Chronic Fluoride Exposure and the Risk of Autism Spectrum Disorder

Abstract

The continuous rise of autism spectrum disorder (ASD) prevalent in the past few decades is causing an increase in public health and socioeconomic concern. A consensus suggests the involvement of both genetic and environmental factors in the ASD etiopathogenesis. Fluoride (F) is rarely recognized among the environmental risk factors of ASD, since the neurotoxic effects of F are not generally accepted. Our review aims to provide evidence of F neurotoxicity. We assess the risk of chronic F exposure in the ASD etiopathology and investigate the role of metabolic and mitochondrial dysfunction, oxidative stress and inflammation, immunoexcitotoxicity, and decreased melatonin levels. These symptoms have been observed both after chronic F exposure as well as in ASD. Moreover, we show that F in synergistic interactions with aluminum’s free metal cation (Al3+) can reinforce the pathological symptoms of ASD. This reinforcement takes place at concentrations several times lower than when acting alone. A high ASD prevalence has been reported from countries with water fluoridation as well as from endemic fluorosis areas. We suggest focusing the ASD prevention on the reduction of the F and Al3+ burdens from daily life.

Full text

International Journal of
Environmental Research
and Public Health
Review
Chronic Fluoride Exposure and the Risk of Autism
Spectrum Disorder
Anna Strunecka * and Otakar Strunecky
The Institute of Technology and Business, Okružní517/10, 370 01 ˇ
CeskéBudˇejovice, Czech Republic;
otakar.strunecky@gmail.com
*Correspondence: anna.strunecka@gmail.com; Tel.: +420-272743573
Received: 7 July 2019; Accepted: 12 September 2019; Published: 16 September 2019


Abstract:
The continuous rise of autism spectrum disorder (ASD) prevalent in the past few decades
is causing an increase in public health and socioeconomic concern. A consensus suggests the
involvement of both genetic and environmental factors in the ASD etiopathogenesis. Fluoride (F)
is rarely recognized among the environmental risk factors of ASD, since the neurotoxic effects of
F are not generally accepted. Our review aims to provide evidence of F neurotoxicity. We assess
the risk of chronic F exposure in the ASD etiopathology and investigate the role of metabolic and
mitochondrial dysfunction, oxidative stress and inflammation, immunoexcitotoxicity, and decreased
melatonin levels. These symptoms have been observed both after chronic F exposure as well as in
ASD. Moreover, we show that F in synergistic interactions with aluminum’s free metal cation (Al
3+
)
can reinforce the pathological symptoms of ASD. This reinforcement takes place at concentrations
several times lower than when acting alone. A high ASD prevalence has been reported from countries
with water fluoridation as well as from endemic fluorosis areas. We suggest focusing the ASD
prevention on the reduction of the F and Al3+burdens from daily life.
Keywords:
autism spectrum disorder; ASD prevalence; aluminum; endemic fluorosis; chronic
fluoride exposure; immunoexcitotoxicity; neurotoxicity; socioeconomic status; water fluoridation
1. Introduction
Autism spectrum disorder (ASD) covers a range of heterogeneous neurodevelopmental conditions,
characterized by persistent deficits in social and communication interactions and presenting with
repetitive, stereotypic interests and behaviors. ASD incorporates autistic disorder, Asperger’s disorder,
childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified [
1
–
4
].
The word autism is traditionally used interchangeably with ASD. ASD begins early in childhood and
tends to persist into adolescence and adulthood.
ASD is the fastest-growing developmental disability in the last two decades in all countries over
the world. While in the period 1960–1989 autism was a rare disease with one to four per 10,000 in
both Europe and America, the WHO estimates that 62 per 10,000 children (one in 160) in the world
have ASD currently [
2
]. The rise in the ASD rate has sparked fears of an ASD epidemic, mainly in
the United States (US). In US children aged 3–17 years, the estimated prevalence of ASD diagnosis,
based on parental reports, is one in 40 [5].
Understanding of the ASD etiopathogenesis is therefore urgently warranted to find an effective
prevention. A consensus suggests that the ASD etiopathogenesis is presumably multifactorial and
results from very complex interactions between genetic and environmental factors [
6
]. The high ASD
prevalence has been reported from countries with artificial water fluoridation as well as from countries
with endemic fluorosis areas [
7
]. On the contrary, a lower ASD prevalence has been reported from
some member states of the European Union (EU) [
8
–
10
], where water fluoridation was banned in
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Int. J. Environ. Res. Public Health 2019,16, 3431 2 of 21
the 1970s–1990s. Therefore, in our previous papers, we suggested that fluoride (F) might be among
the key environmental factors in the ASD etiopathogenesis [
7
,
11
]. F is not currently recognized as
a causative culprit of ASD, since its neurotoxic effects are not generally accepted. We assessed the risks
of chronic F exposure in the ASD etiopathology regarding the F-induced effects on metabolism and
mitochondrial dysfunction, oxidative stress and inflammation, immunoexcitotoxicity, and melatonin
levels. Such effects have all been observed in people with ASD. Our review aims to provide evidence of
F neurotoxicity. Moreover, F in the synergistic action with aluminum’s free cation (Al
3+
) in molecules of
aluminofluoride complexes can trigger various metabolic and pathological symptoms. This triggering
takes place at F concentrations several times lower than F acting alone [12–14].
Fluoridation of drinking water in addition to the extensive use of F in medicine and industry,
started the era of supplementation of the living environment with these ions as never before in the
history. It must be pointed out that F does not have any known essential function in human physiology
and development. No signs of F deficiency have been identified [15].
2. Fluoride-Induced ASD Symptoms
F has long been known to influence the activity of various enzymes
in vitro
[
14
]. We present
evidence that F induces oxidative stress, inflammation, and immunoexcitotoxicity. There is a link
between a known effect of F on melatonin synthesis in the pineal gland [
16
] and significantly reduced
melatonin synthesis in ASD [17].
2.1. Enzymes and Mitochondrial Disorders
F alters, in micromolar and millimolar concentrations,
in vitro
activities of many important
enzymes. We reviewed that F has been found to inhibit 22 various enzymes, such as enolase,
pyruvate kinase, lactate dehydrogenase, Na
+
, and K
+
-ATPase [
14
]. On the other hand, 20 enzymes are
stimulated by F, including adenylyl cyclase, alanine transaminase, lactate dehydrogenase, and glycogen
phosphorylase. It is therefore evident that F alters the energy metabolism and might trigger numerous
metabolic disorders.
It might seem that F in whole organisms may not reach the concentrations that were used in the
laboratory experiments
in vitro
. Some studies found that under certain circumstances, the inhibitory
or stimulatory impact of F can be more pronounced at a lower level of intake than at a higher
level (paradoxical dose–response effects) [
18
]. It is therefore difficult to predict the actual effective
concentrations of F in vivo.
However, autistic patients display peripheral markers of mitochondrial energy metabolism
dysfunctions, such as elevated lactate and alanine levels in the blood (Figure 1) [
19
]. Even though
alterations in the mitochondrial and cellular energy metabolism are not specific for ASD, they indicate
the potential pathological events that might be induced by F.
Int. J. Environ. Res. Public Health 2019, 16, x 2 of 21
banned in the 1970s–1990s. Therefore, in our previous papers, we suggested that fluoride (F) might
be among the key environmental factors in the ASD etiopathogenesis [7,11]. F is not currently
recognized as a causative culprit of ASD, since its neurotoxic effects are not generally accepted. We
assessed the risks of chronic F exposure in the ASD etiopathology regarding the F-induced effects on
metabolism and mitochondrial dysfunction, oxidative stress and inflammation,
immunoexcitotoxicity, and melatonin levels. Such effects have all been observed in people with ASD.
Our review aims to provide evidence of F neurotoxicity. Moreover, F in the synergistic action with
aluminum's free cation (Al3+) in molecules of aluminofluoride complexes can trigger various
metabolic and pathological symptoms. This triggering takes place at F concentrations several times
lower than F acting alone [12–14].
Fluoridation of drinking water in addition to the extensive use of F in medicine and industry,
started the era of supplementation of the living environment with these ions as never before in the
history. It must be pointed out that F does not have any known essential function in human
physiology and development. No signs of F deficiency have been identified [15].
2. Fluoride-Induced ASD Symptoms
F has long been known to influence the activity of various enzymes in vitro [14]. We present
evidence that F induces oxidative stress, inflammation, and immunoexcitotoxicity. There is a link
between a known effect of F on melatonin synthesis in the pineal gland [16] and significantly reduced
melatonin synthesis in ASD [17].
2.1. Enzymes and Mitochondrial Disorders
F alters, in micromolar and millimolar concentrations, in vitro activities of many important
enzymes. We reviewed that F has been found to inhibit 22 various enzymes, such as enolase, pyruvate
kinase, lactate dehydrogenase, Na+, and K+-ATPase [14]. On the other hand, 20 enzymes are
stimulated by F, including adenylyl cyclase, alanine transaminase, lactate dehydrogenase, and
glycogen phosphorylase. It is therefore evident that F alters the energy metabolism and might trigger
numerous metabolic disorders.
It might seem that F in whole organisms may not reach the concentrations that were used in the
laboratory experiments in vitro. Some studies found that under certain circumstances, the inhibitory
or stimulatory impact of F can be more pronounced at a lower level of intake than at a higher level
(paradoxical dose–response effects) [18]. It is therefore difficult to predict the actual effective
concentrations of F in vivo.
However, autistic patients display peripheral markers of mitochondrial energy metabolism
dysfunctions, such as elevated lactate and alanine levels in the blood (Figure 1) [19]. Even though
alterations in the mitochondrial and cellular energy metabolism are not specific for ASD, they
indicate the potential pathological events that might be induced by F.
Figure 1. Patients with autism spectrum disorder (ASD) have increased levels of blood lactate,
alanine, and glutamate. LDH—lactate dehydrogenase; ALT—alanine transaminase.
Figure 1.
Patients with autism spectrum disorder (ASD) have increased levels of blood lactate, alanine,
and glutamate. LDH—lactate dehydrogenase; ALT—alanine transaminase.
Evidence of mitochondrial dysfunction was reported among 73 Egyptian children with autism
who were compared with another 73 healthy age-matched children. Serum lactate, pyruvate, creatine

Int. J. Environ. Res. Public Health 2019,16, 3431 3 of 21
kinase, L-carnitine, lactate dehydrogenase, pyruvate kinase, alanine transaminase, and aspartate
transaminase were measured [
20
]. The plasma levels of lactate and the lactate/pyruvate ratio were
significantly higher among autistic children than in the control group.
Mitochondrial dysfunction and its link to ASD symptoms were investigated [
21
]. The authors
found that overall, 62% of 76 ASD individuals showed mitochondrial enzyme activity outside the
control rates of individuals without significant chronic health conditions. This study demonstrated
for the first time that such variations are related to the social function and behavior of ASD persons.
Clinical aspects of mitochondrial dysfunction in ASD might include unusual neurodevelopmental
regression, especially if triggered by an inflammatory event, gastrointestinal symptoms, seizures,
motor delays, fatigue, and lethargy [22,23].
2.2. A Synergy of F and Al3+in the ASD Etiopathology
Fluoride reacts with trace amounts of Al
3+
within body fluids and produces aluminofluoride
complexes [
12
–
14
,
24
,
25
]. Aluminum tetrafluoride (AlF
4−
) is a molecule whose shape and physical
properties closely resemble those of the phosphate anion, PO
43−
. Since the 1980s, AlF
4−
has been
widely used in laboratory investigations as an analog of phosphate groups to study phosphoryl transfer
reactions and heterotrimeric G protein involvement in signal transduction. However, an important
functional difference between a phosphate group and the structurally analogous AlF
4−
exists [
26
].
In phosphate, oxygen is covalently bound to the phosphorus and does not exchange with oxygen from
a solvent, while in AlF
4−
, the bonding between the electropositive Al
3+
and the highly electronegative
F is more ionic, allowing F in the bound complex to exchange freely with F ions in solution. While the
reaction of a bound phosphate with orthophosphate is endergonic and slow, the corresponding reaction
with AlF4−is rapid and spontaneous.
AlF
4−
binds ionically to the terminal oxygen of ADP or GDP
β
-phosphate. ADP or GDP
could, therefore, form a complex with AlF
4−
that imitates ATP or GTP in their effect on protein
conformation. This effect causes a structural change that locks the site and prevents the release of the
γ
-phosphate. The interactions of AlF
4−
with signaling cascades of G protein-coupled receptors (GPCRs)
have been documented by several authors using both biochemical as well as X-ray crystallographic
analysis [27–29].
Physiological agonists of GPCRs include neurotransmitters and hormones, such as glutamate,
dopamine, serotonin, melatonin, acetylcholine, and neuropeptides [
30
]. AlF
4−
is a molecule sending
a false message. The false signal of AlF
4−
is amplified during its conversion into a functional
response (Figure 2). Even very low F concentration in synergy with Al
3+
can exacerbate alterations
in neurotransmission and hormonal regulation. AlF
4−
can thus evoke a whole network of
pathological events in micromolar concentrations. It has the potential to modulate neurodevelopment,
brain structure, structural plasticity, as well as higher neuronal functions. The detailed description of
molecular targets of AlF
4−
relevant to the ASD pathogenesis was discussed in our previous review [
11
].
Recently, Al
3+
has been regarded as nontoxic, and it is a component of pot water, food, beverages,
medicine, and cosmetics.

Int. J. Environ. Res. Public Health 2019,16, 3431 4 of 21
Int. J. Environ. Res. Public Health 2019, 16, x 4 of 21
Figure 2. Amplification of signaling pathways by AlF4−. Its message is greatly amplified during the
conversion into the functional response of a cell. Effector enzymes are adenylyl cyclase or
phospholipase C. The second messenger molecule could be cAMP, inositol 1,4,5- trisphosphate, and
diacylglycerol.
2.3. The Effects of F in Oxidative Stress, Inflammation, and Immunoexcitotoxicity
Oxidative stress, defined as an imbalance between oxidants and antioxidants in favor of the
oxidants, represents the link between genetic, epigenetic, immunological, and environmental factors
underlying ASD. The glutathione (GSH) redox system is most important for reducing oxidative
stress. A decrease in GSH is one of the best-documented biochemical changes in plasma, immune
cells, and brains of children with ASD [11,20,31–34]. F exposure can reduce the cellular level of GSH
and induce oxidative stress (Figure 3).
Figure 3. The glutathione redox system. Glutathione (GSH), a radical scavenger, is converted to
oxidized glutathione (GSSG) through GSH peroxidase (GPx) and converted back to GSH by GSH
reductase (GR). GSH can detoxify hydrogen peroxide (H2O2), preventing the formation of free radical
generation and lipid peroxidation products.
The oxidative stress in the pathology of endemic fluorosis in the blood of the population living
in the areas with severe coal-burning endemic fluorosis in China was also reported [7,35]. The
Figure 2.
Amplification of signaling pathways by AlF
4−
. Its message is greatly amplified during the
conversion into the functional response of a cell. Effector enzymes are adenylyl cyclase or phospholipase
C. The second messenger molecule could be cAMP, inositol 1,4,5- trisphosphate, and diacylglycerol.
2.3. The Effects of F in Oxidative Stress, Inflammation, and Immunoexcitotoxicity
Oxidative stress, defined as an imbalance between oxidants and antioxidants in favor of the
oxidants, represents the link between genetic, epigenetic, immunological, and environmental factors
underlying ASD. The glutathione (GSH) redox system is most important for reducing oxidative stress.
A decrease in GSH is one of the best-documented biochemical changes in plasma, immune cells,
and brains of children with ASD [
11
,
20
,
31
–
34
]. F exposure can reduce the cellular level of GSH and
induce oxidative stress (Figure 3).
Int. J. Environ. Res. Public Health 2019, 16, x 4 of 21
Figure 2. Amplification of signaling pathways by AlF4−. Its message is greatly amplified during the
conversion into the functional response of a cell. Effector enzymes are adenylyl cyclase or
phospholipase C. The second messenger molecule could be cAMP, inositol 1,4,5- trisphosphate, and
diacylglycerol.
2.3. The Effects of F in Oxidative Stress, Inflammation, and Immunoexcitotoxicity
Oxidative stress, defined as an imbalance between oxidants and antioxidants in favor of the
oxidants, represents the link between genetic, epigenetic, immunological, and environmental factors
underlying ASD. The glutathione (GSH) redox system is most important for reducing oxidative
stress. A decrease in GSH is one of the best-documented biochemical changes in plasma, immune
cells, and brains of children with ASD [11,20,31–34]. F exposure can reduce the cellular level of GSH
and induce oxidative stress (Figure 3).
Figure 3. The glutathione redox system. Glutathione (GSH), a radical scavenger, is converted to
oxidized glutathione (GSSG) through GSH peroxidase (GPx) and converted back to GSH by GSH
reductase (GR). GSH can detoxify hydrogen peroxide (H2O2), preventing the formation of free radical
generation and lipid peroxidation products.
The oxidative stress in the pathology of endemic fluorosis in the blood of the population living
in the areas with severe coal-burning endemic fluorosis in China was also reported [7,35]. The
Figure 3.
The glutathione redox system. Glutathione (GSH), a radical scavenger, is converted to
oxidized glutathione (GSSG) through GSH peroxidase (GPx) and converted back to GSH by GSH
reductase (GR). GSH can detoxify hydrogen peroxide (H
2
O
2
), preventing the formation of free radical
generation and lipid peroxidation products.
The oxidative stress in the pathology of endemic fluorosis in the blood of the population living in
the areas with severe coal-burning endemic fluorosis in China was also reported [
7
,
35
]. The activities
of superoxide dismutase, GSH reductase (GR), and catalase were significantly decreased in blood
plasma and erythrocytes of children and adults with a low level of GSH.

Int. J. Environ. Res. Public Health 2019,16, 3431 5 of 21
GSH synthesis and intracellular redox balance are linked to folate and methylation metabolism,
metabolic pathways that have also been shown to be abnormal in ASD [
33
,
36
] (Figure 4).
DNA methylation changes in autistic cerebral cortex regions were described [
37
]. Additionally,
along with F, Al3+disrupts enzymes involved in the methylation pathways [11,14].
Int. J. Environ. Res. Public Health 2019, 16, x 5 of 21
activities of superoxide dismutase, GSH reductase (GR), and catalase were significantly decreased in
blood plasma and erythrocytes of children and adults with a low level of GSH.
GSH synthesis and intracellular redox balance are linked to folate and methylation metabolism,
metabolic pathways that have also been shown to be abnormal in ASD [33,36] (Figure 4). DNA
methylation changes in autistic cerebral cortex regions were described [37]. Additionally, along
with F, Al3+ disrupts enzymes involved in the methylation pathways [11,14].
Figure 4. Methylation and transsulfuration metabolism in ASD. SAM—S-adenosyl methionine,
SAH—S-adenosylhomocysteine, GSH—glutathione, GSSG—oxidized glutathione, ATP—adenosine
triphosphate.
Reduced levels of GSH greatly increase the sensitivity of neurons and astrocytes to oxidative
stress and excitotoxicity. Oxidative stress induces the secretion of many vasoactive and
proinflammatory molecules, which leads to neuroinflammation. The initial reaction to inflammation
may be the activation of microglia, the resident immune cells in the brain [11,38]. Several groups of
researchers and clinicians have reported inflammation in the brain of both young and old individuals
with ASD. Vargas et al. [39] demonstrated an active neuroinflammatory process in brain tissues and
the cerebrospinal fluid (CSF) in autistic patients. Brain tissues obtained at autopsy from 11 patients
with autism were used for morphological studies. Fresh-frozen tissues available from seven patients
and CSF from six living autistic patients were used for cytokine protein profiling.
Immunocytochemical studies showed marked activation of microglia and astroglia, and cytokine
profiling in the cerebral cortex, white matter, and notably in the cerebellum. CSF showed a unique
proinflammatory profile of cytokines. These findings indicated that innate neuroimmune reactions
play a pathogenic role, at least in some ASD patients.
Blaylock was the first to explain that excitotoxicity is the central mechanism of F neurotoxicity
and the central mechanism in the ASD pathogenesis [40]. Excitotoxicity is caused by excess levels
of glutamate and over-activation of ionotropic glutamate receptors on neuronal membranes, leading
to ionic influx, disruption of the energy metabolism, and potential neuronal death. In 2008, Blaylock
coined the term immunoexcitotoxicity to describe the link between inflammation and excitotoxicity
[41]. The complex network of immunoexcitotoxic processes in the pathogenesis of ASD has been
explained further in detail [11,42]. Both F and Al3+ induce oxidative stress, microglial activation, and
Figure 4.
Methylation and transsulfuration metabolism in ASD. SAM—S-adenosyl
methionine, SAH—S-adenosylhomocysteine, GSH—glutathione, GSSG—oxidized glutathione,
ATP—adenosine triphosphate.
Reduced levels of GSH greatly increase the sensitivity of neurons and astrocytes to oxidative stress
and excitotoxicity. Oxidative stress induces the secretion of many vasoactive and proinflammatory
molecules, which leads to neuroinflammation. The initial reaction to inflammation may be the
activation of microglia, the resident immune cells in the brain [
11
,
38
]. Several groups of researchers
and clinicians have reported inflammation in the brain of both young and old individuals with
ASD. Vargas et al. [
39
] demonstrated an active neuroinflammatory process in brain tissues and the
cerebrospinal fluid (CSF) in autistic patients. Brain tissues obtained at autopsy from 11 patients with
autism were used for morphological studies. Fresh-frozen tissues available from seven patients and
CSF from six living autistic patients were used for cytokine protein profiling. Immunocytochemical
studies showed marked activation of microglia and astroglia, and cytokine profiling in the cerebral
cortex, white matter, and notably in the cerebellum. CSF showed a unique proinflammatory profile of
cytokines. These findings indicated that innate neuroimmune reactions play a pathogenic role, at least
in some ASD patients.
Blaylock was the first to explain that excitotoxicity is the central mechanism of F neurotoxicity
and the central mechanism in the ASD pathogenesis [
40
]. Excitotoxicity is caused by excess levels of
glutamate and over-activation of ionotropic glutamate receptors on neuronal membranes, leading to
ionic influx, disruption of the energy metabolism, and potential neuronal death. In 2008, Blaylock
coined the term immunoexcitotoxicity to describe the link between inflammation and excitotoxicity [
41
].
The complex network of immunoexcitotoxic processes in the pathogenesis of ASD has been explained
further in detail [
11
,
42
]. Both F and Al
3+
induce oxidative stress, microglial activation, and the

Int. J. Environ. Res. Public Health 2019,16, 3431 6 of 21
production of inflammatory cytokines and affect neurotransmission. AlF
4−
molecules are triggers of
immunoexcitotoxicity and might have a key role in ASD pathogenesis.
2.4. Decreased Melatonin as the Potential Marker of ASD
Sleep problems and the early onset of puberty in patients with ASD suggest abnormalities in
the melatonin physiology and dysfunctions of the pineal gland. F accumulates in the pineal gland of
gerbils treated with fluoridated water for 16 weeks until the time of sexual maturation [
16
]. Animals
excreted less melatonin metabolite in their urine and took a shorter time to reach puberty. The human
pineal gland avidly attracts F from the bloodstream because the gland calcifies physiologically with
hydroxyapatite. This change takes place even in childhood. F in the apatite crystals averaged about
9000 ppm and in one case went as high as 21,000 ppm in the pineal glands from 11 human corpses [
43
].
Decreased levels of melatonin in the blood or the urine have been reported as very common
features in individuals with ASD compared to typically developing controls. Researchers estimate that
50%–80% of children with ASD suffer from sleep disorders, particularly insomnia [44].
The team of twelve French researchers assessed plasma melatonin, whole-blood serotonin,
and platelet N-acetyl serotonin in 278 patients with ASD, their 506 first-degree relatives, and 416
sex- and age-matched controls [
17
]. They confirmed a deficit in melatonin in 51% (45%–57%) as well
as hyperserotonemia in 40% (35%–46%) of ASD patients. The melatonin deficit was significantly
associated with insomnia. Biochemical impairments were also observed in the first-degree relatives
of patients.
The disruption of the serotonin-N-acetyl serotonin-melatonin pathway (Figure 5) was therefore
suggested as a biomarker for ASD [
17
]. To support this hypothesis, another study also investigated the
melatonin synthesis in post-mortem pineal glands of 9 ASD patients and 22 controls, in gut samples
(11 patients and 13 controls), and blood platelets from 239 ASD patients and 278 controls. The results
confirmed the enzymatic mechanism for melatonin deficit in ASD in the pineal gland as well as in
the gut and platelets of patients [
45
]. In a cohort of children drinking water containing 2.5 ppm F,
serum serotonin was also increased as compared to controls [46].
Int. J. Environ. Res. Public Health 2019, 16, x 6 of 21
the production of inflammatory cytokines and affect neurotransmission. AlF4− molecules are triggers
of immunoexcitotoxicity and might have a key role in ASD pathogenesis.
2.4. Decreased Melatonin as the Potential Marker of ASD
Sleep problems and the early onset of puberty in patients with ASD suggest abnormalities in the
melatonin physiology and dysfunctions of the pineal gland. F accumulates in the pineal gland of gerbils
treated with fluoridated water for 16 weeks until the time of sexual maturation [16]. Animals excreted less
melatonin metabolite in their urine and took a shorter time to reach puberty. The human pineal gland
avidly attracts F from the bloodstream because the gland calcifies physiologically with hydroxyapatite.
This change takes place even in childhood. F in the apatite crystals averaged about 9000 ppm and in one
case went as high as 21,000 ppm in the pineal glands from 11 human corpses [43].
Decreased levels of melatonin in the blood or the urine have been reported as very common
features in individuals with ASD compared to typically developing controls. Researchers estimate
that 50%–80% of children with ASD suffer from sleep disorders, particularly insomnia [44].
The team of twelve French researchers assessed plasma melatonin, whole-blood serotonin, and
platelet N-acetyl serotonin in 278 patients with ASD, their 506 first-degree relatives, and 416 sex- and
age-matched controls [17]. They confirmed a deficit in melatonin in 51% (45%–57%) as well as
hyperserotonemia in 40% (35%–46%) of ASD patients. The melatonin deficit was significantly
associated with insomnia. Biochemical impairments were also observed in the first-degree relatives
of patients.
The disruption of the serotonin-N-acetyl serotonin-melatonin pathway (Figure 5) was therefore
suggested as a biomarker for ASD [17]. To support this hypothesis, another study also investigated
the melatonin synthesis in post-mortem pineal glands of 9 ASD patients and 22 controls, in gut
samples (11 patients and 13 controls), and blood platelets from 239 ASD patients and 278 controls.
The results confirmed the enzymatic mechanism for melatonin deficit in ASD in the pineal gland as
well as in the gut and platelets of patients [45]. In a cohort of children drinking water containing
2.5 ppm F, serum serotonin was also increased as compared to controls [46].
Figure 5. Serotonin is converted to melatonin in dark through the action of two enzymes: serotonin
N-acetyltransferase and hydroxyindole O-methyltransferase. F inhibits hydroxyindole O-
methyltransferase [47].
Some studies have observed a correlation between abnormal melatonin concentrations and the
severity of autistic behaviors [44,48]. Babies with the lowest melatonin production had the most
severe neurobehavioral problems. Nocturnal excretion of melatonin was negatively correlated with
the problems in the level of verbal language, imitative social play, and repetitive use of objects.
Melatonin exhibits extraordinary diversity in terms of its functions (Figure 6).
Figure 5.
Serotonin is converted to melatonin in dark through the action of two enzymes:
serotonin N-acetyltransferase and hydroxyindole O-methyltransferase. F inhibits hydroxyindole
O-methyltransferase [47].
Some studies have observed a correlation between abnormal melatonin concentrations and the
severity of autistic behaviors [
44
,
48
]. Babies with the lowest melatonin production had the most
severe neurobehavioral problems. Nocturnal excretion of melatonin was negatively correlated with
the problems in the level of verbal language, imitative social play, and repetitive use of objects.
Melatonin exhibits extraordinary diversity in terms of its functions (Figure 6).

Int. J. Environ. Res. Public Health 2019,16, 3431 7 of 21
Int. J. Environ. Res. Public Health 2019, 16, x 7 of 21
Figure 6. Most important physiological actions of melatonin. Reducing the level of melatonin, F (in
yellowgreen) interferes with all indicated events. Image used from Wikimedia Commons according
to GNU Free Documentation License.
Melatonin is an important modulator of mitochondrial metabolism, digestive functions, and
immunity. It also has a powerful antioxidant effect and increases the levels of several antioxidant
enzymes in the brain. F entering the pineal gland and reducing the melatonin synthesis can thus evoke
several disruptions of homeostasis, development, and behavior. When combined with the reduced energy
production, one can reasonably expect an increase in the vulnerability of neurons and astrocytes to
excitotoxicity and oxidative stress.
The recent data for 2065 ASD children aged 4–18 years with sleep disturbance were analyzed to
investigate the variation in genes around melatonin synthesis [49]. No significant associations were found
between 25 circadian gene variants and sleep problems in this sample of children with ASD. This study,
therefore, does not show genetic abnormalities, suggesting that the changes in melatonin synthesis are
either secondary to alterations in regulatory pathways or due to F-induced epigenetic changes.
3. Fluoride as an Environmental Neurotoxin in the ASD Etiopathogenesis
Research into environmental risk factors for ASD has risen dramatically. According to recent
evidence, up to 40%–50% of the variance in ASD liability might be determined by new
ecotoxicological factors [6]. How these toxicant exposures may contribute to ASD remains a
significant knowledge gap. According to a report by UNICEF in December 1999, fluorosis is endemic
in at least 25 countries, such as China and India, Indonesia, South Africa, Iran, and others [50].
F has been linked to neurological and psychiatric disturbances since the 1930s [51]. A sharp
decline in mental activity, memory impairment, difficulties with concentration and thinking, and
reduced ability to write were observed in aluminum smelter workers and persons living near a
factory where F was in high concentrations in the atmosphere [52]. A review of medical evidence in
500 people affected by chronic F intake from artificially fluoridated water appeared in 1977 [53]. The
authors made a list of the clinical features, such as chronic fatigue, headaches, loss of mental acuity
and the ability to concentrate, depression, a diminished ability to focus, gastrointestinal symptoms
and deterioration of muscular coordination. Carlson's concerns about what increased F levels would
do to the developing brain of newborn infants [54] led to the refusal of water fluoridation in most of
the European countries. A comprehensive historical review and over 50 papers regarding F
neurotoxicity have been provided in the e-book Fluoride Fatigue [51].
The surprising observation brought the study of behavior and brain F levels in rats after sodium
fluoride (NaF) exposures during late gestation, at weaning, and in adults [55]. Rats exposed
Figure 6.
Most important physiological actions of melatonin. Reducing the level of melatonin, F (in
yellowgreen) interferes with all indicated events. Image used from Wikimedia Commons according to
GNU Free Documentation License.
Melatonin is an important modulator of mitochondrial metabolism, digestive functions,
and immunity. It also has a powerful antioxidant effect and increases the levels of several antioxidant
enzymes in the brain. F entering the pineal gland and reducing the melatonin synthesis can thus evoke
several disruptions of homeostasis, development, and behavior. When combined with the reduced
energy production, one can reasonably expect an increase in the vulnerability of neurons and astrocytes
to excitotoxicity and oxidative stress.
The recent data for 2065 ASD children aged 4–18 years with sleep disturbance were analyzed
to investigate the variation in genes around melatonin synthesis [
49
]. No significant associations
were found between 25 circadian gene variants and sleep problems in this sample of children with
ASD. This study, therefore, does not show genetic abnormalities, suggesting that the changes in
melatonin synthesis are either secondary to alterations in regulatory pathways or due to F-induced
epigenetic changes.
3. Fluoride as an Environmental Neurotoxin in the ASD Etiopathogenesis
Research into environmental risk factors for ASD has risen dramatically. According to recent
evidence, up to 40%–50% of the variance in ASD liability might be determined by new ecotoxicological
factors [
6
]. How these toxicant exposures may contribute to ASD remains a significant knowledge
gap. According to a report by UNICEF in December 1999, fluorosis is endemic in at least 25 countries,
such as China and India, Indonesia, South Africa, Iran, and others [50].
F has been linked to neurological and psychiatric disturbances since the 1930s [
51
]. A sharp decline
in mental activity, memory impairment, difficulties with concentration and thinking, and reduced
ability to write were observed in aluminum smelter workers and persons living near a factory where F
was in high concentrations in the atmosphere [
52
]. A review of medical evidence in 500 people affected
by chronic F intake from artificially fluoridated water appeared in 1977 [
53
]. The authors made a list
of the clinical features, such as chronic fatigue, headaches, loss of mental acuity and the ability to
concentrate, depression, a diminished ability to focus, gastrointestinal symptoms and deterioration of
muscular coordination. Carlson’s concerns about what increased F levels would do to the developing
brain of newborn infants [
54
] led to the refusal of water fluoridation in most of the European countries.
A comprehensive historical review and over 50 papers regarding F neurotoxicity have been provided
in the e-book Fluoride Fatigue [51].

Int. J. Environ. Res. Public Health 2019,16, 3431 8 of 21
The surprising observation brought the study of behavior and brain F levels in rats after
sodium fluoride (NaF) exposures during late gestation, at weaning, and in adults [
55
]. Rats exposed
prenatally had dispersed behaviors typical of hyperactivity, whereas rats exposed as adults displayed
behavior-specific changes typical of cognitive deficits. The accumulations of F were found in all the
regions of the brain, with the highest levels in the hippocampus.
Most of the evidence of F as a developmental neurotoxin in humans has been gathered in China.
A study [
56
] revealed adverse effects of F on the brains of 15 aborted fetuses between five and
eight months of gestation from an endemic fluorosis area compared with those from a non-endemic
area. This study showed poor differentiation of brain nerve cells and delayed brain development.
Purkinje cells of fetuses from the endemic fluorosis area were abnormally disorganized, and a higher
nucleus-cytoplasm ratio was observed in brain cones and hippocampus cones. F passing through the
placenta of mothers with chronic fluorosis and its accumulation within the brain of the fetus impact
the developing central nervous system. A meta-analysis of 16 studies carried out in China between
1998 and 2008 found that children living in an area with a high fluorosis occurrence have five times
higher odds of developing a statistically lower IQ than those who live in a low F level area [57].
Choi et al. [
58
] performed a systematic review and meta-analysis of 27 studies published over
22 years. The authors investigate the effects of increased F exposure and delayed neurobehavioral
development of children in China and Iran. Their results revealed the adverse effects of F exposures on
children’s neurodevelopment, its potential neurotoxicity, particularly during fetal development and
early childhood. These meta-analyses suggest an inverse association between high F exposure and
children’s intelligence. From the geographic distribution of the studies, the authors concluded that it
seems unlikely that F-attributed neurotoxicity could be attributable to other water contaminants [58].
Based on this study, F was included among the most important developmental neurotoxicants [
59
].
F differs from most of the current environmental chemicals with impacts on children’s intellectual
development in that children are intentionally exposed to it because of its role in the prevention
of caries.
The effect of chronic F exposure on children’s intelligence, measured as intelligence quotient (IQ),
has been traditionally investigated as an indication of the neurotoxic effect of F in various geographical
areas. Over 40 studies published in China, Iran, India, and Mexico found an association between
lowered IQ and exposure to F [
60
–
64
]. The strength of association between higher F concentrations in the
water and children’s reduced intelligence was further supported by a dose-response meta-analysis [
65
].
These authors evaluated 26 studies of 7258 children and suggested that exposure to F in water should
be controlled in areas with high F levels in the water.
The effects of prenatal F exposure and development of children
´
s cognitive abilities were followed
in 299 Mexican mother–children pairs of the Early Life Exposures to Environmental Toxicants birth
cohort study [
66
]. The children’s cognitive ability was evaluated at four years of age using the McCarthy
Scales of Children’s Abilities and at 6–12 years of age. Children completed an IQ assessment and
provided urine samples for biochemical investigation. Higher levels of F in mothers’ urine during
pregnancy were associated with lower cognitive and IQ scores in their children. The authors estimated
that each 0.5 mg F/L increase in maternal urinary concentration was associated with an average
decrease of 3.15 and 2.50 points in cognitive and IQ scores, respectively.
The prospective multicenter birth cohort study included 601 mother–child pairs recruited from six
major cities in Canada [
67
]. Children were born between 2008 and 2012, and 41% lived in communities
supplied with fluoridated municipal water. Children were between the ages of three and four years
at the time of testing IQ scores. Data were analyzed between March 2017 and January 2019. A 1 mg
higher daily intake of F among pregnant women was associated with a 3.66 lower IQ score (95% CI,
−
7.16 to
−
0.14) in boys and girls. This study found a significant interaction between child sex and
maternal urinary F, indicating a differential association between boys and girls. A 1 mg/L increase in
maternal urinary F was associated with a 4.49-point lower IQ score (95% CI,
−
8.38 to
−
0.60) in boys,
but there was no statistically significant association with IQ scores in girls.

Int. J. Environ. Res. Public Health 2019,16, 3431 9 of 21
We found that 315 laboratory, clinical, epidemiological, and ecological studies over the whole
world bring evidence about F neurotoxicity. This is in good agreement with the findings of other
authors, who introduced that over 300 animal and human studies indicate that F is neurotoxic [
68
].
Recently, F has been added to the WHO’s list of Ten chemicals of greatest public health concern. Bellinger
review brings surveys about how these chemicals adversely affect the brain [
69
]. To support the
case that F may induce neurotoxicity, Bellinger refers to the concern raised by basic neuroscience
and ecological studies about the potential effects of excessive F exposure in developing animals
and children.
4. Is There A Link between ASD Prevalence and a Chronic F Exposure?
In the past few decades, studies have demonstrated that ASDs occur globally and that the numbers
of recorded cases are rising. However, determining the true prevalence figures is still a major challenge.
The awareness of ASD, redefinition of diagnostic criteria, the age of investigated children, and the high
costs of such surveys have a lot of impact on prevalence figures.
There has been a growing interest in possible environmental factors involved in the
etiopathogenesis of ASD. An increasing number of epidemiological reports highlighted the potential
link between ASD and chronic F exposure. We attempted to compare the current rates of the ASD
prevalence from countries with artificial water fluoridation and the available rates from geographic
regions with endemic fluorosis and evaluate the regions with low F supply. While a lot of progress had
been made in the global awareness of ASD, much remains to be done to have a more accurate picture
of the trend or global burden of the disorder [
70
]. Our comparison is therefore affected and limited by
the available reports. Despite these limitations, the available data are a warning.
The review of Elsabbagh et al. [
8
] presents the comprehensive tables of available figures of the
autism and other pervasive developmental disorders prevalence in various geographic regions over the
world in the period of the 1960s–2010. Fifty more studies from 21 countries published during 2000–2016
were analyzed [
9
], and 27 eligible studies from 18 countries on 5 continents were identified [
71
].
These reviews demonstrate that the prevalence rates of autism/ASD have increased over time in all
investigated regions.
4.1. The ASD Prevalence in Countries with Fluoridated Water
The US is at the top of the list of the current ASD prevalence in developed countries with an average
value of 250 per 10,000 (one in 40) children aged 3–17 years [
5
] (Table 1). It is a further increase since
the previous survey in 2014, when one in 59 among eight-year-old children was diagnosed with
ASD in the US [
72
]. CDC reports that 74.4% of the population has been supplied with fluoridated
water for 70 years. A large increase in dental fluorosis prevalence was observed in the US [
73
,
74
].
In the last survey (2010–2012), dental fluorosis was found in 65 % of adolescents aged 12–15 years.
The prevalence rates of ASD are on the rise in Canada. While in 2003, one in 204 children had
a diagnosis of autism/ASD, this rate rose to approximately one in 66 in 2018 [
75
,
76
]. As of 2007, 45.1% of
the Canadian population had access to fluoridated water supplies and water fluoridation remains
a contentious issue. The Canadian Health Measures Survey found that 16% of children may have
very mild or mild dental fluorosis. Australia, where 80% of the population had access to fluoridated
water has revised its ASD prevalence rates from one in 200 in 2012 to an estimated one in 150 people in
2015 [
77
]. New Zealand reports one person in 66 having ASD [
78
], and 62% of the total population has
fluoridated water supply [79].

Int. J. Environ. Res. Public Health 2019,16, 3431 10 of 21
Table 1. Current ASD prevalence in countries with water fluoridation.
Country Year Prevalence per
10,000
This is 1 in X
Children
Water Fluoridation% of
Population Reference
US 2014 169 1:59 70% for 70 y [72]
US 2016 250 1:40 70% for 70 y [5]
Canada 2018 152 1:66 45% for 12 y [75,76]
New Zealand 2016 152 1:66 62% for 50 y [78,79]
Australia 2015 144 1:150 80% for 35 y [77]
In countries with fluoridation of public water, it is vital to account additionally for other sources
of F intake to assess the public health risk of its chronic exposure. Regarding the potential contribution
of F in the ASD etiopathogenesis, one must include that infants and toddlers up to three years receive
significantly more F than they should from infant formula. The recommended water F level in the US
(0.7–1.2 mg/L) is several times higher than the F level found in the breast milk (0.001–0.004 mg/L) [
68
].
F intake from other sources including swallowing toothpaste or the use of F tablets in children might
further increase the daily F dose.
Tea plants (Camellia sinensis) are well known for their ability to accumulate high concentrations of
both F and Al
3
[
80
]. A cup of black tea could contain 1.4 mg of F. Evidence suggests that the culture of
tea drinking in the US, the UK, and New Zealand, contributes significantly to the total body burden of
F [80]. In developed countries a high intake of Al3+from food and medicine products is obvious.
4.2. The ASD Prevalence in Countries with Endemic Fluorosis
Endemic fluorosis occurs widely in the world and is characterized by skeletal and dental fluorosis
and a vast array of bodily pathological changes. Endemic fluorosis has been regarded as a severe
public issue in China since the 1960s. There, endemic fluorosis consists of three types: drinking water
type, coal-burning type, and drinking brick-tea type. The latter two types only exist extensively
in China, but these types are overlapping in some of 34 Chinese provinces [
81
]. Dental fluorosis
occurred in 43%–63% of children aged 8–12 years in endemic areas where the total F intake was
2.7–19.75 mg/day [82].
Recent data show the alarming increase in ASD in China. The last meta-analysis [
83
] included
44 studies with 2,337,321 children aged 1.6–8 years, covering 30 of the 34 provinces of the country.
These studies were conducted between 2000 and 2016. Based on diagnostic criteria, the pooled
prevalence of ASD from 16 studies was 39.23 per 10,000, which is lower than in other countries
worldwide. However, based on screening tools, the prevalence of ASD ranged from 33 to 1853 per
10,000 with a pooled figure of 429 per 10,000 (one in 23.3). For children in the age group
≤
4 years,
the ASD prevalence was 530 per 10,000 (one in 19) in China (Table 2). Despite the many limitations for
the comparison of various ASD prevalence studies that exist, the reports from the last decade revealed
that autism prevalence in China is comparable to the Western prevalence [84].
A high prevalence of ASD has been reported from Japan (161/10,000) [
76
]. Recently, the prevalence
of ASD in a total population sample of 5-year-old children was estimated at 3.22 % (2.66–3.76) [
85
].
Dental fluorosis in 18 endemic areas in Japan was described in 1931 by Dean in his classical report [
86
],
which formed the foundation of the concept that the ingestion of F will harden the surface of teeth
and make them less susceptible to dental caries. It is interesting that Japan has been practicing
a school-based F mouth-rinse program (S-FMR) in nursery schools until graduating from junior high
school since 1970 to prevent dental caries [87]. The prevalence of fluorosis is reported as 1.7%–15.4 %
of the population in Japan. It seems very probable that children, who use the mouth-rinse every day,
swallow F from the mouth-rinse and that this adds to the other F sources from drinking tea, natural
waters, and the typical Japanese diet.

Int. J. Environ. Res. Public Health 2019,16, 3431 11 of 21
The last available study reports the prevalence of ASD in South Asia ranging from 0.09% in India
to 1.07% in Sri Lanka [
88
]. Preliminary data on the prevalence ASD in a population sample of school
children in Eastern India [89] estimate 0.23% (0.07%–0.46%).
An alarmingly high ASD prevalence of 300 per 10,000 (one in 33) was found in Dhaka, the capital
city of Bangladesh, which is known to have vast water contamination by various neurotoxicants [88].
In a 2011 survey, South Korea also had a high rate of prevalence (220 per 10,000) [90] (Table 2).
Table 2. ASD prevalence in countries with endemic fluorosis.
Country Year Prevalence per 10,000 This is 1 in X Children Reference
Bangladesh 2016 15; 80 1:666; 1:125 [88]
Dhaka 2016 300 1:33 [88]
China 2013–2016 19; 42 1:526; 1:238 [9,83]
China 2016 429; 530 1:23; 1:19 [83]
China 2013 Jilin 108 1:92.5 [84]
Japan 161 1:62 [76]
Japan 2018 322 1:31 [85]
India 2016 9 1:1111 [88]
India 2017 23 1:435 [89]
South Korea 2011 220 1:45 [90]
Sri Lanka 2016 93 1:107 [88]
4.3. The ASD Prevalence in the EU Member States
Most of the European states rejected water fluoridation shortly after its introduction in the
1970s–1990s. The Republic of Ireland (Ireland) is the only country in the EU with mandatory artificial
fluoridation of drinking water. On the contrary, Northern Ireland, which is the part of the United
Kingdom (UK), does not add F to drinking water since the local supply may contain naturally occurring
F [
91
]. In some parts of England, the level of F in the public water supply already reaches 1 mg F/L as
a result of the geology of the area. In other areas of the UK, the F concentration has been adjusted by
artificial fluoridation. Currently, around six million people in the UK live in areas with fluoridation
schemes. Many schemes have been operating there for over 50 years. A high ASD prevalence has been
estimated to have been present in the UK since the 1990s (16.8–116 per 10,000) [
8
,
9
,
71
]. The National
Autistic Society informs that there are around 700,000 people with ASD in the UK, which is more than
one in 100 [92].
Unfortunately, there is no central recording of ASD cases in any EU Member State. The European
Commission (EC) considered ASD as a rare disease until 2005. The prevalence rates for autism in the
EU could be estimated as varying from 3.3 to 16.0 per 10,000 [10,93]. The first pilot project funded by
the European Parliament and managed by the EC (ASDEU) provided an estimate of ASD prevalence
in 14 European countries in the period 2015–2018 [
93
]. The program scrutinized 631,619 children aged
between seven and nine years. Overall, ASD prevalence estimates varied among European countries
from 44 to 197 per 10,000 according to the ASDEU project [
93
]. However, the estimations from this
project are higher than the lower estimates made for the Czech Republic, Poland, Portugal, Finland,
France, and Germany [9,71] (Table 3).
A recent survey of ASD prevalence in the Irish population was performed in 2017 and 2018 as
part of an ASDEU project [
91
,
93
]. This report indicates an estimated prevalence of autism of 2.9% in
school-aged children in Northern Ireland, significantly higher than the 1.5% estimated in the Ireland
in the same age group but several years earlier. Waugh et al. showed that the culture of habitual
tea drinking in the Ireland could readily exceed the levels known to cause chronic F intoxication in
the general population [
94
], and this is probably relevant to the Irish population in Northern Ireland
as well.
Similarly, an increasing ASD prevalence to 1% was reported from Spain, where fluoridated water
has been provided for 10% of the population [
9
,
71
]. There are substantial intra-regional differences in

Int. J. Environ. Res. Public Health 2019,16, 3431 12 of 21
ASD prevalence in Sweden, where differences in the F content in drinking water (0.8–1.4 mg F/L) are
also well documented [95].
Table 3. The ASD prevalence in the EU member states according to the available last reports.
Country Prevalence per 10,000 This is 1 in X Children Reference
EU total 2015–2018 44–197, average 122 1:82 [93]
Belgium 60 1:167 [96]
Czech Republic 12 1:833 (pers. comm.)
Denmark 34; 68 1:294; 1:147 [76,96]
Finland 77 1:130 [91]
France 27; 36 1:370; 1:277 [97]
Germany 38 1:263 [96]
Ireland 150 1:66 [91]
Italy Pisa 86 1:116 [91,98]
Netherland 57, 84 1:175; 1:119 [9,71]
Northern Ireland 290 1:35 [91]
Norway 12; 70 1:833; 1:142 [9,91,96]
Poland 3 1:3333 [96]
Portugal 9.2 1:1086 [9,71,76]
Spain 13; 100 1:769; 1:100 [9,71,76]
Sweden 71; 115 1:141; 1:87 [9,71,91]
UK 100 1:100 [92]
4.4. The Definition of a Safe Concentration of F for Humans
An awareness of the potential role of F in the ASD pathogenesis could contribute to the qualified
reassessment of its widespread use in the health practice of water fluoridation, which is regarded as
a valuable and safe method for reducing dental caries.
We cannot analyze the history of water fluoridation in the US and the discussion considering F
standards. Under the Safe Drinking Water Act, the US Environmental Protection Agency (USEPA)
sets the standards for drinking water quality. Currently, the enforceable F standard is set at 4.0 mg/L.
The most commonly cited health concerns about F were assessed in the 2006 National Research Council
(NRC) report. No evidence substantial enough to support the negative health effects of F at levels below
4.0 mg F/L other than severe dental fluorosis was found [
99
]. Since 1962, the US Public Health Service
(PHS) has recommended an optimal F concentration of 0.7 mg/L and considers that this provides the
best balance of protection from dental caries while limiting the risk of dental fluorosis [100].
Table 4provides the latest values for adequate intakes (AI) and upper tolerable intake levels (UL)
for F (in mg/day) developed by the National Academies of Sciences, Engineering, and Medicine for
the US, updated on 3/27/2019 [
101
]. The F AI and UL for 0–8-year children were updated in 2017
by the National Health and Medical Research Council (NHMRC) for Australia and New Zealand.
This update was intended to prevent dental caries without exceeding intakes that are associated with
severe dental fluorosis [
102
]. Dental fluorosis is caused by overexposure to F during the first eight
years of life. The NHMRC did not establish an AI for infants less than six months of age. The review of
evidence did not find a reduction in dental caries with F intake in the first six months of life [102].
As F is not an essential nutrient, the panel of the European Food Safety Authority (EFSA) considered
that no average requirement for the performance of its essential physiological functions could be
defined (12/4/2017) [
103
]. However, the panel considered that data on the dose–response relationship
between caries incidence and the consumption of drinking water with different F concentrations are
adequate to set an AI of 0.05 mg F/kg body weight per day for children. This can also be applied to
adults, including pregnant and lactating women. However, the AI covers F intake from all sources,
including toothpaste and other dental hygiene products. The available data on F intake of the European
population is variable but generally at or below 0.05 mg/kg/day [
103
]. For younger children (1–6 years
of age) the UL was exceeded when consuming more than one liter of water at 0.8 mg F and F from

Int. J. Environ. Res. Public Health 2019,16, 3431 13 of 21
other sources. For infants up to six months old receiving infant formula, if the water F level was higher
than 0.8 mg/L, the intake of F exceeded 0.1 mg/kg/day, and this level is 100 times higher than the level
found in breast milk [104].
Table 4.
Adequate intake (AI) and upper tolerable intake levels (UL) for F (in mg/day). According to
the National Academies of Sciences, Engineering, and Medicine, US [
101
]; NHMRC, Australia and
New Zealand [102]; and EFSA, EU [103].
Age US Australia, NZ EU
AI UL AI UL Age AI
0–6 m 0.01 0.7 – 1.2 0–6 m –
7–12 m 0.5 0.9 0.5 1.8 7–11 m 0.4
1–3 y 0.7 1.3 0.6 2.4 1–3 y 0.6
4–8 y 1.0 2.2 1.1 4.4 4–6 y 1.0
9–13 y 2.0 10 2.0 *; 3.0 +10 7–10 y 1.5
14–18 y 3.0 10 2.0 *; 3.0 +10 11–14 y 2.2
Males 4.0 10 4.0 10 15–17 y 3.2
Adult females 3.0 10 3.0 10 Adults 3.4
The following reference body weights were used when the AI and UL were expressed in mg F/day: 0–6 months, 6 kg;
7–12 months, 9 kg; 1–3 years, 12 kg; 4–8 years, 22 kg; 9–13 years, 40 kg; boys aged 14–18 years, 64 kg; 14–18 year-old
girls, 57 kg; 76 kg for adult men; and 61 kg for adult women. * girls, +boys.
The analysis of the literature regarding the loss of IQ after F chronic exposure [
68
] shows that to
protect against a five-point IQ loss, the benchmark dose (BMD) is between 0.0014 and 0.05 mg/day for
children. It means that the protective daily dose should be no higher than 0.05 mg/day for children.
The information now available supports a reasonable conclusion that F exposure of the developing
human organism should be minimized. Prolonged exposure to F in the prenatal as well as postnatal
stages of development might have neurotoxic effects on the development and metabolism of the brain.
Infantile autism develops most likely during the second trimester of prenatal development and results
in significant abnormalities in the development of the brain, including the cerebellum, brainstem,
and cerebrum. The regressive autism appears to be the result of postnatal events. This form of autism
is responsible for the rising ASD incidence in the last decades. Autism-specific brain imaging features
were identified at six months of age. Age-specific brain and behavior changes were demonstrated
across the first two years of life [
105
]. This time is a period of intense postnatal synaptic development.
The human cerebellum matures postnatally, with the greatest acceleration of growth and neural
organization during the first two years after birth [11].
However, the dose, at which dental caries reduction is expected, is not far away from the one that
might cause chronic pathological effects. It is evident that the definition of a safe concentration of F for
humans must consider the fact that in synergy with trace amounts of Al
3+
, every concentration of F
might be dangerous for the developing brain.
4.5. Socioeconomic Status
ASD may significantly limit the capacity of some individuals to conduct daily activities. Since it is
a life-long condition, it often imposes a significant socioeconomic burden on society, people with these
disorders, and their families. In the US, the annual societal costs per the year 2011 for children with
ASD were estimated to be between $11.5–60.9 billion, including a variety of direct and indirect costs,
from medical care to special education and lost parental productivity [
3
]. Children and adolescents
with ASD had average medical expenditures that exceeded expenditures of those without ASD by
$4110–6200 per year.
According to estimates, annual costs due to ASD in the US in 2015 were around $268 billion [
106
].
This figure is estimated to increase to about $461 billion by the year 2025. If the prevalence of ASD
continues to grow as it has in recent years, ASD costs will likely exceed those of diabetes and ADHD

Int. J. Environ. Res. Public Health 2019,16, 3431 14 of 21
by far by 2025 [
106
]. The economic impact of IQ loss among US children is the loss of tens of billions of
dollars [68].
The costs of autism for individuals (lifetime) and society (annual) in the UK was calculated in 2005.
The average additional lifetime cost for an individual with autism and additional learning disability
was estimated to be £2,940,538. For people with high-functioning autism, the additional lifetime cost is
estimated to be £784,785 [92].
One of the ASDEU’s key aims was to estimate the economic burden of ASD in the EU. The type
of ASD, age, and comorbidities are important drivers of the costs. Estimations of direct costs ranged
from
€
1594 in Romania to
€
22,378 in Denmark per individual annually. Lifetime costs of caring for
a person with autism without an intellectual disability were estimated at
€
766,865 based on an average
life expectancy of 70 years for people with ASD in Germany. The costs of productivity losses among
carers range yearly from
€
615 per carer in Poland to
€
8934 per carer in Austria [
93
]. ASDEU also found
that the total cost of universal screening of ASD prevalence would range from
€
43,000 per year in
Iceland to €5 million per year in France [93].
These significant health and socioeconomic concerns could probably be lowered by focusing more
on ASD prevention through the elimination of F from daily life.
5. Discussion
A vast array of observations has been made at the cellular and the molecular levels of the ASD
etiopathogenesis. Yet, a unifying mechanism to explain the various etiological factors and different
symptoms of ASD has not been proposed. There is a consensus of several researchers that causal
processes in the ASD etiopathogenesis involve the interactions of multiple genetic and environmental
risk factors. We reviewed literature showing that F is an environmental neurotoxin. F induces symptoms
which are observed in people with ASD, such as mitochondrial dysfunction and impairment of energy
metabolism, oxidative stress and inflammation, immunoexcitotoxicity, and decreased melatonin levels.
We showed that all these symptoms have also been observed in endemic fluorosis areas in China [
7
],
where daily intake of F exceeds the national standard 2 mg per day and might reach up to 20 mg per
day [82].
Based on the ASD prevalence in various countries and cities, we suggest that F might be the
significant culprit in the ASD etiopathogenesis both in areas with artificial water fluoridation as well
as in F endemic areas. Nevertheless, F is not yet included among environmental risk factors in the
ASD pathogenesis in countries with fluoridated water and a high rate of dental fluorosis. CDC named
community water fluoridation one of ten great public health achievements of the 20th century.
Three main categories of toxicants have been suggested as contributing to the ASD pathogenesis:
heavy metals, persistent organic pollutants, and emerging new endocrine disruptors. For example,
Modabbernia et al. conducted a review of the 80 studies, nine systematic reviews, and 23 meta-analyses
of environmental risk factors for ASD [
107
]. These authors pointed out that the association between
environmental factors and ASD might include gene-related epigenetic effects, oxidative stress,
inflammation, endocrine disruption, neurotransmitter alterations, and interference with signaling
pathways. Nevertheless, an assessment of F participation in these events was not suggested [107].
Ng et al. reviewed 315 articles regarding environmental factors associated with ASD in the years
2003–2013. Research has been conducted worldwide; many studies were concentrated in the US,
the UK, Australia, and Japan [
108
]. However, F was not involved in any of the 315 reviewed articles
examining chemical factors [
108
]. Concerning neurotoxicants, Schofield referred to the current medical
databases for 100 molecules or elements that can be listed as developmental neurotoxicants [
109
].
She reviewed knowledge of the six neurotoxicants, which could be involved in the growing epidemic
of neurological illnesses, including autism, over the last 20–25 years. F was not included among
the referred neurotoxicants. F did not receive any attention or suspect for participation among
environmental factors in the ASD etiopathogenesis, nor in recent reviews of Almandil et al. [
6
] nor
Bjørklund et al. [110].

Int. J. Environ. Res. Public Health 2019,16, 3431 15 of 21
In countries of the EU, where F daily intake is low, F does not probably work as the trigger in
the complicated network of pathogenetic events, and ASD is often still considered a rare disease.
The document of the EC also concluded that available human studies do not support the conclusion
that F in drinking water impairs children’s neurodevelopment at levels permitted in the EU [104].
Many other environmental neurotoxins, exogenous excitotoxic amino acids, and endocrine
disruptors can disturb both prenatal as well as postnatal brain development [
11
,
111
,
112
]. Moreover,
F from natural waters (e.g., in Sweden and the UK), tea, and non-dietary sources, such as F tablets,
toothpaste, and other dental hygiene products, might play a role in the ASD pathogenesis. The severity
and the development of ASD symptoms depend on genetics, nutritional status, immune system,
and the presence of recurrent infections [
11
]. Nevertheless, an increase in the ASD prevalence is also
observed in some states of the EU with water fluoridation, such as the RoI, the UK, and partially
in Spain.
We showed that even very low F concentration in synergy with Al
3+
can exacerbate alterations
in neurotransmission and hormonal regulation. Under such circumstances, a “safe” F concentration
might induce pathological effects in children with a genetic susceptibility. The heterogeneity of mutual
dynamic interactions can explain the clinically heterogeneous symptoms of ASD and contribute to
an understanding of the various responses in any given child to identical environmental neurotoxins.
Anderson [
113
] suggested in his review that the novel, “emergent” phenomena may arise in
individuals with ASD from underlying and interacting genetic and environmental factors. Some of the
ASD behavioral symptoms, including delayed stereotyped language, rigid preferences for routines,
and repetitive motor mannerisms, among others, can be discussed as potentially being emergent.
The rise of ASD in the last decades challenges us to change the research from a reductionistic approach
to an understanding of underlying integrative networks. Our review shows how diverse molecules
and biological processes can be affected by F.
In the Czech Republic, artificial water fluoridation was stopped in the 1990s for economic reasons,
since tap water is used for many purposes. There are many discussions and analyses regarding the
efficiency and costs of water fluoridation in the prevention of dental caries over the world. However,
the possibility that chronic F intake could evoke chronic diseases with high health and socioeconomic
impact would also be involved. The information now available supports a reasonable conclusion that
economic losses associated with ASD may be quite large.
Moreover, while ASD is often considered purely behavioral, it comes with many different
comorbidities, like gut imbalances, seizures, hormonal disorders, obesity, and sleep problems. Disturbed
sleep potentially exacerbates ASD-related symptoms such as impaired social interactions, deficits in
nonverbal intelligence and communication, repetitive behaviors, and mood disorders. Poor sleep may
have adverse effects on children’s attention, memory, learning, and conduct aggression.
The ASD prevalence is consistently estimated as a ratio of approximately 4.5 male:1 female during
2006–2014 in the US [
3
,
70
]. For example, the pooled ASD prevalence was one in 38 boys and one in 152
girls. However, the male-to-female prevalence ratios ranged from 2.7 in Utah to 7.2 in Alabama [
72
].
The perspective that ASD could impact around 30% of young men in the next decades is very alarming
and requires urgent solutions. The reduction of F and Al
3+
consumption of pregnant women and
developing children could be a very easy and inexpensive way to prevent ASD.
6. Conclusions
The rise in the ASD prevalence in countries with water fluoridation as well as in endemic fluorosis
areas supports a view that F is an important environmental factor in the ASD etiopathogenesis.
Our suggestion of the important role of F in the ASD etiopathogenesis is supported by the observation
that a high ASD rate is found in countries with a high occurrence of dental fluorosis.
F neurotoxicity has been demonstrated in many laboratory studies with cells and animals, as well
as in human epidemiological studies. We present evidence that F induces mitochondrial dysfunction,
oxidative stress, inflammation, and immunoexcitotoxicity. There is a link between a known effect of F

Int. J. Environ. Res. Public Health 2019,16, 3431 16 of 21
on melatonin synthesis in the pineal gland and the finding that melatonin synthesis is significantly
reduced in ASD. Moreover, understanding F-induced pathways in the ASD etiopathology may lead
to novel treatments. All these F-induced symptoms could evoke several disruptions of the brain
development, alter neurotransmission and hormonal regulations, deficits in social interactions and
induce repetitive, stereotypic interests, and behaviors resulting in ASD.
At present, there is a divergence between public health practice of water fluoridation, which is
regarded as valuable and safe for reducing dental caries, and current scientific evidence, which indicates
that F is a neurotoxin disturbing prenatal as well as postnatal brain development, eroding intelligence,
and behavior. The potential neurotoxicity associated with exposure to F, which has generated
controversy about community water fluoridation, remains unclear. In the recent Canadian study,
maternal exposure to higher levels of F during pregnancy was associated with lower IQ scores in
children aged 3 to 4 years [
67
]. These findings support the possible need to reduce F intake during
pregnancy. Intellectual disability is present in 65%–75% of individuals with a diagnosis of autistic
disorder and in 30%–55% of all ASD [3].
F is not an essential nutrient as no physiological function can be defined for which F is required.
The presence of trace amounts of Al
3+
strongly potentiates the neurotoxic effects of F. AlF
4−
can trigger
the pathological symptoms of ASD at concentrations several times lower than those for F acting
alone. In synergy with Al
3+
every concentration of F might be dangerous for the developing brain.
Our review suggests that the reduction of F exposure in daily life might be an efficient way to prevent
an ASD epidemic soon. Monitoring of the ASD prevalence in children born after the removal of F from
drinking water will provide relevant information for our hypothesis.
Author Contributions:
Both authors are responsible for the intellectual content, literature review and drafting of
the manuscript. Both authors reviewed and approved the final manuscript.
Funding:
This work was undertaken without funding for the benefit of public health and the advancement of
science education in the fields of health promotion and disease prevention. The authors did not receive any
financial support.
Acknowledgments:
Authors thank Paul Grof, MD, Ph.D., FRCP, Professor of Psychiatry, University of Toronto
and Director Mood Disorders Center of Ottawa for careful reading of our manuscript, stimulating discussion,
and valuable comments. Authors thank Bruce James Spittle, MB ChB with distinction, DPM (Otago), a Fellow of
the Royal Australian and New Zealand College of Psychiatrists, and the managing editor (since 1999) of Fluoride,
for his critical comments and stimulation. We appreciate the excellent cooperation of Hana Kruž
í
kov
á
in the
preparation of figures.
Conflicts of Interest: The authors declare no conflict of interest.
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