Fluoride Metabolism

Abstract

Fluoride is widely used as an anticariogenic agent but excessive ingestion may lead to systemic toxicity. Its biological effects are dependent on the amount, time of exposure and the metabolic handling of ingested fluoride. After being ingested, fluoride is first absorbed in the stomach, followed by its distribution through soft and hard tissues and urine excretion. These events occur in a pH-dependent manner because the coefficient of permeability of lipid bilayer membranes to hydrogen fluoride is much higher than that of ionic fluoride. Therefore, fluoride drives through cell membranes as HF, in response to a pH gradient between adjacent compartments, going from more acidic to more alkaline compartments. The fluoride plasma peak is quickly reached after ingestion, as result of a rapid pH-dependent absorption in the stomach. The small intestine also contributes to fluoride absorption but this event is not pH dependent. Plasma fluoride levels are rapidly decreased mainly due to fluoride uptake in hard tissues and renal excretion, while the nonabsorbed fluoride is excreted in feces. Thus, plasma fluoride concentration is a result of the relation between the levels of ingestion, and metabolism accounted by its deposition in calcified tissues and excretion. Additionally, the latter is modified by several factors that will be discussed in detail in this chapter.

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54
CHAPTER 4
Fluoride Metabolism
CAMILA PERES BUZALAFa, ALINE DE LIMA LEITEb, AND
MARÍLIA AFONSO RABELO BUZALAF*b
aPró-Reitoria de Pesquisa e Pós-Graduação, Universidade do Sagrado
Coração, Bauru, São Paulo 17011-160, Brazil; bDepartment of Biological
Sciences, Bauru Dental School, University of São Paulo, Bauru, São Paulo
17012-901, Brazil
*E-mail: mbuzalaf@fob.usp.br
4.1 Introduction to Fluoride Metabolism
Fluoride is an inorganic anion of fluorine, which is naturally found in the
biosphere with ubiquitous presence in the environment and is the most
electronegative and reactive among all elements. This latter characteristic
explains why fluorine is mainly found as inorganics instead of the elemen-
tal form. Its well-known cariostatic effect led to the implementation of fluo-
ride in systemic (such as water, salt, sugar, milk and supplements) and topic
sources (such as toothpastes, gels, mouth rinses and varnishes). The ben-
efits of fluoride include both caries control and bone formation (Bratthall
et al. 1996; Kobayashi et al., 2014). However, it can negatively affect the qual-
ity of the developing mineralized tissues, causing dental and skeletal fluoro-
sis. The differential responses seem to be determined by the dose and time
of exposure (Everett, 2011). Thus, the knowledge of fluoride metabolism
is crucial not only to understand its biological effects but also to optimize
fluoride-driven preventive and therapeutics strategies.
Food and Nutritional Components in Focus No. 6
Fluorine: Chemistry, Analysis, Function and Effects
Edited by Victor R Preedy
© The Royal Society of Chemistry 2015
Published by the Royal Society of Chemistry, www.rsc.org

55
Fluoride Metabolism
4.2 General Aspects of Fluoride Metabolism
Fluoride metabolism includes absorption, distribution and excretion,
where each step depends on the pH. Hydrogen fluoride (HF) is a weak acid
with a pKa of 3.4. By definition, at pH 3.4, 50% of fluoride is in the undis-
sociated form (HF), while the remaining 50% is in the ionic form (F–).
As pH decreases from 3.4, the concentration of HF increases, and as pH
increases, the concentration of F– increases (Whitford, 1996) (Figure 4.1).
This means that fluoride permeates cell membranes as HF, in response to
a pH gradient between adjacent body-fluid compartments, going from the
more acidic to the more alkaline compartment (Figure 4.1). Aer inges-
tion, plasma fluoride reaches a peak within 20–60 min, due to its pH-de-
pendent gastric absorption, followed by a rapid decline as a result of both
uptake in calcified tissues and urine excretion (Figures 4.2 and 4.3). The
small intestine also contributes to fluoride absorption in a pH-indepen-
dent mechanism. Nonabsorbed fluoride is excreted in feces (Figure 4.3).
From plasma, fluoride is distributed to both hard and so tissues followed
Figure 4.1 Metabolism of fluoride is dependent on pH. HF is a weak acid with pKa
of 3.4, meaning that at pH 3.4, half of fluoride is in the nonionic form
(HF) and half is in the ionic form (F−). As pH decreases from 3.4, the con-
centration of HF enhances, while as pH increases, the concentration of
F− increases. Besides, the coefficient of permeability of cell membranes
to HF is much higher than that of F−. Thus, fluoride drives through cell
membranes as HF, in response to a pH gradient between adjacent com-
partments, going from more acidic to more alkaline compartments.

Chapter 4
56
by its renal excretion (Figure 4.3). A minor portion of absorbed fluoride
is found in so tissues through a steady-state distribution between extra-
cellular and intracellular fluids. However, about 50% of the absorbed flu-
oride is incorporated in calcified tissues, mainly in bone, where 99% of
the fluoride content in the body is found (Whitford, 1994). Its incorpora-
tion in calcified tissue is not irreversible, from where it can be released
back to plasma compartment. The fluoride excreted in urine is mostly a
result of the total absorbed amount subtracted by the incorporated quan-
tity in hard tissues. Each step related to the fluoride metabolism will be
described in sequence.
Figure 4.2 Plasma fluoride levels aer a small amount of fluoride ingested. (A)
Aer ingestion, plasma fluoride reaches a peak within 20–60 min, due
to its pH-dependent gastric absorption, followed by a rapid decline as
a result of both uptake in calcified tissues and urine excretion. The flu-
oride plasma level returns to baseline levels within 3–4 h. The small
intestine also contributes to fluoride absorption in a pH-independent
mechanism. (B) From plasma, fluoride is distributed to both hard and
so tissues followed by its renal excretion. Plasma fluoride levels are
rapidly decreased mainly due to fluoride uptake in hard tissues and
renal excretion, while the nonabsorbed fluoride is excreted in feces (A
and B).

57
Fluoride Metabolism
4.3 Fluoride Absorption
Approximately 80–90% of fluoride ingested is absorbed from the gastro-
intestinal tract by passive diffusion and does not seem to require specific
transporters (Whitford, 1996). However, “Fluc” proteins-based channel was
recently purified in phospholipid bilayers with extreme selectivity for F–
over Cl–, but its expression and role in fluoride absorption in mammalian
is still unknown (Stockbridge et al., 2013). Recently, selective bacterial CLC
anion-transporting proteins that catalyze transport of fluoride in liposomes
were discovered (Stockbridge et al., 2012). In addition, a novel fluoride
Figure 4.3 General aspects of fluoride metabolism. Aer infestion, fluoride is
absorbed in the stomach and small intestine by a pH-dependent and
-independent mechanisms, respectively. Nonabsorbed fluoride is excreted
in feces. A minor portion of absorbed fluoride is found in so tissues
through a steady-state distribution between extracellular and intracellu-
lar fluids. However, about 50% of the absorbed fluoride is incorporated
in calcified tissues, mainly in bone, where 99% of the fluoride content in
the body is found. Its incorporation in calcified tissue is not irreversible,
from where it can be released back to the plasma compartment. From
systemic circulation, fluoride content is followed by renal excretion. The
fluoride excreted in urine is mostly a result of the total absorbed amount
subtracted by the quantity incorporated in hard tissues.

Chapter 4
58
exporter named FEX functioning as fluoride efflux was recently described
in eukaryotic cells (Li et al., 2013). This exporter allows organisms such as
fungi to evoke from fluoride-mediated toxicity but has not been identified
in mammalian cells as yet. Together, these novel findings open new per-
spectives regarding fluoride-mediated inter- and intracellular transport.
While about 20–25% of the total ingested fluoride is absorbed in the stom-
ach, the remainder is absorbed in the proximal small intestine (Nopakun et
al., 1989; Whitford, 1996). Its absorption occurs in a half-life of 30 min, reach-
ing a peak within 20–60 min (Whitford, 1996), independently of the amount
ingested. Aer that, the fluoride concentrations are rapidly decreased and
correlate to both uptake in calcified tissues and renal excretion (Whitford,
1996). The gastric absorption is very quick and is determined by acidity and
content gastric and fashion of gastric emptying (Whitford and Pashley, 1984).
This is explained by the fact that the gastric absorption is inversely related
to the pH. i.e., the higher the gastric acidity, the higher the fluoride absorp-
tion (Whitford and Pashley, 1984). When ionic fluoride enters the acid gastric
lumen environment, it is converted into HF, which is an uncharged molecule
that readily crosses cell membranes, including gastric mucosa (Gutknecht
and Walter, 1981). Thus, peak plasma fluoride concentration is higher in
acidic compared to neutral environments. Thus, the pH dependency of fluo-
ride absorption has a direct implication not only for the treatment in cases of
acute fluoride toxicity but also in therapeutic applications. Moreover, delayed
gastric emptying may promote both slower and smaller fluoride absorption
leading to a decreased plasma concentration (Nopakun et al., 1989).
About 75% of the fluoride not absorbed in the stomach is absorbed
predominantly as ionic fluoride by the proximal small intestine in a
pH-independent mechanism. In this case, F– crosses the tight junctions
of intestine epithelia arranged between cells and paracellular channels
(Nopakun et al., 1989). The greatest absorption in the small intestine may
be a consequence of about 100-thousand times more [F–] compared to
[HF] in neutral pH (Messer et al., 1989).
The fluoride absorption is also affected by the diet and other foods with
which fluoride interacts. Sodium fluoride (NaF) in water solutions is 100%
absorbed (Whitford, 1996). However, when in combination with foods or
drinks containing calcium, aluminum and magnesium (bi- and -trivalent
cations), fluoride forms insoluble complexes that decrease fluoride absorp-
tion (Trautner and Siebert, 1986). This explains the use of calcium-containing
solutions in acute fluoride toxicity (Li, 2003).
The absorption is also altered by the type of administered fluoride. So far,
there are divergences in the literature about similarities in fluoride absorp-
tion when it comes from NaF or disodium monofluorophosphate (Na2PO3F,
SMFP) (Whitford et al., 1990). The fact is that the absorption of fluoride
from SMFP requires phosphatases-mediated enzymatic hydrolysis, leading
to lower and delayed peak plasma fluoride levels compared to those seen
from NaF. However, the bioavailability of fluoride from both compounds is
roughly the same (Buzalaf et al., 2008).

59
Fluoride Metabolism
4.4 Fluoride Distribution
Aer ingestion, plasma fluoride levels rapidly increase until a peak is reached
(around 20–60 min). From then on, the levels start declining to baseline levels
within 3 to 11 h, depending on the dose that is ingested (Whitford, 1996). Blood
plasma is considered the central compartment from which fluoride is distrib-
uted to so and mineralized tissues and specialized body fluids, followed by its
elimination or clearance (Whitford, 1996). In so tissues, fluoride establishes
a steady-state distribution between extracellular and intracellular fluids. Vari-
ations in the plasma fluoride concentrations lead to proportional alterations
in intracellular compartments (Ekstrand, 1996). In mineralized tissues, fluo-
ride is reversibly incorporated mainly in bone, being released back to plasma
during bone remodeling. Hereaer, detailed aspects related to each compart-
ment involved in the fluoride distribution will be described.
4.4.1 Plasma Fluoride
Plasma fluoride is found both as inorganic (ionic or free fluoride) and
organic (nonionic) forms and together they represent the total of plasma flu-
oride (Ekstrand, 1996; Whitford, 1996). Inorganic fluoride is not bound to
plasmatic macromolecules and can be quantified by a specific electrode. Its
biological importance in dentistry, medicine and public health is well estab-
lished. Instead, organic fluoride is composed chiefly of different types of lip-
id-like molecules that bind to plasmatic proteins. Although present in greater
amounts, the biological role of organic fluoride is still unknown. In contrast
to other ions, such as calcium, sodium, phosphorus and chlorine, plasma
fluoride is not homeostatically regulated but reflects the balance between
the amount and time of intake, absorption, distribution and uptake, and
renal filtration and excretion (Ekstrand, 1996; Whitford, 1996). In humans
living in areas with fluoridated water (containing 1 ppm F or ∼52.6 µmol L−1),
plasma fluoride concentrations ranged between 0.5 and 1.5 µmol L−1 (Whit-
ford, 1996). Thus, although plasma fluoride levels are modulated by several
physiological factors regardless of fluoride intake, they constitute an import-
ant biomarker of past and present exposures (Ohmi et al., 2005).
4.4.2 Fluoride in So Tissues
The rate of fluoride distribution from plasma to so tissues is determined
by the velocity of blood flow to different tissues and organs. It is influenced
by both the magnitude and direction of transmembrane pH gradient. This
means that the proportion of fluoride levels in plasma or tissue is dependent
on the ratio between the extracellular pH (epH) and intracellular pH (ipH).
It is important to consider that fluoride accumulates in the more alkaline
compartment in response to a pH gradient (Figure 4.1) and that the extra-
cellular fluid is usually more alkaline than the cytosol of mammalian cells.
Thus, it is reasonable to expect higher fluoride concentration in plasma and

Chapter 4
60
extracellular compared than those found in intracellular fluids (ranging
between 10 and 50%) (Whitford et al., 1979). Moreover, as the pH gradient
between membranes can be changed by altering extracellular pH, a flux of
fluoride into and out of cells may be achieved. When epH increases, fluoride
leaves the cell. Inversely, when epH decreases, fluoride goes into the cell. This
is the basis for the alkalinization in cases of acute fluoride toxicity, in order to
promote releasing of fluoride out of cells (Whitford et al., 1979). Besides pH
gradient control, a complex molecular mechanism involved in fluoride toxic-
ity was recently described. It was shown that many species present fluoride
RNA-based riboswitches to control the expression of proteins that soen the
deleterious effects of this anion (Baker et al., 2012). Thus, differential degrees
of fluoride toxicity may be due to differences in expression and/or usage of
those fluoride-sensing RNAs, rather than only the pH gradient.
Fluoride concentrations were screened and compared among tissues and
organs using 18F, revealing that the kidney presents the greatest ratio in fluo-
ride concentration, followed by liver, lung, spleen and others (Whitford et al.,
1979). This may explain why these organs are targets for the fluoride-mediated
toxicity (Guo et al., 2003; Pereira et al., 2013). In this context, some compounds
have been used to ameliorate the fluoride-driven toxic response by decreasing
its accumulation in so tissues and organs (Blaszczyk et al., 2012). Reduced
distribution of fluoride in so tissue is accompanied of a greater fluoride
uptake in bone by uncertain mechanisms (Blaszczyk et al., 2012).
4.4.3 Fluoride in Specialized Body Fluids
Specialized body fluids do not present the same fluoride concentration
observed in plasma but changes in fluoride levels are directly modulated
according to plasma concentration. From those, cerebrospinal fluid presents
50% less fluoride from those seen in plasma (Whitford, 1996). Moreover,
gingival crevicular present fluoride levels in a slightly greater fashion com-
pared to plasma (Whitford, 1994). Instead, concentrations in parotid and
submandibular ductal saliva are slightly decreased, showing ductal saliva
and plasma fluoride concentrations ratios around 0.8–0.9 (Whitford, 1994).
Similarities with plasma allowed them to be used as a biomarker of fluoride
exposure aiming to analyze fluoride bioavailability from fluoridated prod-
ucts (Olympio et al., 2007). Fluoride levels in whole saliva are predominantly
more variable and higher compared to ductal saliva because they are oen
contaminated by exogenous fluoride sources such as food, water and fluori-
dated dental products (Whitford et al., 1999).
4.4.4 Fluoride in Mineralized Tissues
About 99% of total fluoride retained in the organism is found in calcified tis-
sues, mainly in bone. Enamel and dentin also contribute but in a lesser extent
(Whitford, 1994). Age seems to be a factor that alters fluoride concentration
in bone. About 36% and 55% of the fluoride daily absorbed in incorporated in

61
Fluoride Metabolism
healthy adults (18–75 years) and children (<7 years), respectively (Villa et al.,
2010). This is due to the continuous fluoride intake throughout life (Richards
et al., 1994). Chronic intake of fluoride negatively impacts mineralized tissues,
promoting dental and skeletal fluorosis dependent on dose, time and duration
of exposure. Controversy persists concerning the impact of community water
fluoridation on bone health in adults, and few studies have assessed relation-
ships with bone at younger ages. Recently, bone in adolescents measurements
were performed aer life-long fluoride intake through a prospective cohort
study (Levy et al., 2014). Daily fluoride intake was 0.66 mg for females and 0.78
mg for males and no significant relationships between daily fluoride intake
and adolescents’ bone measures we found. The findings suggest that fluoride
exposures at the typical levels for most US adolescents in fluoridated areas do
not have significant effects on bone mineral measures (Levy et al., 2014).
Retention of fluoride in bone is not uniform and is more pronounced in
the periosteal and endosteal regions and in cancelous rather than contact
bone (Richards et al., 1994). Bone fluoride is divided into surface and inner
compartments. The first is rapidly exchangeable while the latter is not. In
surface and inner bone, fluoride uptake occurs in different steps. First, iso-
and heteroionic exchanges on the hydration shells of bone crystallites take
place in contact with extracellular fluids. This proximity allows a steady-state
relationship between the extracellular fluid (that represents physiological
plasma fluoride concentration) and hydration shells of bone regulating fluo-
ride concentration. This characteristic corroborates the use of bone surface
as a terminal biomarker of acute fluoride exposure (Buzalaf et al., 2005). Sec-
ondly, fluoride incorporates into precursors of hydroxyfluorapatite followed
by the apatitic lattice itself (Neuman and Neuman, 1958).
Fluoride associated with the nonexchangeable inner compartment, is irre-
versibly bound. Instead, it is continuous mobilized during bone remodeling
and bone absorption (Rao et al., 1995).
Levels of fluoride in dentin are quite similar to bone and increase aer
long-term intake. Higher concentration is found in pulp from which it starts
to reduce as it approaches the dentin-enamel junction (Hallsworth and
Weatherell, 1969). Enamel fluoride content also enhances with age but is
comparatively lower and unrelated to dentin (Vieira et al., 2006). Usually, flu-
oride concentration in tooth enamel reflects fluoride exposure during amelo-
genesis (Hallsworth and Weatherell, 1969). However, although enamel is the
target tissue for dental fluorosis, correlations between fluoride content in
enamel and fluorosis occurrence were not found (Vieira et al., 2006).
4.5 Kidney Excretion
Fluoride renal excretion is one of the most important mechanisms for the
regulation of fluoride levels in the body (Buzalaf and Whitford, 2011). About
60% and 45% of the daily ingested fluoride is excreted in urine of healthy
adults and children, respectively (Villa et al., 2010). Increases in urinary flu-
oride were observed in children aged 5–8 years living in a nonfluoridated

Chapter 4
62
community aer using fluoride containing-dental varnishes (Garcia-Hoyos
et al., 2012). Thus, plasma and the kidney excretion rate constitutes the
physiologic balance determined by fluoride intake, uptake to and removal
from bone and the capacity of fluoride clearance by the kidney. Fluoride
is freely filtered through the glomerulus and undergoes a variable degree
of proximal tubular reabsorption, directly related to glomerular ultrafil-
trate pH (Whitford, 1994). Aer fluoride enters the renal tubules, a variable
amount (from 10 to 90%) is reabsorbed to systemic circulation, depending
on the urinary pH because transmembrane migration occurs by diffusion of
HF (Whitford et al., 1976). When the pH of tubular fluid is relatively high,
the amount of fluoride as HF is lower, while it is higher as F–. Thus, low flu-
oride concentration is available to be reabsorbed crossing the epithelium of
the epithelium renal tubule into the interstitial fluid. Instead, the majority
of nonreabsorbed is excreted in urine as F– (Ekstrand, 1996) (Figure 4.4).
In addition, any factor that affects urinary pH will have an impact on the
amount of fluoride that is excreted in urine (Buzalaf and Whitford, 2011).
Urinary F excretion is also influenced by the glomerular filtration rate since
its reduction, as occurs in chronic renal dysfunction as well as in the last
decades of life, results in lower excretion and increased plasma F levels
(Ekstrand, 1996).
Fluoride causes kidney injury seen by tubular dysfunction at plasma con-
centrations of fluoride 0.625 µg ml−1 and renal cell apoptosis (Bai et al.,
Figure 4.4 Fluoride reabsorption from renal tubule to systemic circulation. The
extent of fluoride reabsoption is depedent on urine pH. When the
urine is alkaline, the concentration of HF, the most permeable form
of fluoride, is lower and most of the fluoride remains in the tubule to
be excreted (on the le). Conversely, when the urine is acidic, the con-
centration of HF is higher that crosses to the renal tubule membrane
towards interstitium. Herein, it dissociates forming F– that diffuses into
the peritubular capillaries returning to systemic circulation (on the
right).

63
Fluoride Metabolism
2010). Several urine markers such as urinary kidney injury molecule (Kim-1),
clusterin (Clu), osteopontin, β-2-microglobulin, cystatin-C and heat shock
protein (HSP)-72 were shown to be upmodulated in 50 ppm fluoride-treated
rats compared to untreated rats (Cardenas-Gonzalez et al., 2013), while a tox-
icity marker (aflatoxin B1 aldehyde reductase) was up-modulated (Kobayashi
et al., 2011).
4.6 Modulators of Fluoride Metabolism and Their
Implications
Any systemic, metabolic and genetic alteration can modify fluoride metab-
olism. They interfere with absorption, excretion and so, with fluoride fate
in the body. Imbalances may lead to pathological conditions such as acute
and chronic toxicities, dental and skeletal fluorosis. They include acid–base
disturbances, physical activity, circadian rhythm and hormones (Whitford,
1996). Other factors such as nutritional status, diet composition, renal
impairment and genetic predisposition modify fluoride metabolism. They
are described in detail below.
4.6.1 Acid–Base Disturbances
Considering that urinary pH is a limitant factor in determining fluoride reab-
sorption and/or renal excretion (Figure 4.4), it seems obvious that acid–base
disturbances promote variations in fluoride tissue concentration. The acid–
base disturbances are usually induced by diet composition. In this sense,
vegetarian-based diet turns towards alkaline pH, while a meat-rich diet turns
towards acidic pH. Acute respiratory disorders similarly impact renal excre-
tion as well as metabolic disorders (Whitford, 1996).
4.6.2 Physical Activity, Circadian Rhythm and Hormones
Physical activity is associated with either decreased or increased circulating
fluoride levels (Whitford, 1996). Prolonged exercise causes enhancement of
diffusion of HF from extracellular to intracellular fluid in skeletal muscle
cells, a phenomenon caused by reduction in the pH gradient between cell
membranes.
A biological rhythmicity of fluoride was found to be opposite to that of
calcium and phosphate, discouraging the role of bone as a regulator. The
mean fluoride peak is reached around 9 a.m., and decreased around 9 p.m
in animals (Whitford, 1996). In humans, plasma fluoride concentrations
were positively correlated with urinary fluoride excretion rates and serum
parathormone levels, suggesting that both the renal system and hormones
regulate fluoride rhythmicity (Cardoso et al., 2008). In agreement with this,
Villa et al. (2008) demonstrated that fluoride urinary excretion is lower in the
diurnal compared to nocturnal period.

Chapter 4
64
4.6.3 Nutritional Status
There is a consensus that malnutrition and dental fluorosis prevalence are
related. However, this association still needs to be scientifically proven. The lack
of evidence so far is due to difficulties in data interpretations. A relationship
between water fluoride concentration, socioeconomic status, nutritional status
and prevalence of enamel lesions in child from Saudi Arabia was demonstrated
(Rugg-Gunn et al., 1997). In Brazil, using assessment of malnutrition by height-
for-age and weight-for-age indexes, dental fluorosis was shown to be dependent
on water fluoride concentration but independent of malnutritional status (Cor-
reia Sampaio et al., 1999). To clarify this controversy, longitudinal studies should
be conducted where variables such as nutritional status, diet composition and
fluoride intake are all implicated and controlled during tooth formation.
4.6.4 Diet Composition
Diet that interferes in the acidification and alkalinization of urine promotes
significant differences in urinary fluoride clearance and plasma half-lives. As
described above, this principle applies for protein-rich and vegetarian diets.
Long-term diet-induced changes in urinary pH could decrease (vegetari-
an-induced alkaline urine) or increase (protein-induced acidic urine) the risk
of dental fluorosis (Whitford, 1997). Thus, vegetarianism is inversely related
to dental fluorosis prevalence (Awadia et al., 1999).
High dietary concentrations of calcium also reduce fluoride absorption
(Whitford, 1994). Its impact over dental fluorosis prevalence was also evi-
denced (Chen et al., 1997). The rate of dental fluorosis of a milk-drinking
group was 7.2%, whereas that of the nonmilk-drinking group was 37.5%
(Chen et al., 1997). This and other data support the use of calcium supple-
mentation in endemic fluorosis areas.
Regular diets also seem to regulate fluoride retention in the body. Intake of
sorghum was associated with higher fluoride-induced toxicity compared to
rice- and wheat-based diets. These data were accompanied by less and more
fluoride urinary excretion, respectively (Lakshmaiah and Srikantia, 1977).
Also, a multigrain diet enriched with protein was showed to attenuate fluoro-
sis toxicity (Vasant and Amaravadi, 2013).
4.6.5 Renal Impairment
Greater plasma fluoride was observed in patients with renal failure (Schmidt
and Leuschke, 1986). However, no differences were observed in plasma insu-
lin level, as a result of fluoride intake, when control and renal-deficient rats
were compared (Lupo et al., 2011). Moreover, renal impairment in children
has been associated with tooth defects that include enamel pitting and hypo-
plasia. Intake of fluoride by nephrectomized rats impaired fluoride clearance
from plasma and worsened tooth development (Lyaruu et al., 2008). This
relationship was also demonstrated in humans (Farge et al., 2006).

65
Fluoride Metabolism
4.6.6 Genetic Predisposition
Increasing evidence has led to the affirmative that dental fluorosis is geneti-
cally influenced (Polzik et al., 1994). This is clarified through observation that
not all children living in the same fluoridated area develop dental fluorosis
(Yoder et al., 1998). The genetic character is reinforced when differences in
urinary fluoride levels and meal fluoride values are not found among these
children. The genetic predisposition to dental fluorosis is also well established
in mice (Everett et al., 2002; Everett, 2011). Examination of 12 inbred strains of
mice showed differences in susceptibility to dental fluorosis. The A/J strain is
“susceptible”, with a rapid onset and severe development of dental fluorosis,
while the 129P3/J is “resistant”, with minimum development of dental fluoro-
sis (Everett et al., 2002). These strains also differ regarding their susceptibili-
ties to the effects of fluoride in bone (Mousny et al., 2006; Everett, 2011). To
determine whether such differences were due to differences in F metabolism,
a metabolic study was conducted in which total F intake and excretion were
measured. The data showed that, compared to A/J mice, 129P3/J mice ingested
less water, excreted less urine, had lower urinary fluoride excretion and conse-
quently had higher F retention and plasma and femur fluoride levels (Carvalho
et al., 2009). Despite this, the amelogenesis in 129P3/J mice was remarkably
unaffected by fluoride (Carvalho et al., 2009). Recently, the molecular mecha-
nisms underlying the renal fluoride metabolism in A/J and 129P3/J mice that
may account for their differential metabolic handling of fluoride were deter-
mined using a proteomic approach (Carvalho et al., 2013). In F-treated groups,
Na(+)/H(+) exchange regulatory cofactor NHE-RF3, a protein involved in the
regulation of renal tubular reabsorption capacity was downmodulated in the
kidney of 129P3/J mice (Carvalho et al., 2013). Exclusive proteins expressed in
A/J or 129P3/J mice exhibited the same profile, regardless of exposure to fluo-
ride, suggesting that genetic background per se accounts for such differences
between these two strains of mice. In addition, some proteins that are codified
by chromosomes 2 and 11, loci previously characterized to determine suscep-
tibility and resistance to dental fluorosis in A/J and 129P3/J mice, respectively
(Everett et al., 2009; Everett et al., 2011), were found and may disclose the
molecular mechanism of dental fluorosis (Carvalho et al., 2013).
Besides, several gene polymorphisms have been addressed to explore pos-
sible correlation with dental fluorosis in humans. They include parathyroid
hormone (Wen et al., 2012), myeloperoxidase (Zhang et al., 2013), collagen1A2
(Huang et al., 2008) and estrogen receptor (Ba et al., 2011) gene polymor-
phisms. Except for parathyroid hormone gene, the others showed an associa-
tion between polymorphisms and risk and/or development of dental fluorosis.
4.7 Conclusion
Fluoride amounts in the body are tightly regulated and have a direct impact
on cellular and systemic levels. It is clear that the beneficial as well as the
adverse impact can be attributed to the magnitude and duration of fluoride

Chapter 4
66
exposure. In addition to the individual physiological differences in han-
dling fluoride, fluoride levels are also modulated by environmental, bio-
chemical, physiological, pathological and molecular factors. In the past
few years, this area of research was aimed to understand the molecular
mechanisms involved in fluoride metabolism and its biological effects.
Although considerable progress has been achieved, there is still much to
be explored.
Summary Points
● Fluoride is a cariostatic agent and is implemented in several systemic
and local sources.
● Fluoride presents both positive and negative impacts on hard tissues
depend on the dose and time of exposure.
● Fluoride metabolism includes its absorption in the gastrointestinal
tract, distribution through so and hard tissues followed by renal
excretion.
● The coefficient of permeability to HF is much higher compared to ionic
fluoride.
● Absorption of fluoride in the stomach occurs in a pH-dependent man-
ner and the tubular reabsorption depends on the urinary pH.
● Plasma is an important biomarker of fluoride exposure.
● Most fluoride absorbed is incorporated in bone.
● Several conditions affect fluoride metabolism including acid–base
disturbances, physical activity, circadian rhythm and hormones,
nutritional status, diet composition, renal impairment and genetic
predisposition.
Definitions and Explanation of Key Terms
Systemic toxicity: Requires absorption and distribution of the toxicant to a
site distant from its entry point, at which point effects are produced. Most
chemicals that produce systemic toxicity do not cause a similar degree of
toxicity in all organs, but usually demonstrate major toxicity to one or two
organs.
Coefficient of permeability: A rate of particle diffusion through cell
membranes that depends on the particle and total area through which it
traverses.
pH gradient: A gradient of hydrogen ion concentration between extracel-
lular and intracellular compartments.
Weak acid: An acid that is partially dissociated in an aqueous solution. It
is measured by the acid dissociation constant (Ka).

67
Fluoride Metabolism
Steady-state distribution: A condition of a physical system or device
that does not change over time, or in which any one change is contin-
ually balanced by another, such as the stable condition of a system in
equilibrium.
Fluc proteins-based channel: Fluoride ion exporters formed by a group of
small membrane proteins known as the “crcB” family or Fluc, a protein
that is widespread among unicellular organisms.
CLC anion-transporting proteins: A subclass of the widespread CLC super-
family of anion-transport proteins.
Fluoride exporter named FEX: A widespread family of fluoride export pro-
teins that mediated eukaryotic resistance to fluoride toxicity.
Tight junctions: Form the closest contact between adjacent cells known
in nature.
Bone remodeling: A lifelong process where mature bone tissue is
removed from the skeleton, a process called bone resorption, and
new bone tissue is formed, a process called ossification or new bone
formation.
RNA-based riboswitches: Riboswitches are specific components of an
mRNA molecule that regulates gene expression. The riboswitch is a part
of an mRNA molecule that can bind and target small target molecules.
Glomerular filtration rate: The flow rate of filtered fluid through the
kidney.
Amelogenesis: The formation ofenamel onteeth and begins when the
crown is forming during the bell stage oooth development aer dentino-
genesis, which is the formation of dentin. It has three stages: the induc-
tive, the secretory, and the maturation stages.
Dental fluorosis: A developmental disturbance of dental enamel caused
by excessive exposure to high concentrations of fluoride during tooth
development. It presents different appearances, varying from tiny white
streaks or specks in the enamel to discoloration or brown markings in the
tooth. The enamel may be pitted and rough.
Skeletal fluorosis: A bone disease caused by excessive consumption of flu-
oride that causes pain and damage to bones and joints.
List of Abbreviations
HF hydrogen fluoride
pKa the logarithmic form of acid dissociation constant
F– fluoride ion
NaF sodium fluoride
SMFP disodium monofluorophosphate
epH extracellular pH
ipH intracellular pH

Chapter 4
68
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