environmental exposures. Although much information has become available in recent decades, the molecular mechanisms of fluoride inhibition of NKA remain to be defined. The lack of detailed information on the molecular mechanisms underlying how fluoride inhibits NKA activity impedes our understanding of how fluoride exposure may also contribute to pathological states. Consequently, the objective of this current study is to elucidate the molecular mechanisms and biological pathways by which fluoride inhibits NKA activity. In addition, this study examines the potential implications of fluoride induced loss of NKA activity on human health and disease inequalities.

A relationship between glutamate excitotoxicity and neuroinflammation in autism has also been ascertained [ 103 104 ]. Moreover, in animal studies, loss of AMPA receptors have been found to result in early-onset motor deficits, hyperactivity, cognitive defects, behavioural seizures and sleep disorders [ 105 ]. Taken together, these findings suggest a possible causal link between loss of NKA activity and childhood neurodevelopmental disorders that present with motor deficits, hyperactivity and cognitive defects including attention deficit hyperactivity disorder (ADHD) and autism spectrum disorders (ASD). Moreover, it is important to note that ADHD is highly comorbid with other psychiatric or neurodevelopment disorders associated with loss of NKA including, major depressive disorder and schizophrenia [ 106 107 ], which again supports the hypothesis that loss of NKA is implicated in the pathophysiology of ADHD. Importantly, NKA is also critical for sodium iodide symporter (NIS) functionality and iodine transport [ 108 110 ]. Thus, NKA inhibition may contribute to iodine deficiency [ 110 ]. Iodine deficiency can lead to hypothyroidism [ 111 112 ]. Interestingly, hypothyroidism has also been found to decrease NKA activity [ 113 114 ]. Crucially, Schmitt et al. found that hypothyroidism at a critical period of development in utero can result in permanent inhibition of NKA activity [ 114 ]. Moreover, Ahmed et al. showed that the hypothyroid status during pregnancy and lactation produced inhibitory effects on NKA as well as Ca(2+)-ATPase and Mg(2+)-ATPase in different brain regions of the offspring [ 115 ]. Further studies have established that hypothyroidism leads to marked reduction in dendritic branching in the rat brain [ 116 ], which is consistent with loss of NKA activity as previously described.

Furthermore, a great body of evidence associates neurotoxicity with a reduction of NKA activity, suggesting that reduction in NKA activity may be a link between several common neurotoxic mechanisms [ 14 79 ]. Loss of NKA is associated with autism [ 65 69 ]; Alzheimer’s disease [ 66 81 ]; Parkinson’s disease [ 82 ]; amyotrophic lateral sclerosis [ 70 82 ]; Down syndrome and Huntington’s disease [ 69 ]; depression and mood disorders [ 71 74 ]; bipolar disorder [ 14 76 ] and schizophrenia [ 15 79 ], as well as in animal models of depression [ 83 84 ]. Interestingly, NKA activity has been found to be significantly lower in subjects with phenylketonuria [ 85 ], a disease associated with intellectual disability, seizures, behavioural problems and mental disorders. Of note, loss of NKA activity has also been found to be associated with neonatal seizures and epilepsy [ 86 87 ]. It is also known that recovery of NKA activity in the hippocampus is responsible for neuroprotection [ 88 ]. Experimental studies have also shown that impairment of NKA activity in neonatal brain of rats leads to increased anxiety-like behaviour and memory impairment. Interestingly, in this study treatment with folic acid was found to reverse the inhibition of NKA and alleviated cognitive deficits associated with enzyme inhibition [ 89 ]. As NKA activity is essential for synaptic and neural functionality [ 3 ] and since NKA has been shown to trigger dendritic growth [ 90 ]; hence, it is possible that reduction in the activity of this enzyme may disrupt normal brain development. In this context, it has been demonstrated in-vivo that NKA inhibition causes selective neuronal loss in rodents [ 91 ]. In addition, it has been demonstrated that the extent of neuronal loss observed roughly paralleled inhibition of the enzyme [ 92 ]. It has been reported loss of NKA activity causes impairments in the sodium pump leading to neuronal hyperexcitability in the CNS [ 10 94 ]. NKA inhibition also increases neuronal susceptibility to glutamate excitotoxicity contributing to neurotoxicity [ 95 96 ]. Furthermore, NKA impairment has been found to downregulate the synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, leading to synaptic transmission defects and cognitive impairment [ 97 ]. Of note, researchers have also identified glutamatergic neurotransmission dysregulation and cognitive dysfunctions in major psychiatric disorders: including, schizophrenia, depressive disorders and suicidal behaviours [ 98 101 ]. Excess extra-cellular glutamate leading to excitotoxicity has also been suggested to play a role in in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer’s disease and Huntington’s disease [ 102 ].

Previous studies have shown that inhibition of NKA activity has been found to accelerate depletion of adenosine triphosphate (ATP), induce mitochondrial depolarization, suppress reactive oxygen species (ROS) scavenging, and enhance ROS production and oxidative stress [ 15 17 ]. It is known that a causal relationship has been identified between NKA enzyme inhibition and airway hyperreactivity [ 18 ]. Consistent with this, NKA inhibition is associated with asthma [ 19 20 ] and chronic obstructive pulmonary disease (COPD) [ 21 ]. Furthermore, loss of NKA activity is associated with allergic diseases [ 22 23 ] including allergic rhinitis [ 24 ] and blood diseases including thalassemia and sickle cell anaemia [ 25 ]. Extensive studies show that NKA is essential for sperm mobility and male fertility [ 26 29 ]. In addition, loss of NKA activity is also associated with rheumatoid arthritis [ 30 31 ], metabolic syndrome [ 32 ], including; chronic kidney disease [ 33 35 ]; diabetes mellitus [ 36 37 ]; diabetic nephropathy and cardiomyopathy [ 38 40 ]; cardiovascular complications [ 32 43 ]; hypertension [ 40 52 ] and obesity [ 11 ]. Loss of NKA activity is also implicated in degenerative eye diseases including cataract formation and age related macular degeneration [ 53 ]. In addition to inflammatory disorders as noted previously, loss of NKA activity has been found to be associated with tumour invasiveness, metastasis, and tissue fibrosis [ 54 ], kidney cancer [ 54 ], prostate cancer [ 55 ], bladder cancer [ 56 ] and urothelial cancer [ 57 ]. Consistent with these findings showing an association between loss of NKA in carcinoma and cancer progression, an isoform of the β subunit of NKA has been found to be a tumour-suppressor [ 58 ] and its expression along with total NKA activity has been found to be markedly reduced in prostate cancer [ 55 ] and kidney cancer [ 59 ]. Moreover, the adhesion molecule on glia (AMOG), another isoform of β subunit of NKA has been found to inhibit glioma cell invasion, while its downregulation increases invasion in glial cells [ 60 ]. Downregulation of the alpha 1 subunit of NKA has also been implicated in colorectal cancer [ 61 ].

Sodium, potassium-activated adenosine triphosphatase (-ATPase) is an integral protein in the plasma membrane that transports Na-ions to the outside and K-ions to the inside of the cell at the expense of ATP, and thus maintains sodium and potassium homeostasis in animal cells [ 1 2 ].-ATPase (NKA) is responsible for the electrochemical gradient across the plasma membrane, a prerequisite for electrical excitability and secondary transport in neurons, as well as for the transport of other ions and metabolites necessary for the regulation of the cellular ionic homeostasis [ 3 ]. In addition, to its function in maintaining cell homeostasis, NKA activity plays a crucial role in the function of neurotransmitter transporters essential for regulating neurotransmitter signaling and homeostasis [ 4 ]. By using the energy from ATP to establish asymmetric distributions of ions across the cell membrane, NKA couples metabolic energy to cellular functions and to signaling events both between and within cells [ 5 ].

Dental-care products are also a major source of Fexposure especially for children, because many tend to use more toothpaste than is advised, their swallowing control is not as well developed as that of adults, and many children under the care of a dentist undergo fluoride treatments [ 150 ]. The use of topical fluoride gels which contain high concentrations of Fhave been reported to significantly increase children’s Fplasma levels up to 79 μM after treatment. Among adults the use of Fgels can result in peak plasma Flevels of 51 μM [ 185 186 ]. Researchers found that the peak concentration was normally reached within 1 to 2 h of treatment and remained significantly elevated for up to 14 h [ 185 ]. The most commonly used fluoride-containing dental product is toothpaste. The vast majority of toothpastes sold today contains between 1000 ppm and 1500 ppm. Human studies have demonstrated that ingested Fin toothpaste is readily absorbed into systemic circulation resulting in rapid rise in plasma ionic Flevels [ 187 ]. A recent study involving healthy adults aged between 20–35y residing in a non-fluoridated community in England observed that when subjects were given a non-fluoridated toothpaste plasma Flevels, as measured in the morning after toothbrushing declined significantly from approximately 3.2 µM to 0.67 µM, respectively [ 188 ]. In another study conducted among adults residing in non-fluoridated community in Scotland, it was found that brushing twice a day (morning and evening) with toothpaste containing 1000 ppm and 1500 ppm for four weeks was found to increase plasma Flevels at midday to 1.18 and 1.33 µM respectively, compared to 0.7 µM in subjects using non-fluoride toothpaste [ 189 ]. The differences in plasma Flevels observed in these latter studies can be accounted for the half-life of Fin systemic circulation. After a single dose of Fplasma concentrations rise to a peak within one hour and decrease back to baseline with a half-life of two-three hours [ 176 ]. Therefore, taking the time differences in sampling of blood the results observed by Zohoori et al. and Jacobson are almost identical. Taken together, this data shows that a single brushing with Ftoothpaste in the morning can provide a dose of Fcomparable to consuming a cup of black tea with a Fcontent of 1.4–2.0 mg/L [ 175 176 ]. Similar to tea, case reports also indicate that skeletal fluorosis can occur from excessive use of Ftoothpaste [ 190 ]. Kurtlan et al. reported a case study of an adult American male aged 52 yrs who developed skeletal fluorosis from brushing his teeth six times per day. Laboratory evaluation of blood found serum Flevels ranged from 15 to 18.0 µM [ 190 ]. The subject did not reside in a fluoridated community and had no known occupational or environmental exposure to Fapart from toothpaste. The patient stated that he did not swallow toothpaste, used non-fluoridated mouthwash, had semi-annual dental visits, but without Ftreatments, did not drink tea or wine, and had not chewed tobacco, inhaled snuff, or cooked with Teflon pots. Within 8 months of documentation of skeletal fluorosis and after avoiding fluoridated dental products, serum Fdecreased to < 2.5 µM [ 190 ]. It is also important to note that the European Academy of Paediatric Dentistry (EAPD) recommend that children who are below the age of two should use toothpastes with low fluoride concentrations (less than 500 ppm) [ 191 ]. For children aged 2–7 years, a pea sized amount of Fdentifrice (0.25 g) has been recommended [ 192 194 ]. However, several studies examining toothpaste usage by children aged 3–6 years have consistently shown that the amount of toothpaste used on toothbrushes typically varies from 0.35 to 3.5 g which can lead to excessive Fintake [ 194 195 ], particularly among children who brush twice daily or more. A current study in a fluoridated community in England involving young children aged 4 to 6-years of age residing reported that the contribution of toothpaste to total dietary Fintake was 53% [ 196 ]. In this study, the highest total daily Fintake (4.439 mg/day = 0.22 mg/kgbw/day) was for a 5-year-old child of which 72% (3.217 mg/day = 0.16 mg/kgbw/day) was from toothpaste ingestion [ 196 ]. In summary, the above studies illustrate that in evaluating the effects of F, consideration must be given to effects of cumulative exposures and their contribution to total Fintake and ionic plasma Flevels. As NKA is found in plasma and bound to plasma membranes, it is the ionic Flevels in systemic circulation and within cells that interacts with NKA functionality. The remainder of this study addresses NKA regulation and the molecular mechanisms by which Finhibits enzyme activity and the most significant health risks likely to be associated with F-induced inhibition of enzyme activity.

Moreover, studies involving adult human subjects in countries without water fluoridation have demonstrated that the consumption of single cup of tea containing between 1.4 and 2 mg/L Fresult in peak plasma Flevels of approximately 3–4 µM. [ 175 176 ]. It is necessary to note that the frequency of Fdosage is known to affect plasma Flevels due to the terminal plasma half-life of F. Thus, multiple low dose exposures can result in higher steady state plasma Flevels than single dose exposure to higher doses [ 177 179 ]. Hence, habitual consumption of tea can result in skeletal fluorosis [ 153 ]. Moreover, currently about one-fifth of the currently marketed pharmaceuticals are organofluorine compounds and almost one-third of the top 100 top-selling drugs are organofluorine compounds. Fluoridated pharmaceuticals include antidepressants, anti-inflammatory agents, antimalarial drugs, antipsychotics, antiviral agents and steroids [ 180 ]. While there is a paucity of information on the biotransformation of fluoridated pharmaceuticals in general, several synthetic organic fluoride drugs which have been found to undergo high rates of biotransformation and defluorination resulting in significantly elevated plasma Flevels and in some instances chronic Fintoxication in humans. While it is beyond the scope of this present study to review the literature on fluoridated pharmaceuticals, of the fluoridated drugs currently on the market, Voriconazole is acknowledged to cause chronic Fintoxication, resulting in musculoskeletal chronic pain disorders and skeletal fluorosis [ 181 182 ]. Skiles et al. reported that elevated plasma Flevels of 24.3 μM after 6 months of voriconazole treatment resulted in skeletal fluorosis. Once Ftoxicity was confirmed and voriconazole was discontinued, within 3 weeks plasma Flevel declined to 6.7 μM [ 181 ]. Furthermore, the use of fluoridated anaesthesia such as sevoflurane can provide 20 times the total daily dietary intake from all sources of fluoridated food and water combined [ 183 ], resulting in peak plasma Flevels in the range of 50 μM [ 184 ].

A mention should be made that the Flevels in infants residing in a fluoridated community in the USA as measured by Warady and associates are within the range observed to cause inhibition of NKA activity in adults [ 137 138 ]. Indeed, the levels are also higher than what has been observed among workers occupationally exposed to Fin aluminum smelting factories at the end of the working day in Sweden [ 170 ] and Japan [ 171 ]. Notably, Ehrnebo and Ekstrand reported that the mean plasma Flevels in workers at the end of their shift were 2.54 μM [ 170 ], while Kono reported serum Flevels ranging from 2.21 to 3.47 µM among exposed workers [ 171 ]. Moreover, the ionic Flevels in blood in infants reported by Warady and associates are within the range known to be associated with skeletal and non-skeletal fluorosis in humans. A review of literature addressing ionic Flevels associated with skeletal and non-skeletal fluorosis in humans has been the subject of an earlier study by Waugh et al. [ 153 ]. However, it should be also noted that the Flevels reported by Warady and associates for infants aged 4–6 months were two fold higher than those reported for 60-day old non-diabetic weaning Wistar rats fed drinking water with a Fconcentration of 50 mg/L, while the plasma Flevels in infants with chronic kidney disease were comparably to diabetic rats fed drinking water with 50 mg/L [ 172 ]. Toxicity studies using 3-week old weaning male Wistar rats provided Fin drinking water at concentrations of 50 mg/L for 60 days lead to mean ionic plasma Flevels of 3.78 µM [ 173 ], which is similar to reported for (A/J) mice given 50 mg/L Fin drinking water for 11 weeks (mean serum Flevels of 3.31 µM) [ 174 ]. Collectively, these findings suggest that in fluoridated community’s plasma Flevels in maternal cord blood and human neonates in early infancy are comparable to the plasma Flevels in rodents administered drinking water with a Flevel of approximately 50 mg/L.

In areas naturally low in water Fand dietary Fintake exclusively breast-fed infants aged less than 12 months have been reported to have a mean ionic serum F– levels of 0.22 μM [ 163 ]. By contrast, in fluoridated communities in the United States the mean Flevel in infants aged 4–6 months and 7–12 months has been reported to be 4.22 ± 3.7 µM, and 1.56 ± 0.53 µM, respectively [ 164 ]. Moreover, the mean ionic plasma Flevel in infants with renal failure during their first 18 months of life was 6.3 µM compared to 3.16 µM in age matched controls [ 164 ]. As is evident from this study, plasma Flevels varied significantly among infants aged 4–6 months with the maximum Flevels being approximately 8 µM. The variations reported reflect the peak plasma Flevels associated with proximity to feeding and the use of breast milk versus optimally fluoridated water used to reconstitute powdered infant formulas. It must be emphasized that the Flevels reported in this study are not unexpected, as a previous study conducted by Anderson et al. in the Republic of Ireland reported that the consumption of infant formula reconstituted with fluoridated water may result in Fdoses above the recommended tolerable upper intake level for healthy adults [ 165 ]. A current study conducted in the USA further supports this observation. In this study, it was reported that the use of optimally fluoridated water (0.7 mg/L) in the preparation of infant formula resulted in 36.8% of infants exceeding the UL [ 166 ]. Furthermore, in this study, it was reported that among bottle fed infants the highest bioavailability of Foccurs in the first six months of life [ 166 ]. Importantly, this also coincides with the period when Fexcretion is impaired in infants due to immature kidney function [ 167 168 ]. Thus, the decline in plasma Fconcentrations after 6 months of age observed by Warady and associates coincides with the development of renal function and increased urinary excretion of F. Further studies conducted in another fluoridated city in the USA reported that when infants were fed milk-based formula reconstituted with non-fluoridated water, the mean plasma Fconcentrations two hrs post feeding was 0.77 μM, despite the lack of direct exposure to fluoridated water [ 169 ]. This finding reflects the higher plasma Flevels in mothers in fluoridated than non-fluoridated communities. Furthermore, this study demonstrated that a dose of 0.25 mg of Fadministered to twenty infants aged one to eighteen months two hours after their last feed resulted in mean peak plasma Flevels of 3.3 µM (range 2.52–4.85 µM). Consistent with the findings of Warady and associates, the authors of this latter study acknowledged that the Fintake and exposure for infants fed powdered infant formula reconstituted with fluoridated water would be significantly higher than those reported in their study, though for some unexplained reason they did not attempt to measure ionic plasma Flevels in infants provided powdered infant formula reconstituted with tap water in a community with fluoridated drinking water [ 169 ].

The Flevel in breast milk from mothers in a low Fcommunity where drinking water Flevels less than 0.16 mg/L are 0.004 mg/L and 0.009 mg/L in breast milk from mothers residing in communities where drinking water Flevels are 1.0 mg/L [ 154 ]. Elsewhere it has been reported that the concentration of Fin cows’ milk is ten-fold higher than human milk and typically ranges from 0.03 to 0.06 ppm [ 155 ]. However, past studies have shown that Fconcentrations in cow’s milk can vary significantly depending on the Flevel of water provided to dairy herds. For example, Gupta et al. demonstrated that when the Fconcentration in drinking water was 0.47, 0.82 and 1.32 mg/L the Fconcentrations in cow’s milk was 0.016, 0.074 and 0.18 mg/L respectively [ 156 ]. Interestingly, a recent study conducted by researchers at Newcastle University and Teesside University in England reported that the mean Fcontent in whole milk products available in a fluoridated region of the UK was 0.08 ppm [ 157 ]. This data indicates that dairy herds in the region were provided with mains fluoridated water as a source of drinking water. Moreover, previous studies have reported that Fvalues in cow’s milk ranging from 0.1–0.4 mg/L are consistent those found in Fpoisoned dairy herds [ 158 ]. From the above data it is obvious that the source of F– in drinking water provided to dairy herds can influence the Fcontent in milk used for the manufacture of powdered infant formula. It has also been reported that the Fcontent in powdered infant formula products can vary depending on the source of water used in processing and that the use of optimally fluoridated mains can result in higher Flevels in powdered infant formula products. Thus, in certain western countries where drinking water is fluoridated significant variations in the Fcontent in powdered infant formula products have been reported [ 159 161 ]. However, it is widely acknowledged the main contributor to the Fintake in infants is the Fcontent in water used to reconstitute powdered infant formulas. It should also be noted that formulas mixed with optimally fluoridated water provide the highest mean Fdaily intake [ 162 ].

Today, community water fluoridation and Ftoothpaste are considered the most common sources of Fexposure in the USA [ 142 150 ]. In countries such as Ireland, the UK, Australia and New Zealand, where habitual tea drinking is commonplace, the major dietary source of Fis tea consumption [ 151 153 ]. In addition to tea, fluoridated water, and toothpaste other sources of Fexposure include other beverages produced from fluoridated water (powdered infant formula, fruit juices, soft drinks, coffee, beers); pesticide residues in foods, foods processed or cooked in fluoridated water; foods grown in soil containing For irrigated with fluoridated water; consumption of foods with elevated Flevels (i.e., seafood and processed chicken); foods cooked in Teflon cookware; tobacco consumption; use of fluoridated mouthwash; use of medical inhalers containing fluoridated gases, and fluoridated medications, in addition to other environmental or occupational exposures to F 153 ]. Although the amount of Fingested in diet can be theoretically measured in dietary intake studies, by measuring the Fcontent in foods and beverages, the most reliable and accurate method of measuring Fexposure is by measuring the Fcontent in serum/plasma, bone or urinary Flevels. As NKA activity is present in blood and considering the evidence that Flevels in blood inhibit NKA activity, it is therefore necessary to learn more about how different sources of Fexposure contribute to blood Flevels in humans. Moreover, understanding the routes of Fexposure in infancy is essential when considering the long-term health implications of chronic Fexposure on NKA activity and implications for health and well-being long term. Furthermore, the magnitude of exposure can be examined by comparing breast fed infants in low Fcommunities to formula fed infants in fluoridated communities, as shown in Table 1 below.

It is acknowledged that Fhas no known essential function in human growth and development and no signs of Fdeficiency have been identified [ 141 ]. However, Fis considered to have played a major role in the reduction of dental caries in the past decades in the industrialized countries. It is added as an anti-caries agent to a variety of vehicles, particularly drinking water and toothpastes. Though Fis not essential nutrient, it has been recognized for some time that topical, rather than systemic exposure of Fcontrols carious lesion development [ 142 145 ]. However, caries development is not a Fdeficiency disease [ 141 ]. Interestingly, a key mechanism in the anti-caries effect of Fis the direct inhibition of ATPases activity in oral bacteria [ 146 149 ].

NKA is a plasma membrane embedded protein in all animal cells. NKA consists of two noncovalently linked α and β subunits. The alpha subunit is also known as the catalytic or functional subunit, since it contains the binding sites for protein kinase, protein phosphatase and transmembrane ion transport activities [ 197 199 ]. The catalytic α-subunit is responsible for conversion of ATP energy to transport of Naand Kacross cell membranes and has ATP and cardiac glycosides binding sites. The β-subunit is responsible for delivery and insertion of alpha one in cell membranes [ 200 202 ]. Thus, plasma membrane expression of the NKA requires the assembly of its alpha- and β- subunits [ 3 ] and the β subunit must interact with α subunit in order to accomplish ion transport [ 203 ]. The presence of magnesium facilitates the binding of ATP to NKA thereby providing the chemical energy required for ion channel function and secondary active transport [ 2 ]. Functionally, it has been identified that the activity of NKA is inhibited by phosphorylation. Furthermore, cyclic adenosine monophosphate (cAMP)-dependent protein kinase, (PKA) and protein kinase C (PKC) catalyse the phosphorylation of the enzyme [ 204 ]. It is well proven that PKC phosphorylates the NKA α subunit, leading to a decrease in enzyme activity [ 205 213 ].

Finally, it is known that the inhibition of enolase results in the formation of advanced glycation end products (AGEs) [ 442 ] and AGEs inhibit NKA enzyme activity [ 443 ]. Again, the mechanistic pathway has been elucidated to involve activation of AA metabolism via PLA2 activation [ 443 ]. It has been known for many decades that enolase is particularly sensitive to Finhibition [ 275 277 ]. Furthermore, recent in vivo rodent studies demonstrated that chronic long-term exposure for six months to Fvia drinking water significantly increased expression of receptors for advanced glycation end products (RAGE), increased RAGE proteins and increased levels of AGEs in cells. A significant increase in the expression NADPH oxidase 2 (NOX2) was also observed among specimens exposed to fluorine for 6 months. Notably these effects were found to occur at concentrations of just 5 mg/L in drinking water, which is the equivalent to approximately 0.5 mg/L in drinking water for humans. Simultaneous in vitro research with SH-SY5Y cells originating from human neuroblastoma confirmed these results [ 444 ]. Taken together these observations provide a basis for the hypothesis that F-induced inhibition of enolase contributes to NKA inhibition by activation of AGEs, which in turn leads to activation of PLA2 and the synthesis of AA which is metabolised to PGE2.

It is also important to point out that nuclear transcription factor kappa-B (NF-κB) has been previously reported to be involved in the up-regulation of COX-2 and generation of PGE2 [ 425 426 ]. It is well established that Factivates the NF-κB mRNA expression in a wide variety of cell types and including monocytes, macrophages as well as lung, kidney and brain tissue [ 427 434 ]. This stimulatory effect has been observed at Fconcentrations of 2.5 µM [ 427 ]. However, the seminal study by Misra et al. demonstrated that beryllium fluorides at concentrations as low as 0.002 uM significantly upregulated the activation of NF-κB in macrophages [ 435 ], indicating that beryllium fluoride complexes have much greater cytotoxicity and genotoxicity than Falone. With an increasing interest in beryllium, concern has raised specifically about the risks of co-exposure to beryllium and F 435 ]. Human biomonitoring studies have determined that the mean concentration of beryllium in breast milk is 0.008 µg/L [ 436 ]. However, a UK study found that the mean concentration of beryllium in ten different brands of powdered infant formula products was 1.1 µg/L [ 437 ]. In view of the fact that infant formula products contain such high levels of beryllium and that beryllium is known to bind with high affinity to the electronegative F 441 ] raises particular concerns considering that infant formula products are reconstituted with fluoridated tap water in countries with water fluoridation.

It is known that NKA activity is inhibited by elevated glucose concentrations although the mechanism of suppression remains largely unknown [ 401 405 ]. It has also been reported that inhibition of NKA activity by hyperglycaemia could be an important etiological factor of chronic complications in diabetic patients [ 406 ]. Importantly, a large number of human and animal studies have demonstrated that F exposure can induce hyperglycaemia [ 363 420 ]. Consistent with this, the U.S. National Research Council (NRC) reported that the conclusions from available studies is that sufficient Fexposure appears to bring about increases in blood glucose or impaired glucose tolerance in some individuals and the increase the severity of some types of diabetes [ 150 ]. Again, it has been shown that mechanistic pathway by which hyperglycaemia inhibits NKA activity is via activation of PKC and phospholipase A2 (PLA2), resulting in the liberation of arachidonic acid (AA) and increased the production of prostaglandin E2 (PGE2), which are known inhibitors of NKA activity [ 406 422 ]. Importantly, several in vitro human tissue models have consistently demonstrated that Fin micromolar concentrations of 1–10 µM significantly increases the synthesis of cAMP, AA, PGE2 and PLA2 in a dose dependent manner [ 249 424 ]. Taken together these observations provide a basis for the hypothesis that F-induced hyperglycaemia contributes to NKA inhibition. Furthermore, evidence suggests that Fmay also inhibit NKA activity directly via stimulation of PLA2 synthesis, leading to increased production of AA and PGE2.

One of the major regulators and inhibitors of proximal renal tubule NKA activity is parathyroid hormone (PTH) [ 374 396 ]. Further research has shown that PTH inhibits NKA through activation of PKC [ 393 398 ], cAMP [ 395 ] and phospholipase A2 (PLA2), pathways [ 397 ]. Furthermore, human studies have shown that when urinary Flevels are in excess of 1 mg/L PTH expression is significantly enhanced compared to subjects with urinary F levels less than 0.5 mg/L [ 399 ]. It is important to point out that Schwartz et al. demonstrated that lowering of blood ionized calcium by an amount as low as 0.02 mmol/L (0.08 mg/L) within 30 min can elicit an immediate large, transient peak release of PTH amounting to 6–16 times the baseline concentration [ 400 ]. Of critical importance, Karademir et al. demonstrated that serum calcium levels were 0.075 mmol/L and 0.1 mmol/L lower among subjects with urinary Flevels of 0.70 mg/L and 0.90 mg/L respectively, compared to controls with urinary Flevels of 0.20 mg/L [ 260 ]. Taken together, these findings provide a basis for the hypothesis that F- induced PTH release contributes to NKA inhibition by a mechanistic pathway that involves PKC, cAMP and PLA2. The association between Fand PLA2 expression will be discussed in the following section.

Several experimental animal and in-vitro studies on tissues/cells have demonstrated that Fstimulates DA release [ 227 383 ], although how this occurs is not well understood. However, other studies suggest that this may be due to the effect of Fon hypothalamus function. It is well established that the hypothalamus regulates pituitary TSH secretion by releasing thyrotropin-releasing hormone (TRH) [ 384 385 ] and excess TSH is associated with iodine deficiency and hypothyroidism [ 110 ]. In addition to TRH induced release of TSH, TRH stimulates DA release [ 386 388 ]. Furthermore, it has also been observed that hypothyroidism increases DA receptor sensitivity by increasing receptor concentration [ 389 ]. As previously described, human studies have consistently found that Fcan stimulate TSH production in humans [ 140 269 ]. Thus, Fmust also induce TRH secretion. Furthermore, the stimulatory effect of Fon DA release has been observed in animal studies with drinking water Flevels of 1 mg/L [ 382 ]. Interestingly, in this study, the highest levels of DA release were observed at 5 mg/L Fin water above which DA release was found to decrease. Consistent with this finding, it has reported that stimulant-induced increases in endogenous DA levels trigger feedback mechanisms that inhibit DA neuron firing [ 390 ]. Interestingly, inhibitors of NKA have also been found to almost completely abolish the TRH-induced DA releasing effect [ 378 ]. Therefore, it is also plausible that the reduction in DA release at higher Fdoses may reflect the enhanced inhibitory of F on NKA activity which in turn inhibits TRH induced DA release. It is also important to understand that biosynthesis of TRH in the hypothalamus is dependent on ATP [ 391 ]. Thus, increased TRH synthesis leads to a reduction in ATP bioavailability. As previously elucidated NKA function requires ATP. In addition to the animal studies which found that Finduces DA release, evidence from human studies have found that the level of DA in foetal brain tissue is elevated in foetuses of F-exposed mothers with dental fluorosis, compared to foetuses from mothers without dental fluorosis living in non-Fendemic areas [ 392 ]. Collectively, these results provide strong evidence that the ability of F to upregulate TRH or TSH secretion may contribute to DA dysfunction leading to enhanced DA release which in turn may contribute to NKA inhibition by a mechanistic action that appears to involve cAMP production and decreased bioavailability of ATP.

It is known that dopamine (DA) inhibits NKA activity [ 231 375 ] though the molecular mechanisms of inhibition remain elusive. Furthermore, it has also been reported that PKC and cAMP signalling contribute to dopaminergic inhibition of NKA activity [ 376 ]. To understand how DA inhibits NKA activity, it is important to point out that DA has been found to directly stimulate cAMP [ 377 379 ]. Furthermore, Vortherms et al. reported that persistent activation of DA receptors results in a compensatory increase in cAMP accumulation [ 378 ]. As previously elucidated, the production of cAMP leads to a reduction in ATP, which is required for NKA enzyme activity.

As previously described, Fis a potent inducer of CT. Moreover, it is also known that the protein RANKL (receptor activator of nuclear factor-KB ligand) increases osteoclast number, bone resorption, and subsequently Pi release [ 354 355 ]. Furthermore, Pi uptake by osteoclasts require sodium-dependent phosphate (Na-Pi) transporters that are dependent on ATPase activity. The energy requirement to drive this process is high, thus, a large amount ATP is required [ 356 ]. Inhibitors of Na-Pi-cotransporters or ATPase or reduced ATP bioavailability result in inhibition of bone Pi resorption [ 356 ], leading to increased plasma Pi levels. As previously described, Freduces ATP bioavailability and impairs glycolysis, and it is well established that Fis an inhibitor of ATPase. Furthermore, recent in vivo studies with rodents and subsequent in vitro studies on bone tissues found that low dose Fexposure stimulates expression of RANKL [ 357 ]. As previously elucidated, RANKL stimulates Pi release. Consistent with the mechanisms elucidated above, several human and animal studies have shown that Fintake can enhance Pi levels [ 349 367 ]. As Pi has been found to inhibit NKA activity, this provides a further mechanistic pathway by which Finhibits NKA activity. Furthermore, as previously elucidated, Finhibition of enolase is also significantly stronger in the presence of Pi [ 278 ] and Finhibition of enolase has previously been found to inhibit NKA activity [ 136 ].

In light of the fact that phosphorylation inhibits NKA activity, rationality postulates that elevated levels of inorganic phosphate (Pi) will also act as an inhibitor of NKA activity. In support of this hypothesis, it has previously been reported that Pi plays a role in phosphorylation of NKA [ 341 ] and high concentrations of Pi inhibit enzyme activity [ 342 ]. Furthermore, previous studies have also shown that Pi inhibits phosphatase activity of calcineurin (Cn) [ 343 ]. As previously described, dephosphorylation of NKA is mediated by Cn [ 2 ]. Hence, evidence suggests that higher levels of Pi may contribute to inhibition of NKA activity by decreasing dephosphorylation activity of Cn. Moreover, it is well established that alkaline phosphatase (ALP) is an enzyme responsible for catalysing dephosphorylation of phosphate esters, which leads to liberating Pi [ 344 ]. As ALP levels rise; more Pi is liberated [ 345 ]. Thus, ALP enzymatic activity plays a role in regulating Pi levels. This also suggests that ALP must play a role in regulating NKA functionality. ALP is found in normal osteoblasts, and the mode of action of Fin stimulating APL activity in humans is well recognized [ 242 351 ]. It is also well established that Facts to increase osteoblast formation [ 150 ]. It has been demonstrated in vitro that Fat concentrations as low as 0.1 μM increase the expression of ALP and stimulate the proliferation of osteoblasts [ 352 ]. Furthermore, it is known that ALP activity increases in the presence of CT [ 353 ].

It has been reported that TGF-β1 plays an important role in fluorosis and increased levels of TGF-β1 have been suggested as an important marker in the evaluation of the pathological action of Fin bone tissue [ 329 330 ]. In vivo and in vitro experimental studies of fluorosis have shown that Fupregulates TGF-β1 protein and mRNA expression in bone cells [ 329 333 ]. Importantly, calcitonin (CT), a hormone that is secreted by parafollicular cells of the thyroid gland, has been found to be a potent stimulator of TGF-β1 protein synthesis as well as TGF-β1 mRNA expression [ 334 ]. Furthermore, a large body of evidence from epidemiological studies has demonstrated that Fis a potent inducer of CT expression in humans [ 335 340 ]. Of fundamental importance, is the seminal research by Chen and associates in providing a biological dose-exposure response relationship for Fexposure in humans on CT expression at relatively low Fintakes. Notably, in this study, it was demonstrated that differential expression of CT occurs when urinary Flevels exceeded 0.38 mg/L [ 338 ]. Taken together, this data suggests that the contribution of Fin enhancing the expression of TGF-β1 via upregulation of CT is another mechanistic pathway whereby Finhibits NKA mRNA expression and protein activity.

It is also known that an important intracellular second messenger cyclic guanosine monophosphate (cGMP) inhibits NKA activity. [ 231 315 ]. The seminal study by de Oliveira et al. demonstrated cGMP inhibited NKA activity and that cGMP activation was induced by nitric oxide (NO) [ 315 ]. Accordingly, NO generating compounds play an important role in regulating NKA activity. Consistent with this finding, several studies have observed that NO inhibits the molecular activity of renal NKA [ 232 319 ]. Several studies have also shown that Finduces NO synthesis in vivo [ 320 326 ]. Furthermore, experimental studies have shown that Fstimulates cGMP in the kidney and thyroid [ 327 328 ]. Hence, the above data suggests that Finhibits NKA activity by stimulating NO expression and cGMP activity.

Furthermore, chronic Fintake has been shown to significantly reduce serum manganese levels in humans along with other trace metals including magnesium, copper and zinc [ 286 ]. Lower manganese accumulation in skeletal tissue has also been observed in humans suffering from fluorosis [ 306 ]. In agreement, several animal studies have also found that excessive Fexposure resulted in decreased manganese in serum [ 292 308 ], as well as in reduced manganese in liver, kidney, heart, lung and muscle tissue [ 309 312 ]. Collectively, this data suggests that Fenhances the activation of adenylate cyclase by CaM leading in increased production of cAMP. Furthermore, the ability of Fto enhance the phosphorylation activity of CaM suggests that Fmay contribute to the inhibitory effects of CaM on NKA enzyme activity. In addition, the contributory effect of Freducing manganese bioavailability suggests that this action may also be a potential mechanism in Finhibition of Cn activity. As previously described, manganese is a crucial activator of Cn and also binds to Cn ensuring structural stability and full enzyme activity. Thus, lower manganese reduces Cn expression. Furthermore, as the dephosphorylation of NKA is mediated by Cn, reduced expression of Cn or inhibition of Cn activity contributes to enhanced phosphorylation, which in turn leads to inhibition of enzyme activity.

It has been shown that Fupregulates the activation of adenylate cyclase by CaM [ 300 ] and activation of adenylate cyclase increases the intracellular level of cAMP [ 301 ]. As previously described, adenylate cyclase is the enzyme which catalyses the conversion of ATP to cAMP, thus increased cAMP synthesis reducing the bioavailability of ATP. As previously noted, cAMP has been observed to inhibit NKA activity and the molecular mechanisms of inhibition have been described. However, CaM has also been found to inhibit NKA activity. in human red blood cells [ 302 ]. Furthermore, previous studies have shown that CaM can mediate the phosphorylation of acetylcholine receptor and CaM stimulation of phosphorylation was enhanced most in the presence of F 303 ]. Revealingly, studies have shown that the presence of Finhibited the activation of phosphodiesterase by CaM [ 304 ]. Interestingly, phosphodiesterase is responsible for the hydrolysis (breakdown) of cAMP [ 305 ], a crucial finding that may elucidate why Fexposure is associated with increased concentrations of cAMP.

It has also been reported that the dephosphorylation (activation) of NKA is mediated by calmodulin-dependent calcineurin (Cn), a serine/threonine phosphatase [ 204 293 ]. Therefore, inhibition of Cn will lead to downregulation of NKA activity. Although the molecular pathway has not been completely elucidated, it has also been reported that Cn is inhibited by F 296 ]. However, it is known that Cn binds to calmodulin (CaM) and the complex of Cn and CaM is the active species of the phosphatase. Furthermore, aside from CaM, Cn requires an additional divalent metal ion such as manganese for structural stability and full activity [ 297 ]. Manganese is also a crucial activator of Cn activity [ 298 299 ].

is known to form complexes with magnesium and increased concentrations of magnesium reduce Fabsorption in the gastrointestinal tract [ 150 ]. This action would also result in reduced absorption of magnesium. Experimental studies have shown that low magnesium diets significantly enhance Fabsorption [ 283 ] and Faccumulation in calcified tissue retards the mobilization of skeletal magnesium [ 284 ]. Furthermore, as magnesium is bound to albumin, loss of albumin in urine can lead to enhanced excretion of magnesium and lower levels of magnesium in systemic circulation. In line with this, I recently elucidated how the loss of albumin in urine and resultant hypoalbuminemia (low levels of albumin in blood) contribute to iodine deficiency and that Facts to induce increased urinary excretion of albumin and hypoalbuminemia [ 110 ]. Therefore, evidence would strongly suggest that Fexposure can lead to magnesium deficiency. Consistent with these hypothesis, human studies have shown that Fsignificantly reduced serum magnesium along with other trace metals including manganese, copper and zinc [ 285 287 ]. In agreement, animal studies have also found that Fexposure results in decreased serum magnesium [ 288 292 ]. Thus, the above data suggests that Fcan inhibit NKA activity by lowering serum magnesium thereby impairing ATP binding to NKA.

The human body contains around 25 g of magnesium [ 280 ]. Magnesium is necessary for the functioning of over 300 enzymes in humans [ 281 ], with 90% of total body magnesium being contained in bones and muscles (~63% and ~27% respectively), 90% of which is bound and with only 10% being free [ 282 ]. In the serum, 32% of magnesium is bound to albumin, whereas 55% is free [ 280 ]. As previously discussed, Jorgensen et al. elucidated that the presence of magnesium facilitates the binding of ATP to NKA, thereby providing the chemical energy required for ion channel function and secondary active transport [ 2 ].

However, in addition to the role of cAMP in depleting ATP, it has also been reported that cAMP inhibits NKA activity by enhancing phosphorylation of the NKA α subunit [ 279 ]. As previously described, it is well proven that phosphorylation of the α subunit, leads to a decrease in its enzyme activity [ 206 213 ]. Therefore, strong evidence indicates that the action of Fin upregulating cAMP may lead to inhibition of NKA activity by two distinct pathways, by reducing ATP bioavailability and increasing phosphorylation of the α subunit of NKA.

In elucidating the molecular mechanisms by which increased production of cAMP inhibits NKA activity, it is necessary to emphasise that the conversion of ATP to cAMP leads to a reduction in ATP molecules. Since energy supply is a limiting factor controlling NKA activity, ATP depletion leads to inhibition of enzyme activity. As described above, Fincreases the conversion of ATP to cAMP. Further studies have shown that Finhibits ATP bioavailability [ 270 271 ]. Indeed, studies have shown that Finduces depletion of ATP in erythrocytes (red blood cells) [ 271 ]. Consistent with these findings, several studies have reported that ATP depletion induced by Fleads to inhibition of NKA in erythrocytes [ 126 128 ]. Furthermore, it is known that the breakdown of glucose by glycolysis is a major source of ATP [ 272 ]. Enolase is required to allow glycolysis to proceed [ 273 ] and inhibition of enolase leads to depletion of ATP [ 274 ]. Given that it is well established that Finhibits glycolysis via inhibition of enolase [ 275 278 ]. it is not surprising that recent in vitro studies found that inhibition of glycolysis by Fcontributed to reduced NKA activity [ 136 ]. However, it is important to also note that Finhibition of enolase is significantly stronger in the presence of phosphate [ 278 ]; the significant of which will be discussed later in this study. Taken together, this data demonstrates that Fstimulation adenylyl cyclase and TSH secretion leads to increased synthesis of cAMP reducing the bioavailability of ATP thereby contributing to inhibition of enzyme activity. Furthermore, it seems reasonable to suggest that the mechanisms outlined above elucidate why previous studies found that hypothyroidism was associated with lower NKA activity [ 113 115 ].

It is also established that Fincreases the conversion of ATP to cAMP by stimulation of adenylyl cyclase [ 235 236 ]. Consistent with this, evidence from human and animal studies show that Fstimulates cAMP production [ 237 248 ] leading to increased concentrations of cAMP in plasma, saliva, urine, and tissues. Moreover, in vitro human tissue models have demonstrated that Fin micromolar concentrations of 1–10 µM significantly increases the synthesis of cAMP in a dose dependent manner [ 249 ]. In addition, thyroid-stimulating-hormone (TSH) is known to be a potent stimulant of adenylyl cyclase activity resulting in increased synthesis of cAMP [ 250 251 ]. Furthermore, early in vitro studies by Wolf and Jones documented the largely identical effect of Fand TSH on adenyl cyclase activity resulting in increased stimulation of cAMP [ 252 ]. Consistent with this, several in vitro studies have shown a synergistic effect of Fand TSH on adenylate cyclase activity resulting in increased cAMP production [ 253 254 ]. Evidence from human studies also show that Finduces TSH production [ 140 269 ]. In this scenario, a positive feedback loop exists that may amplify the effects of TSH and Fon cAMP activity, which may explain the synergistic effect of Fand TSH on cAMP production.

Interestingly, Bocanera et al. also reported that PKC pathway inhibits thyroid iodide uptake in calf thyroid cells by an action distal to cAMP generation and probably because of a decrease in NKA activity [ 222 ]. As previously described, hypothyroidism has also been found to decrease the activity of NKA [ 113 115 ]. and there is evidence from human studies that water fluoridation and elevated Flevels in drinking water may contribute to hypothyroidism [ 139 140 ]. Hypothyroidism has also been shown to significantly upregulate PKC expression and activity [ 223 224 ]. Furthermore, it is also well established that activation of PKC is a physiological action of F-induced cellular toxicity [ 133 230 ]. Overall, these data suggest that Factivation of PKC is a key mechanism underlying Finhibition of NKA activity and that the contributory effect of Fexposure to hypothyroidism may further potentiate inhibition.

As previously described, the β subunit of NKA regulates both the activity and the conformational stability of α subunit and plasma membrane expression of the NKA requires the assembly of its α-and β-subunits. Thus, inhibition of the β subunit leads to reduced expression and lower enzyme activity. It is also important to note that NKA β subunit expression and maturation requires the interaction of Wolfram Syndrome 1 (WFS1) protein [ 215 ]. Furthermore, it has been shown that reduced expression of WFS1 results in reduced expression of NKA β subunit [ 202 ]. It has been reported that WFS1 expression is induced by protein kinase RNA-like ER kinase (PERK) [ 216 ]. Recent in vivo studies examining the role of genetics in response to Fexposure found that chronic Fexposure inhibits the expression of PERK [ 217 ]. Taken together, this data suggests that Fmay also inhibit WFS1 protein expression, leading to inhibition of NKA expression and activity.

Despite the large number of studies demonstrating that Finhibits NKA activity, the molecular mechanisms of down-regulation are not yet clearly understood [ 214 ]. To address the limitations in understanding of the molecular mechanisms of down-regulation it was first necessary to identify modulatory mechanisms from published literature. Thus, a review of literature was undertaken using PubMed and other search engines (Google, Google Scholar, ResearchGate, Yahoo) to source pertinent research articles and publications. Second, in order to elucidate the molecular mechanisms by which Fmay inhibit NKA activity, evidence was sought from published literature including human, animal and in vitro studies to examine how Finteracted with each of the biological pathways identified. As illustrated in Figure 2 and summarized in Table 2 , NKA activity is downregulated by a variety of hormones, proteins, metalloenzymes, neuropeptides and cytokines. Furthermore, regulatory mechanisms governing circulating inorganic phosphate, glucose homeostasis and adenosine-triphosphate production play a crucial role in downregulating enzyme activity.

5. Discussion

As noted from the preceding data, F− inhibition of NKA activity is complex and multifactorial. In summary, evidence is provided to show that activation of PKC, cAMP, cGMP, NO, Pi, PLA2, AA and PGE2 inhibit NKA activity and that F− upregulates PKC, cAMP, cGMP, NO, PLA2, AA, PGE2 and enhances Pi levels in systemic circulation. Furthermore, evidence is presented to show that Cn regulates NKA activity and that F− inhibits CN activity, in part, by regulating the bioavailability of manganese and Pi, as well as by altering the phosphorylation activity of CaM. In addition, evidence is provided to show that F− enhances the activation of adenylate cyclase by CaM, leading to increased levels of cAMP and further evidence is provided to show that F− inhibits the activation of phosphodiesterase by CaM, which is responsible for the breakdown of cAMP. I further elucidate that the presence of magnesium facilitates the binding of ATP to NKA, thereby providing the chemical energy required for ion channel function and secondary active transport. I have described how F− contributes to magnesium deficiency by forming complexes with magnesium thereby lowering the absorption of magnesium and F− in the gastrointestinal tract leading to reduced bioavailability. I have further elucidated that F− retards the mobilization of magnesium in bone and can increase the excretion of magnesium in urine bound to ALB.

In the present study, I have further elucidated the molecular mechanisms by which hypothyroidism lowers NKA activity. In addition, I describe how F− inhibition of enolase leads to inhibition of glycolysis thereby reducing ATP production. As ATP is required for NKA function, lower levels of ATP in turn may lead to inhibition of enzyme activity. Furthermore, evidence is presented to show that NKA activity is inhibited by cAMP and that F− acts to induce cAMP production. Moreover, in this study I have elucidated that there are two distinct mechanisms by which cAMP inhibits NKA activity. This can occur through loss of ATP, as well as the direct effect of cAMP in enhancing phosphorylation of the NKA α subunit. In this study, I also describe how the pituitary hormone TSH increases cAMP production and further evidence is provided to show that F− upregulates TSH secretion leading to a positive feedback mechanism that may result in inhibition of enzyme activity. I have further described how Pi has been found to inhibit NKA activity. I have further elucidated the role of ALP and RANKL in Pi release and the contribution of F− to increased mRNA expression of RANKL and ALP activity, which contributes to increased concentration of Pi in serum. Moreover, I have described how ALP activity is increased in the presence of CT and that F− is a potent inducer of CT activity. I have also described how CT stimulates TGF-β1 protein synthesis as well as TGF-β1 mRNA expression and that TGF-β1 inhibits the expression of the β-subunit of NKA required for enzyme function.

− upregulates dopamine, glucose and PTH activity. Further analysis reveals that the mechanism of dopamine inhibition of NKA is via increased production of cAMP. It has also been elucidated in this study that NKA inhibition by PTH and hyperglycaemia is regulated by activation of PLA2, AA and PGE2. In addition, there is evidence demonstrating that stimulation of AGEs inhibits NKA activity and that F− inhibition of enolase results in the formation of AGEs. Again, the mechanistic pathway by which AGEs inhibit NKA enzyme activity is via activation of the PLA2 pathway leading to enhanced expression of AA and PGE2. I have also elucidated how F− induces NF-κB mRNA expression in a wide variety of cell types and how NF-κB activation leads to increased PGE2 production. Consistent with these findings, evidence is provided from several in vitro studies with human cell lines that have consistently demonstrated that F− at biologically relevant exposures, ranging from 1–10 µM, increases the synthesis of cAMP, PLA2, AA and PGE2 in a dose dependent manner. Moreover, evidence from human studies confirm that F− exposure as measured by serum F− levels in adults, inhibits NKA activity in vivo at levels within the same range observed in human cell in-vitro studies showing increased activation of cAMP, PLA2, AA and PGE2. While this effect on NKA was found to occur in adults at serum F− levels < 5.0 µM, at higher mean serum F− levels of 14.75 µM the activity of NKA declined by approximately 60 per cent compared to controls [− levels observed in the control groups of either of these two studies were comparable to the mean fasting serum F− concentrations recently reported among healthy adults of similar age residing in a non-fluoridated community (drinking water F− level < 0.3 mg/L) in the UK [− intake among adults in western countries where drinking water is fluoridated or where habitual tea drinking is commonplace and where F− toothpaste is widely available would fall within the range observed to cause inhibition of NKA. Furthermore, it is important to acknowledge that in addition to inhibiting NKA activity, Arulkumar et al. also found that the activity of adenosine 5′ triphosphatase (ATPases), Mg(2+) ATPases, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), paraoxonase 1 (PON1) and arylesterase (ARE) declined in a dose dependent manner with increasing serum F− concentrations [− inhibition of these important enzymes. For example, loss of ARE and PON1 is associated with metabolic syndrome and are considered independent risk factors for cardiovascular disease [− downregulates AChE and suggested that this action may contribute to increases the risk of developing Alzheimer’s disease [ Further evidence is presented to show that NKA activity is inhibited by dopamine, PTH and glucose. In addition, evidence is presented demonstrating that Fupregulates dopamine, glucose and PTH activity. Further analysis reveals that the mechanism of dopamine inhibition of NKA is via increased production of cAMP. It has also been elucidated in this study that NKA inhibition by PTH and hyperglycaemia is regulated by activation of PLA2, AA and PGE2. In addition, there is evidence demonstrating that stimulation of AGEs inhibits NKA activity and that Finhibition of enolase results in the formation of AGEs. Again, the mechanistic pathway by which AGEs inhibit NKA enzyme activity is via activation of the PLA2 pathway leading to enhanced expression of AA and PGE2. I have also elucidated how Finduces NF-κB mRNA expression in a wide variety of cell types and how NF-κB activation leads to increased PGE2 production. Consistent with these findings, evidence is provided from several in vitro studies with human cell lines that have consistently demonstrated that Fat biologically relevant exposures, ranging from 1–10 µM, increases the synthesis of cAMP, PLA2, AA and PGE2 in a dose dependent manner. Moreover, evidence from human studies confirm that Fexposure as measured by serum Flevels in adults, inhibits NKA activity in vivo at levels within the same range observed in human cell in-vitro studies showing increased activation of cAMP, PLA2, AA and PGE2. While this effect on NKA was found to occur in adults at serum Flevels < 5.0 µM, at higher mean serum Flevels of 14.75 µM the activity of NKA declined by approximately 60 per cent compared to controls [ 137 138 ]. Moreover, the serum Flevels observed in the control groups of either of these two studies were comparable to the mean fasting serum Fconcentrations recently reported among healthy adults of similar age residing in a non-fluoridated community (drinking water Flevel < 0.3 mg/L) in the UK [ 188 ]. This data suggests that total Fintake among adults in western countries where drinking water is fluoridated or where habitual tea drinking is commonplace and where Ftoothpaste is widely available would fall within the range observed to cause inhibition of NKA. Furthermore, it is important to acknowledge that in addition to inhibiting NKA activity, Arulkumar et al. also found that the activity of adenosine 5′ triphosphatase (ATPases), Mg(2+) ATPases, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), paraoxonase 1 (PON1) and arylesterase (ARE) declined in a dose dependent manner with increasing serum Fconcentrations [ 137 ]. Clearly, further studies are warranted to explain the wider implications associated with Finhibition of these important enzymes. For example, loss of ARE and PON1 is associated with metabolic syndrome and are considered independent risk factors for cardiovascular disease [ 445 ]. Moreover, downregulation of AChE causes inflammatory hyperactivation of the CNS and peripheral nervous system [ 446 447 ]. Moreover, the U.S.A. National Academy of Sciences NRC previously reported that Fdownregulates AChE and suggested that this action may contribute to increases the risk of developing Alzheimer’s disease [ 150 ].

− inhibition of NKA activity on human health and disease inequalities. As I have described, loss of NKA activity has been implicated in many pathophysiological conditions, including asthma and allergic diseases, metabolic disorders, cancer, cardiovascular disease, as well as neurodevelopmental and degenerative brain diseases. Past studies have further suggested that inhibitors of NKA may also contribute to disorders associated with loss of NKA activity [− exposure and pathophysiological states associated with loss of NKA activity including iodine deficiency [− to loss of NKA activity. Returning to the hypothesis/question posed at the beginning of this study, it is important to assess the implications of Finhibition of NKA activity on human health and disease inequalities. As I have described, loss of NKA activity has been implicated in many pathophysiological conditions, including asthma and allergic diseases, metabolic disorders, cancer, cardiovascular disease, as well as neurodevelopmental and degenerative brain diseases. Past studies have further suggested that inhibitors of NKA may also contribute to disorders associated with loss of NKA activity [ 18 ]. This hypothesis is supported by two current studies which infer a causal association between Fexposure and pathophysiological states associated with loss of NKA activity including iodine deficiency [ 110 ] and degenerative eye diseases [ 53 ]. Among the many molecular mechanisms identified in these latter studies was the contribution of Fto loss of NKA activity.

23,− has consistently been found to inhibit NKA activity. Therefore, evidence suggests that F− may also play a role in the pathogenesis of bronchial hyperresponsiveness and inflammatory respiratory diseases. Moreover, earlier occupational epidemiological work by Søyseth et al. suggested that F− exposure was likely to be a causative agent in causing asthmatic symptoms among workers in the aluminium smelting industry [− levels, such that an increase in the plasma F− level of 0.5 µM was associated with an increase in the dose–response slope by a factor of 1.11 (95% CI, 1.05 to 1.17). Furthermore, the authors hypothesized that an increase of plasma F− of 3.4 µM would be associated with a doubling of bronchial responsiveness [− is important, as several studies have found that increased bronchial responsiveness is associated with asthma [451,452,453,456, As I already described, loss of NKA activity has been implicated in asthma [ 19 20 ], and allergic diseases such as allergic rhinitis [ 22 24 ]. Moreover, the seminal study by Gentile et al. provided both correlative and mechanistic evidence for a causal relationship between NKA enzyme inhibition and airway hyperreactivity (or bronchial hyperresponsiveness) among asthmatic and allergic subjects [ 18 ]. Furthermore, Gentile et al. concluded that inhibitors of NKA could play a role in the pathogenesis of AHR in human beings. Indeed, this observation has already been reported in several studies including environmental and occupational exposure studies. For example, Søyseth et al. found that exposure to atmospheric fluorides corresponded to an increase bronchial hyperresponsiveness in children aged 7–13 years [ 448 ]. As described in this current study, Fhas consistently been found to inhibit NKA activity. Therefore, evidence suggests that Fmay also play a role in the pathogenesis of bronchial hyperresponsiveness and inflammatory respiratory diseases. Moreover, earlier occupational epidemiological work by Søyseth et al. suggested that Fexposure was likely to be a causative agent in causing asthmatic symptoms among workers in the aluminium smelting industry [ 449 ]. Of fundamental importance, a positive dose-response association was observed between bronchial responsiveness and plasma Flevels, such that an increase in the plasma Flevel of 0.5 µM was associated with an increase in the dose–response slope by a factor of 1.11 (95% CI, 1.05 to 1.17). Furthermore, the authors hypothesized that an increase of plasma Fof 3.4 µM would be associated with a doubling of bronchial responsiveness [ 449 ]. The association between bronchial responsiveness and elevated plasma Fis important, as several studies have found that increased bronchial responsiveness is associated with asthma [ 450 454 ] and impaired lung function [ 455 457 ].

This leads us to perhaps the most significant evidence revealed in this current study regarding postnatal and early infant chronic F− exposure which has been found to occur in communities with artificially fluoridated drinking water (AFDW). As previously elucidated the plasma F− levels in infants residing in communities with optimally fluoridated water can be extremely high, due to the reconstitution of powdered infant formula with fluoridated tap water. As described, past research has shown that the mean plasma F− levels in infants residing in a community with AFDW during their first 18 months of life was 3.16 μM, with significantly higher plasma F− levels measured in infants aged 4–6 months (mean 4.22 µM with a maximum of ~ 8 µM). I have previously elucidated that this level of exposure is comparable to that observed among adult workers occupationally exposed to F− in the aluminium industry and within the range observed in human studies associated with endemic fluorosis. Importantly, the plasma F− levels are also within the reported range in human studies to cause inhibition of NKA activity in adult subjects.

−. The infant also has an immature blood-brain barrier [− is critical, as it has been shown that beryllium F− complexes are significantly more toxic than F− or beryllium alone, resulting in significantly increased production of NF-κB, which leads to increased production of PGE2. As previously elucidated PGE2 is known to inhibit NKA activity. Therefore, it is plausible that the inhibitory effects of chronic F− exposure on NKA activity may be higher in neonates and infants that adults, particularly in communities where drinking water is artificially fluoridated or where F− levels in drinking water are naturally elevated. Because newborns and infants are the group most different anatomically and physiologically from adults, they exhibit the most pronounced quantitative differences in sensitivity to chemical exposures. For this reason, the U.S.A. National Research Council [− exposure in infancy could have profound implications for neurobehavioral function and later health. Indeed, it would be naive to assume that such exposure during this critical period of development is without negative consequence to long term health. Clearly, further studies are warranted to explore these relationships. Failure to do so leads to conclusions and recommendations regarding water fluoridation that are not reliable, and therefore public health practices that are not reliably safe and effective. In addition, the fact that F has been found to activate NF-κB mRNA expression in a variety of cells types including brain, lung and kidney tissue is highly relevant. As previously described NF-κB increases production of PGE2, and PGE2 inhibits NKA activity. This elucidation may explain why NF-κB activation has been linked to many neurological disorders including autism [465,466,469,470, However, it is not enough to simply extrapolate from research among adults. As previously described, infants have a lower low glomerular filtration rate which results in higher retention of F. The infant also has an immature blood-brain barrier [ 458 459 ] and neonates and infants have lower antioxidant activities than adults [ 460 461 ]. Moreover, as previously described, infants can be exposed to beryllium fluoride complexes from the consumption of powdered in formula reconstituted with fluoridated tap water. The interaction of beryllium with Fis critical, as it has been shown that beryllium Fcomplexes are significantly more toxic than For beryllium alone, resulting in significantly increased production of NF-κB, which leads to increased production of PGE2. As previously elucidated PGE2 is known to inhibit NKA activity. Therefore, it is plausible that the inhibitory effects of chronic Fexposure on NKA activity may be higher in neonates and infants that adults, particularly in communities where drinking water is artificially fluoridated or where Flevels in drinking water are naturally elevated. Because newborns and infants are the group most different anatomically and physiologically from adults, they exhibit the most pronounced quantitative differences in sensitivity to chemical exposures. For this reason, the U.S.A. National Research Council [ 462 ] and others [ 463 ] propose using a 10-fold factor when extrapolating results from studies using adult exposures when estimating safe exposures to chemical toxins for the protection of infants. Given that this exposure has been found to occur at such a sensitive period of development and that loss of NKA activity represents an interconnected molecular function in neurodevelopmental, neuropsychiatric and neurodegenerative disorders, which is also connected with other pathophysiological states, this suggests the possibility that chronic Fexposure in infancy could have profound implications for neurobehavioral function and later health. Indeed, it would be naive to assume that such exposure during this critical period of development is without negative consequence to long term health. Clearly, further studies are warranted to explore these relationships. Failure to do so leads to conclusions and recommendations regarding water fluoridation that are not reliable, and therefore public health practices that are not reliably safe and effective. In addition, the fact that F has been found to activate NF-κB mRNA expression in a variety of cells types including brain, lung and kidney tissue is highly relevant. As previously described NF-κB increases production of PGE2, and PGE2 inhibits NKA activity. This elucidation may explain why NF-κB activation has been linked to many neurological disorders including autism [ 464 467 ], Alzheimer’s disease [ 468 471 ], Parkinson’s disease [ 472 473 ], as well as asthma, COPD, diabetes and cancer [ 474 475 ].

− exposure and loss of NKA plays a role in inflammatory lung diseases, this suggests that the significantly higher burden of childhood respiratory disorders documented in Australia, New Zealand, the Republic of Ireland (RoI) and North America compared to other developed peer countries without AFDW, as noted in large scale epidemiological studies [477,478,− exposure in infancy and F−-induced inhibition of NKA. It is important to note that in Australia, just 15% of infants are fully breastfed to six months of age [477,478, Furthermore, in view of my demonstration that Fexposure and loss of NKA plays a role in inflammatory lung diseases, this suggests that the significantly higher burden of childhood respiratory disorders documented in Australia, New Zealand, the Republic of Ireland (RoI) and North America compared to other developed peer countries without AFDW, as noted in large scale epidemiological studies [ 476 479 ], may be causally associated with chronic Fexposure in infancy and F-induced inhibition of NKA. It is important to note that in Australia, just 15% of infants are fully breastfed to six months of age [ 480 ] with 80% of infants provided with powdered infant formula at 6 months of age and 95% at 12 months [ 481 ]. Moreover, the RoI has the lowest prevalence of breastfeeding internationally [ 482 ]. A recent Irish birth cohort study found that only 14% of babies were exclusively breastfed at two months of age and just 1% at six months [ 483 ]. Similar low breastfeeding prevalence rates to Ireland have been reported among Maori and Pacific Island women in New Zealand [ 484 ] and among mothers from lower socio-economic backgrounds in the United States [ 485 ]. In 2009, Siew et al. measured the F concentrations in 21 milk based powdered infant formula products available in the United States and reported that the F levels ranged from 0.03–0.27 ppm when prepared with deionized distilled water. However, twenty-five per cent of the products were found to contain F levels ranging from 0.22–0.27 ppm F [ 161 ]. Any of these products when reconstituted with AFDW would result in F levels exceeding the UL for healthy adults. It is also important to note that in countries where AFDW is widely available the prevalence of childhood asthma has increased dramatically and disproportionally to other peer countries in recent decades and this increase parallels the increased prevalence of dental fluorosis. For example, in late 1960s and 1970s asthma prevalence among children in Australia [ 486 ] and the USA [ 487 ] was less than 4%. Similarly, in 1983, asthma prevalence among children aged 4–19 years of age was 4.4% in the RoI [ 488 ]. In contrast, Masoli et al. reported that the prevalence of current asthma symptoms among children aged 13–14 years in the USA, RoI, Australia and New Zealand were 21%, 28%, 30% and 32% respectively in 2004 [ 489 ]. Today, the burden of asthma in countries with water fluoridation is of sufficient magnitude to warrant its recognition as a priority disorder in government health strategies. As mentioned, these dramatic changes mirror almost exactly the changes in prevalence of dental fluorosis which occurred during the same period. To illustrate this point, in the RoI, Whelton et al. reported that in 1984 the prevalence of dental fluorosis among 8 and 15-year olds in the RoI was 6% and 5% respectively, increasing to 23% and 36% in 2002 [ 490 ]. Similarly, in the US the prevalence of dental fluorosis was 9% among individuals born in the period 1961–1970, compared to 41% among all US children born between 1984–1985 [ 491 ]. In Australia, the prevalence of dental fluorosis among children born in 1989/90 was reported to be 34.7% [ 492 ]. Interestingly, breastfeeding practices in France are among the lowest in Europe and lower than North America, Australia and New Zealand [ 482 ]. In comparison to the United States, RoI and Australia, a study conducted in France in 1998 reported that 97% of children had no sign of dental fluorosis, and 3% mild, very mild or doubtful fluorosis without aesthetic consequences [ 493 ]. A similar study conducted in Germany in 2007, reported that the prevalence of dental fluorosis among children aged 15 years ranged from 7.1% to 11.3% [ 494 ]. Notably, childhood asthma prevalence in Europe, including Germany and France is significantly lower than Australia, New Zealand, RoI and North America [ 476 479 ]. Despite these obvious associations, no study has ever been conducted to examine the causal association between chronic F intake in infancy and childhood asthma. Clearly, given the burden of childhood asthma in countries with AFDW such studies are warranted.

496,99,100, As previously discussed, I have also elucidated that maternal hypothyroidism in pregnancy can results in loss of NKA activity in offspring that leads to marked reduction in enzyme activity in later life. Animal models of F-induced hypothyroidism have also shown that excessive intake of F in drinking water and prenatal F intoxication of mothers induces hypothyroidism in offspring [ 495 497 ]. Interestingly, a recent animal study also found that maternal exposure to F during pregnancy and early postnatal life exposure had deleterious impact on learning and memory of offspring which was mediated by reduced mRNA expression of glutamate receptor subunits in the hippocampus [ 498 ]. Furthermore, the inhibition of glutamate receptors by F was found to occur in a dose dependent manner. Clearly, inhibition of mRNA expression of glutamate receptors can lead to a loss of glutamate receptors. Loss of glutamate receptors can subsequently lead to excessive activation due to their impaired expression, which can lead to enhanced excitotoxicity from chronic glutamate toxicity. These elucidations strongly suggest that chronic F exposure can attenuate adverse effects associated with glutamate excitotoxicity. Consistent with this hypothesis, an earlier study by Blaylock suggested that F exposure may contribute to glutamate induced excitotoxicity, thought the effects of F on mRNA expression of glutamate receptors were not known at that time [ 499 ]. While these observations may not be part of the original goal of this study, they are clearly important because, as previously discussed, loss of NKA has been suggested to enhance glutamate excitotoxicity [ 95 96 ] and glutamate excitotoxicity is associated with major psychiatric disorders [ 98 101 ], neurodegenerative diseases [ 102 ] and autism [ 103 104 ]. Furthermore, it is also important to note, that in addition to inhibition of glutamate receptors, it has also been found that maternal exposure to F during pregnancy results in inhibition of mRNA levels of M1 and M3-muscarinic acetylcholine receptors (mAChRs) in offspring [ 500 ]. Similar results have been observed in adult rodents chronically exposed to F in drinking water [ 501 ]. Interestingly, in addition to loss of NKA activity, a reduction or deficiency in mAChR have also been implicated in the pathophysiology of many major diseases of the CNS including schizophrenia [ 502 ], bipolar and major depression [ 503 ], Alzheimer’s disease [ 504 ] and ADHD [ 505 ]. Loss of M3 mAChRs has also been found to result in impairments in glucose tolerance and insulin release [ 506 ]. Taken together, these results further strengthen the hypothesis that F exposure can contribute to etiology and pathophysiology of a diverse range of disorders.

508,511,512,513,514,520,521,524,525,526, Furthermore, in this study I have described how F exposure can result in increased TSH and higher TSH is associated with iodine deficiency and hypothyroidism. Consistent with these findings, I have discussed how evidence from human studies indicate that water fluoridation is associated with increased prevalence of hypothyroidism [ 139 ]. Moreover, it is well acknowledged that iodine deficiency and maternal hypothyroidism is associated with increased risk of cognitive impairment in offspring [ 507 509 ], along with increased risk of ASD and ADHD [ 510 515 ], schizophrenia [ 516 ], epilepsy and seizures [ 517 ]; and asthma [ 518 ]. As elucidated in this study, loss of NKA activity has been found to play a central role in these disorders. This evidence further supports the hypothesis that prenatal loss of NKA is implicated in disorders associated with maternal hypothyroidism. Taken together, these findings suggest the possibility that paternal exposure to F can have epigenetic transgenerational effects on future generations. Indeed, this observation has already been observed in several rodent studies [ 519 522 ]. It is not known however, whether inhibition of NKA activity during the early postnatal period and early infancy can persist during the entire lifespan. Nonetheless, this possibility clearly exists, as several studies have found that early life exposure to environmental chemicals and stress can result in epigenetic changes by reprogramming gene expression patterns, which persist into adulthood [ 523 527 ]. Clearly, further research is warranted to elucidate whether chronic F exposure in early infancy results in epigenetic changes in gene expression. This is particularly important given the seminal research of Liu et al. where they found that chronic F exposure resulting in dental fluorosis, altered the expression of over 960 genes in children compared to controls without dental fluorosis, including 71 robustly up-regulated genes and 60 robustly down-regulated genes [ 528 ].

530,− levels and incidence of childhood-onset type 1 diabetes has been observed in Canada [− exposure may contribute to these disorders. Moreover, in this study I have elucidated that chronic F exposure has been found to inhibit the expression of PERK, which is required to stimulate WFS1 expression. I have further described how loss of WFS1 expression leads to reduced expression of the NKA β sub unit which is required for expression of NKA on plasma membranes and for enzyme activity. Thus, F inhibition of PERK can lead to reduced NKA expression and lower enzyme activity. Interestingly, loss of WFS1 activity is also associated with increased risk of psychiatric disorders [ 529 531 ], as well as juvenile-onset diabetes, progressive neurologic degeneration, and endocrine dysfunction [ 532 533 ]. The association between loss of WFS1 and juvenile diabetes is particularly interesting considering the dramatic increase in juvenile diabetes in the United States in recent decades [ 534 ], which also happens to coincide with the dramatic rise in dental fluorosis observed in the United States in recent decades [ 535 536 ]. Moreover, it should be noted that an association has been found between drinking water Flevels and incidence of childhood-onset type 1 diabetes has been observed in Canada [ 537 ]. Furthermore, a recent ecological study in the USA reported an association between water fluoridation and diabetes [ 538 ]. Revealingly, studies have also shown that PERK protects pancreatic β-cells from ER stress [ 532 ] and PERK deficiency is associated with hyperglycaemia and increased apoptosis in β-cells [ 539 ]. Taken together, these observations may explain why loss of NKA activity has been found to be associated with psychiatric disorders, metabolic syndrome and diabetes. They also provide insights into molecular mechanisms by which chronic Fexposure may contribute to these disorders.

55,56,57,58,59,− intake may be a contributory factor to the high burden of COPD and cancer in countries with artificial water fluoridation. While these associations are self-evident, they are merely observations and do not prove causality, nevertheless a causal biological mechanism exists, making the hypotheses plausible. Clearly, additional studies in this important area of investigation are also warranted. In this present study, I have also elucidated that evidence from human studies implicate loss of NKA with the pathogenesis of COPD [ 21 ]. According to WHO, COPD will move from fifth leading cause of death in 2002, to fourth place in the rank projected to 2030 worldwide [ 540 ]. Notably, in the USA, death rates for COPD doubled between 1970 and 2002 [ 541 ]. It is also evident that the RoI, New Zealand and Australia, have by far the highest prevalence rates for COPD among developed countries despite having an adult smoking prevalence well below the OECD average [ 542 ]. Interestingly, Australia, New Zealand and the RoI, also have the highest age-standardised incidence rates of cancer worldwide for men and women together being ranked 1st, 2nd and 3rd, with the USA in 5th place [ 543 544 ]. As previously described, several studies have also found that loss of NKA is implicated with carcinoma and cancer progression [ 54 60 ]. Since loss of NKA has been found to be associated with both cancer risk and inflammatory respiratory lung diseases, this suggests a plausible scenario that Fintake may be a contributory factor to the high burden of COPD and cancer in countries with artificial water fluoridation. While these associations are self-evident, they are merely observations and do not prove causality, nevertheless a causal biological mechanism exists, making the hypotheses plausible. Clearly, additional studies in this important area of investigation are also warranted.

45,46,47,48,49,50,51,52,547,548,549,552,553,554,555,− can induce TSH secretion. Therefore, F− must also induce TRH secretion. This elucidation may explain why increased TRH release is associated with lower NKA activity. Moreover, these findings are consistent with the hypothesis that F exposure contributes to pathological states associated with loss of NKA activity. Past studies have also shown that reduced NKA activity may underlie the pathophysiological aspects linked to the prehypertensive status in humans [ 40 545 ]. Additionally, it has been widely documented that inhibition of NKA activity is associated with hypertension [ 44 53 ]. Again, several human studies have found that exposure to excessive F is closely associated with hypertension [ 546 550 ]. Similar observations have been observed in experimental studies with rodents [ 551 556 ]. Interestingly, activation of the TRH system, with increased production of TRH and an upregulation of its receptors has also been implicated in the pathogenesis of hypertension [ 557 ]. As previously, described, TRH regulates the secretion of TSH and stimulates the secretion of DA, which can lead to inhibition of NKA activity. As previously described, evidence from human studies have shown that Fcan induce TSH secretion. Therefore, Fmust also induce TRH secretion. This elucidation may explain why increased TRH release is associated with lower NKA activity. Moreover, these findings are consistent with the hypothesis that F exposure contributes to pathological states associated with loss of NKA activity.

15,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,−-induced loss of NKA activity could lead to pathological states or further contribute to the severity of a diverse range of inflammatory diseases/disorders associated with loss of enzyme activity. Of particular note, while loss of NKA activity has been shown to be associated with autism spectrum disorder’s, research has also shown that inhibition of NKA activity can ultimately lead to a leaky and dysfunctional epithelium associated with chronic inflammation [− exposure may include contributing to the burden and severity of ASD. In this study, I have also elucidated how loss of NKA leads to downregulation of AMPA receptor, which leads to synaptic transmission defects, and consequently cognitive impairment [− has recently been found to be associated with cognitive impairment and increased risk of ADHD in offspring [ Furthermore, in this current study, I have provided compelling evidence that NKA activity is vital for normal brain development and loss of NKA activity is associated with cognitive impairment, neurological and developmental disorders [ 14 86 ]. Indeed, loss of NKA activity represents an interconnected molecular function in neurodevelopmental and neuropsychiatric and neurodegenerative disorders including; Down syndrome, Alzheimer’s, Parkinson’s and Huntington’s disease, as well as epilepsy, autism, schizophrenia, mood and depressive disorders. Furthermore, evidence has been presented that loss of NKA activity is also associated with allergic diseases, blood diseases, autoimmune diseases, metabolic disorders, male infertility and cardiovascular disease. Therefore, it is plausible that F-induced loss of NKA activity could lead to pathological states or further contribute to the severity of a diverse range of inflammatory diseases/disorders associated with loss of enzyme activity. Of particular note, while loss of NKA activity has been shown to be associated with autism spectrum disorder’s, research has also shown that inhibition of NKA activity can ultimately lead to a leaky and dysfunctional epithelium associated with chronic inflammation [ 558 ]. Accumulating evidence demonstrates that gastrointestinal inflammation and increased permeability of the intestinal tract, referred to as a “leaky gut” is a hallmark of ASD and the severity of gastrointestinal symptoms relate to the severity of ASD [ 559 ]. This suggests that a potential adverse effect of chronic Fexposure may include contributing to the burden and severity of ASD. In this study, I have also elucidated how loss of NKA leads to downregulation of AMPA receptor, which leads to synaptic transmission defects, and consequently cognitive impairment [ 97 ]. I have further described how loss of AMPA receptors have been found to result in early-onset motor deficits, hyperactivity, cognitive defects and behavioural seizures [ 105 ]. I have further elucidated that these findings suggest a possible causal mechanism explaining how loss of NKA activity is associated with childhood neurodevelopmental disorders such as ADHD and ASD. Consistent with this, several studies have already demonstrated an association between cognitive impairment and loss of NKA activity and between loss of NKA and ASD. Importantly, these findings may also explain why exposure to fluoridated water has been found to be associated with increased prevalence of ADHD in the USA [ 560 ]. Interestingly, one of the most common difficulties in children with epilepsy is ADHD. Indeed, in children with epilepsy (seizures), ADHD has been found to be present in 20–50% of patients [ 561 ]. These findings further support the hypothesis that downregulation of AMPA receptors which results from loss of NKA activity is a factor in the pathogenesis of ADHD disorders. Furthermore, these findings may elucidate a key mechanism by which prenatal exposure to Fhas recently been found to be associated with cognitive impairment and increased risk of ADHD in offspring [ 562 563 ].

512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,567, Increasing evidence also suggests that maternal iodine deficiency and hypothyroidism is associated with increased risk of cognitive impairment, neurodevelopmental and neuropsychiatric disorders in offspring including ADHD [ 511 565 ], ASD [ 566 568 ], behavioural seizures [ 517 ] and schizophrenia [ 516 ]. Moreover, maternal iodine deficiency and hypothyroidism is also associated with increased risk of asthma [ 518 ] and hypertension [ 569 ] in offspring. As elucidated in this study, loss of NKA activity appears to be a critical contributor to the pathophysiological underpinnings of these disorders. These findings suggest that loss of NKA activity is associated with iodine deficiency disorders. Consistent with this, I have previously elucidated that NKA is essential for NIS functionality, iodine uptake and metabolism. Indeed, I recently reported that inhibition of NKA contributes to iodine deficiency disorders [ 110 ]. As highlighted above, there is also an association between hypothyroidism and loss of NKA activity, suggesting a negative feedback mechanism that may further decrease enzyme activity. As elucidated in this current study, this mechanism appears to be driven by increased TRH secretion and DA release which leads to inhibition of enzyme activity.