Biological Trace Element Research

https://doi.org/10.1007/s12011-019-01964-4

Fluoride-Free Diet Stimulates Pineal Growth in Aged Male Rats

Aaron Mrvelj 1,2 & Mark D. Womble 1

Received: 29 July 2019 / Accepted: 29 October 2019

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
The pineal gland is a naturally calcifying endocrine organ which secretes the sleep-promoting hormone melatonin. Age-related
changes of the pineal have been observed, including decreased pinealocyte numbers, increased calcification, and a reduction in
melatonin production. Since fluoride is attracted to calcium within the pineal gland, this study sought to examine the effects of a
fluoride-free diet on the morphology of the pineal gland of aged male rats (26 months old). All animals had previously been raised
on standard fluoridated food and drinking water. These control animals were compared to other animals that were placed on a
fluoride-free diet (Bfluoride flush^) for 4 or 8 weeks. At 4 weeks, pineal glands from fluoride-free animals showed a 96% increase
in supporting cell numbers and at 8 weeks a 73% increase in the number of pinealocytes compared to control animals. In contrast,
the number of pinealocytes and supporting cells in animals given an initial 4-week fluoride flush followed by a return to
fluoridated drinking water (1.2 ppm NaF) for 4 weeks were not different from control animals. Our findings therefore demon-
strate that a fluoride-free diet encouraged pinealocyte proliferation and pineal gland growth in aged animals and fluoride
treatment inhibited gland growth. These findings suggest that dietary fluoride may be detrimental to the pineal gland.

Keywords Pineal gland . Pinealocytes . Melatonin . Calcification . Fluoride . Fluoridation . Drinking water . Aging .
Neurodegenerative disease

Introduction Aging is associated with morphological changes in the pi-

Pineal Morphology and Aging
The pineal gland is a neuroendocrine organ that secretes the
sleep-regulating hormone melatonin (MEL). The secretory
pinealocyte cells of the rat and human pineal gland are classi-
fied as either light (type I) or dark (type II) cells based on their
light microscopic staining characteristics, with the majority of
pinealocytes being light cells [1–6]. More recently, single-cell
analysis of rat pinealocytes transcriptomes has described α- and
β-pinealocytes which together contribute to the production of
MEL [7, 8]. Pinealocytes constitute approximately 95% of the
gland, with the remainder consisting of interstitial and
supporting cells, such as astrocytes, microglia, and capillary
endothelial cells, within a network of nerve fibers [6, 9, 10].
* Mark D. Womble
mdwomble@ysu.edu
1
Department of Biological Sciences, Youngstown State University, 1
University Plaza, Youngstown, OH 44555, USA
2
Department of Anatomy and Neurobiology, Northeastern Ohio
Medical University, Rootstown, OH, USA
neal gland and a decline in MEL production. Pineal parenchy-
mal volume is directly proportional to both pinealocyte num-
bers and melatonin production [11–14]. The size and weight
of the human pineal declines with age [15] and glands from
aged rats and humans show increased fibrosis and elevated
glial cell numbers [1, 15, 16]. In both rats and humans, levels
of MEL as measured in the saliva [17], blood serum [18], or
urine [19, 20] also decline with increasing age.
The decline in MEL production with aging is due to the
loss of pinealocytes. Several studies have found that the pineal
glands of aged (> 22 months) rats [16, 21, 22] or shrews [23]
have decreased numbers of pinealocytes. Although the total
number of pinealocytes declines with age, glands from elderly
animals have elevated numbers of dark pinealocytes, but these
cells display several indicators of degeneration, including or-
ganelle swelling, increased numbers of cytoplasmic vacuoles
and dense bodies, and increased plasma membrane permeabil-
ity [1, 21–23], suggesting that dark cells may be in the process
of undergoing cell death. Pinealocyte precursor cells have the
morphological features of type II pinealocytes (dark cells) and
are found dispersed throughout the gland of adult rats [10].
Observations of dying dark cells in aged animals may there-
fore indicate the selective loss of these precursor cells, thereby
reducing the regenerative ability of the pineal.

The pineal also has naturally occurring intracellular and
extracellular calcium phosphate concretions known as
acervuli (corpora arenacea or brain sand) [1, 24–29]. In rats,
shrews, and humans, the degree of pineal calcification in-
creases with age [1, 15, 23, 25, 27, 29–32]. An analysis of
light and dark cells in the rat pineal found elevated levels of
intracellular calcium and signs of cellular degeneration only in
dark cells [22], suggesting that calcification is related to both
the age-dependent loss of pinealocytes and the decline in
MEL production in older humans [29]. Decreased pineal vol-
ume and increased calcification have also been correlated with
the cognitive decline of Alzheimer’s disease but not other
dementias [13, 33], a finding that may be related to a decline
in MEL secretion and the loss of its neuroprotective anti-
inflammatory and anti-oxidative actions [34, 35].
Fluoride
Due to the reduction in dental caries [36], water fluoridation is
regarded as one of the ten greatest public health achievements
of the twentieth century [37]. The American Dental
Association advocates for the use of fluoridated drinking wa-
ter because it benefits developing teeth by incorporating fluo-
ride into the hydroxyapatite crystals of the tooth enamel, thus
making it harder and more resistant to decay [38]. The US
Environmental Protection Agency currently allows drinking
water to be treated with fluoride up to a maximum level of 1.2
parts per million (ppm). However, based on comparisons of
populations exposed to fluoridated water versus non-
fluoridated water, some authors have argued that systemic
fluoridation via the water supply does not have an effect on
the prevalence of dental caries [39–41]. Since in adults, fluo-
ride must be applied directly to the surface of the teeth to be
effective in preventing dental caries [38], it has been suggested
that fluoride-treated drinking water may not bestow an anti-
caries effect for adults, with fluoridated toothpaste and regular
dental checkups instead serving as the primary factors contrib-
uting to observed decreased rates of tooth decay.
The federally required Material Safety and Data Sheet clas-
sifies fluoride as a mutagen, and studies have suggested that
dietary fluoride has the potential to cause human health prob-
lems [40]. In support of this, one study found that rats given
drinking water with moderate levels of fluoride (2 or 10 ppm)
resulted in morphological alterations in hippocampal neurons
[42]. After 30 days of treatment, neurons showed decreased
cell diameters and exhibited several indicators of cell death,
including increased cytoplasmic vacuoles, nuclei showing
signs of pyknosis or karyolysis, and elevated expression of
cathepsin D, an intracellular protease. At higher levels (100–
200 ppm), fluoridated drinking water can also impair repro-
ductive functions in male and female rats [43–45].
As an anion, fluoride tends to concentrate in calcium-
containing areas such as the pineal gland. Elevated fluoride
Mrvelj and Womble
has been found in the pineal glands of elderly humans [25,
30]. In these glands, fluoride was positively correlated with
calcium levels and the fluoride/calcium ratio of the pineal
exceeded that of bone. Similarly, ducks exposed to elevated
environmental levels of fluoride show significantly higher
concentrations of fluoride in their pineal gland compared to
bone tissue [46].
Fluoridated public drinking water is widely used as a sys-
temic treatment for dental caries, but few studies have been
conducted to assess the effects of chronic dietary fluoride ex-
posure. The objective of this study was to examine the effect
of removing fluoride from the diet of aged rats (starting age 26
months) on the cellular structure of the pineal gland and com-
pare this to pineal glands from animals exposed to dietary
fluoride. Glands obtained from animals previously raised with
fluoridated food and drinking water were compared to animals
placed on a fluoride-free diet for 4 or 8 weeks. Additional
animals were maintained fluoride-free for the initial 4-week
period and then switched back to fluoridated drinking water
(1.2 ppm) for the final 4 weeks. It was found that the fluoride-
free diet resulted in increased numbers of supporting cells and
pinealocytes, suggesting growth of the pineal gland. In com-
parison, glands from both groups of fluoride-treated animals
showed fewer pinealocytes, suggesting that dietary fluoride
may have a detrimental effect on the pineal gland.
Methods
Animal use adhered to the current Guide for the Care and Use
of Laboratory Animals [47] and was approved by the
Youngstown State University’s Institutional Animal Care
and Use Committee (protocol no. 04-16). Animals were
housed in the Department of Biological Sciences animal fa-
cility and at the end of the study were sacrificed by carbon
dioxide asphyxiation and decapitation.
Population
Twenty male Sprague-Dawley rats (Charles River, Ohio), ini-
tially 26 months old and weighing an average of 582.75 g,
were used for this study. The animals had previously been
used in a behavioral research project unrelated to this study.
Animals were housed in clear plastic cages (50 cm × 40 cm ×
21 cm) with metal cage tops. Each cage had bedding and
contained three to four animals. The animal facility was on a
reverse 12-h light-dark schedule, with light periods being
10:30 a.m.–10:30 p.m. Animal well being was monitored on
a daily basis. Fresh water was supplied daily and ad libitum
dry rat food was provided weekly. Body weight and food and
water consumption were recorded weekly, and no significant
differences were found between the experimental groups in
these measures during the course of the study.
Fluoride-Free Diet Stimulates Pineal Growth in Aged Male Rats
Experimental Groups
The animals were divided into four groups, consisting of two
to seven animals per group (Fig. 1). All animals had previous-
ly been raised on standard rat food (Prolab Isopro RMH 3000,
LabDiet, St. Louis Missouri; 16 ppm fluorine) and fluoridated
tap water (0.9 ppm fluoride; Aqua America, Youngstown
Ohio). To determine the effects of this previous fluoride ex-
posure, the animals in group 1 were sacrificed at the beginning
of the experiment (day 0).
All other animals were then placed on a fluoride-free diet
(fluoride Bflush^) consisting of rat food with 0 ppm fluoride
(AIN-76A Rodent Diet; Bio Serv, Fleming NJ, USA) and
Milli-Q filtered water (Laboratory standard type I ultrapure
water; 18.2 MΩ*cm at 25 °C). After 4 weeks of fluoride flush
(day 28), animals in group 2 were sacrificed. Animals in group
3 also followed the fluoride-free flush for 4 weeks (days 0–
28), but at the beginning of week 5 (day 29), their drinking
water was changed to water containing 1.2 ppm NaF.
Fluoridated water was prepared weekly using NaF powder
(> 99%, Sigma-Aldrich Corp.) dissolved into Milli-Q filtered
water. Group 3 animals were maintained on 1.2 ppm fluori-
dated water and fluoride-free food for the remaining 4 weeks
(days 29–56) and then sacrificed at the end of week 8 (day 56).
Animals in group 4 were maintained on the fluoride-free food
and water diet for the entire 8 weeks of the study and were
sacrificed at the end of week 8 (day 56).
Morphological Analysis
Pineal glands from all animals (groups 1–4) were harvested,
and 17 glands were used for morphological measurements.
Glands were fixed overnight in 10% buffered formalin and
then processed for paraffin embedding, sectioning (7 μm),
and hematoxylin and eosin (H&E) staining using standard
histological protocols [48].
Fig 1 Experimental design.
Twenty animals were divided into
four treatment groups. At the
onset of the experiment, group 1,
which had been raised with
fluoridated tap water and food,
was sacrificed. Groups 2, 3, and 4
then began a 4-week diet of
fluoride-free food and water.
Group 2 was sacrificed at the end
of week 4. For the final 4 weeks,
group 3 received drinking water
with 1.2 ppm NaF, while group 4
continued the fluoride-free diet.
Groups 3 and 4 were sacrificed at
the end of week 8
A minimum of three randomly selected tissue sections
from each pineal gland were used for analysis. Within each
tissue section, 3–5 separate areas were selected and cell counts
were done for each of these areas using a calibrated reticle grid
in the eyepiece of an Olympus CX22LED light microscope at
× 400 total magnification. All cells within the grid were count-
ed, but any cell whose nucleus touched the outside border of
the grid was excluded. Each view was counted twice and
averaged. The grid was 7656 μm2 in size, and all cell counts
reported here are expressed as the number of cells per this unit
area.
Cells were characterized as pinealocytes or supporting cells
using cell size, intensity of cytoplasmic staining, and nuclear
characteristics as distinguishing features [1–6] (Fig. 2).
Pinealocytes displayed a single cellular process, a round to
oval cell body, and a characteristically round to ovoid nucleus.
Pinealocytes were also categorized as light or dark cells. Light
cells were more abundant, with a lighter staining cytoplasm
and a round or oval nucleus with less dense chromatin and a
prominent nucleolus. Dark cells were round, oval, or elongat-
ed, with irregularly shaped nuclei containing clusters of con-
densed chromatin. All other cells, including glial cells, endo-
thelial (capillary) cells, and fibroblasts, were collectively clas-
sified as supporting cells.
Statistical Analyses
All data are reported as the mean ± standard deviation (SD).
Data that violated homoscedasticity were log-transformed and
Tukey honest significant difference was used for all post hoc
tests. The effect of fluoride treatment on dependent measures
of cell number per unit area was analyzed using a one-way
ANOVA to determine if there were differences based on treat-
ment. Levene’s test for equality of error variance was signifi-
cant; thus, the data was log10 transformed, but this did not
affect the outcome of analyses.

Fig 2 Light micrographs of
pineal glands, showing
representative examples of light
pinealocytes (circle), dark
pinealocytes (square), and
supporting cells (pentagon). a
Group 1 (baseline control,
previously raised on fluoridate
food and water; sacrificed day 0).
b Group 2 (4 weeks fluoride
flush; sacrificed day 28). c Group
3 (4 weeks fluoride flush then 4
weeks 1.2 ppm fluoride;
sacrificed day 56). d Group 4 (8
weeks fluoride flush; sacrificed
day 56)
Results
Effects of Previous Fluoride Diet
To provide baseline control measurements for the morphology
of pineal glands, we began by examining glands from control
animals previously raised on standard rat food (16 ppm fluo-
ride) and fluoridated tap water (0.9 ppm fluoride) and
sacrificed at day 0 (group 1). Glands from these animals
(Fig. 2a) had 26.41 ± 4.46 total cells/unit area (n = 24), which
included both supporting cells (12.21 ± 3.40 supporting cells/
unit area, n = 24) and pinealocytes (14.20 ± 3.90 pinealocytes/
unit area, n = 24) (Fig. 3; Table 1). Classification of
pinealocytes as either light or dark cells found that these
glands had 9.09 ± 2.66 light cells/unit area (n = 24) and 5.11
± 1.68 dark cells/unit area (n = 24) (Fig. 4; Table 1).
Effects of Fluoride-Free Diet
We next sought to assess the effects of changing animals
to a fluoride-free diet (fluoride flush) on the morphology
of the pineal gland. Since no background information was
found in the scientific literature which described the peri-
od of time necessary to remove fluoride from the pineal,
flush periods of 4 weeks (group 2) or 8 weeks (group 4)
were used (Fig. 2b, d).
Mrvelj and Womble
The total number of cells per unit area significantly in-
creased with time during the fluoride flush (Fig. 3; Table 1).
Both group 2 (40.78 ± 8.88 total cells/unit area, n = 24, p <
0.001; 54% increase) and group 4 (41.13 ± 8.38 total cells/unit
area, n = 50, p < 0.001; 56% increase) had significantly more
cells compared to group 1. There was no significant difference
between groups 2 and 4 in total cell numbers per unit area.
The increase in total cells (pinealocytes plus supporting
cells) observed in group 2 after 4 weeks of a fluoride-free flush
was largely due to a significant increase in the number of
supporting cells rather than pinealocytes (Fig. 3; Table 1).
Thus, group 2 (23.99 ± 9.18 supporting cells/unit area, n =
24) displayed a 96% increase (p < 0.002) in supporting cells
compared to group 1. In contrast, there were no significant
differences between group 1 and group 2 in the total number
of pinealocytes (group 2: 16.79 ± 4.98 pinealocytes/unit area,
n = 24) or in the number of light pinealocytes (group 2: 10.25
± 3.14 light cells/unit area, n = 24) (Fig. 4; Table 1). However,
there was a small but significant increase in the number of
dark pinealocytes in group 2 (6.54 ± 2.65 dark cells/unit area,
n = 24; p = 0.026; 28% increase) compared to group 1.
Pineal glands from animals provided a fluoride-free diet for
8 weeks (group 4) also showed a significant increase in total
cell numbers compared to group 1 (Fig. 3; Table 1). But, in
contrast to group 2 (4-week flush) which showed increased
supporting cell numbers, group 4 (8-week flush) showed no
Fluoride-Free Diet Stimulates Pineal Growth in Aged Male Rats

Fig 3 Fluoride-free diet increases

and fluoride treatment decreases
pineal cell numbers as seen with
changes in the number of total
cells, pinealocytes, and
supporting cells per unit area
between group 1 (baseline
controls), group 2 (4 weeks
fluoride flush), group 3 (4 weeks
fluoride flush then 4 weeks
1.2 ppm fluoride), and group 4 (8
weeks fluoride flush).
*Significant difference from
group 1 (p < 0.05); **significant
difference from group 2 (p <
0.001); ***significant difference
from group 3 (p < 0.001)

difference in supporting cell number (16.44 ± 5.23 supporting
cells/unit area, n = 50) compared to group 1. Thus, supporting
cell density (cells per unit area) declined during the additional
4 weeks of fluoride-free flush.
The increase in total cell numbers observed after 8 weeks of
flush was instead due to a significant increase in the number of
pinealocytes per unit area (Fig. 3; Table 1). Thus, total
pinealocytes for group 4 (24.69 ± 8.60 pinealocytes/unit area,
n = 50) showed a 73% increase compared to group 1 (p <
0.001) and a 47% increase compared to group 2 (p < 0.001)
(Fig. 3). Similarly, glands from group 4 had significantly larg-
er numbers of light pinealocytes (15.15 ± 6.00 light cells/unit
area, n = 50) and dark pinealocytes (9.54 ± 4.17 dark cells/unit
area, n = 50) compared to both group 1 (light cells/unit area, p
= 0.006; dark cells/unit area, p < 0.001) and group 2 (light
cells/unit area, p < 0.001; dark cells/unit area, p = 0.001) (Fig.
4; Table 1). Thus, pineal glands from group 4 had 67% and
87% more light and dark cells respectively than found in
group 1 control glands. It therefore appears that an 8-week
fluoride-free flush encouraged the proliferation of both light
and dark pinealocytes.
Together, our observations of pineal glands obtained from
animals provided a fluoride-free diet indicates that a 4-week
fluoride flush initially encouraged the proliferation of
supporting cells, but an additional 4 weeks of flush allowed
for the subsequent preferential proliferation of pinealocytes,
thus decreasing (Bdiluting^) the density of supporting cells
observed per unit area. These findings suggest that removal
of dietary fluoride promotes growth of the pineal gland in
elderly animals.
Effects of Fluoride
As a final test for the effects of fluoride on pineal gland mor-
phology, animals in group 3 were initially placed on a
fluoride-free diet for 4 weeks, but then for the final 4 weeks
were switched to fluoridated drinking water containing
1.2 ppm NaF, the maximum fluoride concentration allowed
by the EPA in U.S. drinking water (Fig. 2c).
After 4 weeks of fluoride treatment, total cell numbers for
group 3 animals (29.87 ± 7.10 cells/unit area, n = 49) were
not different from group 1 animals previously raised with

Table 1 Summary of experimental findings. All measurements are the number of cells per unit area (mean ± SD)

Group 1 Group 2 Group 3 Group 4

Total cells 26.41 ± 4.46 (n = 24) 40.78 ± 8.88a,c (n = 24) 29.87 ± 7.10 (n = 49) 41.13 ± 8.38a,c (n = 50)
Pinealocytes 14.20 ± 3.90 (n = 24) 16.79 ± 4.98 (n = 24) 14.60 ± 5.58 (n = 49) 24.69 ± 8.60a,b,c (n = 50)
Supporting cells 12.21 ± 3.40 (n = 24) 23.99 ± 9.18a,c (n = 24) 15.27 ± 6.39 (n = 49) 16.44 ± 5.23b (n = 50)
Light pinealocytes 9.09 ± 2.66 (n = 24) 10.25 ± 3.14 (n = 24) 9.59 ± 3.75 (n = 49) 15.15 ± 6.00a.b.c (n = 50)
Dark pinealocytes 5.11 ± 1.68 (n = 24) 6.54 ± 2.65a,c (n = 24) 5.02 ± 3.06 (n = 49) 9.54 ± 4.17a,b,c (n = 50)
a
Significantly different from group 1
b
Significantly different from group 2
c
Significantly different from group 3

Fig 4 Fluoride-free diet increases
and fluoride treatment decreases
pinealocyte numbers. Changes in
the number of total pinealocytes,
light pinealocytes, and dark
pinealocytes per unit area
between group 1 (baseline
controls), group 2 (4 weeks
fluoride flush), group 3 (4 weeks
fluoride flush then 4 weeks
1.2 ppm fluoride), and group 4 (8
weeks fluoride flush).
*Significant difference from
group 1 (p < 0.05); **significant
difference from group 2 (p <
0.01); ***significant difference
from group 3 (p < 0.001)
fluoridated food and tap water (Fig. 3; Table 1). Compared to
group 1, the pineal glands from group 3 also showed no
significant differences in the numbers of pinealocytes
(14.60 ± 5.58 pinealocytes/unit area, n = 49), light or dark
pinealocytes (9.59 ± 3.75 light cells/unit area, n = 49; 5.02 ±
3.06 dark cells/unit area, n = 49), or supporting cells (15.27
± 6.39 supporting cells/unit area, n = 49) (Fig. 4; Table 1).
Thus, the increases in total cell, supporting cell, and dark cell
numbers observed in group 2 (after 4 weeks of fluoride
flush) were completely reversed by an additional 4-week
treatment with fluoridated drinking water (group 3).
Not surprisingly, when compared to group 4 (8-week
flush), glands from group 3 showed significantly fewer total
cells (p < 0.001) (Fig. 3; Table 1). Since there was no signif-
icant difference between these groups in the number of
supporting cells per unit area (Fig. 3; Table 1), the difference
in total cell numbers was due to a significantly lower density
of pinealocytes for group 3 (p < 0.001; 41% fewer). This
difference in pinealocytes resulted from lowered numbers of
both light and dark pinealocytes per unit area (Fig. 4; Table 1),
with group 3 showing significant decreases in light cells (p <
0.001; 37% fewer) and dark cells (p < 0.001; 47% fewer)
compared to group 4.
Despite these differences in pinealocyte numbers, it was
found that light cells were more abundant than dark cells in
glands obtained from both fluoride-treated animals (group 3)
and fluoride-free animals (group 4), which agrees with earlier
reports of light and dark pinealocyte numbers and ratios [1, 2,
5, 6]. Thus, there was no difference in the ratio of light to dark
pinealocytes between group 3 (2.25±1.49 light/dark
pinealocyte) and group 4 (1.83 ± 0.99 light/dark pinealocyte),
suggesting that the proliferation of pinealocytes observed after
8 weeks of a fluoride-free diet involved both types of
pinealocytes approximately equally.
In sum, our findings suggest that the removal of dietary
fluoride promotes growth of the pineal gland in aged rats.
Mrvelj and Womble
This growth initially involves an increase in supporting cell
numbers, followed by subsequent increases in the numbers of
both light and dark pinealocytes.
Discussion
Previous research has found significantly higher concentra-
tions of fluoride within the elderly human pineal gland com-
pared to muscle, bone, or brain tissue [25, 30]. Electron-probe
microscopy has shown high concentrations of calcium within
the mitochondria and dense vacuoles of pinealocytes [22, 24],
and a correlation between fluoride levels and calcium concen-
trations in pinealocytes has been found [25, 30]. This suggests
that fluoride preferentially accumulates within the pineal
where it may be attracted by calcium. The pineal also exhibits
intracellular and extracellular calcium phosphate deposits
known as acervuli [29], which are thought to initially begin
as intracellular vacuoles containing high calcium ion concen-
trations that grow until the cell disintegrates, thereby releasing
the calcium concrements into the extracellular space [24, 26,
27]. Studies in rats and humans have shown that the degree of
pineal gland calcification and acervuli volume increases with
age [1, 15, 25, 27, 29–32], and these changes correlate with
decreased levels of MEL measured in the saliva, urine, or
blood [17–20, 29]. Thus, there appears to be correlations be-
tween increasing age, the degree of pineal calcification, fluo-
ride levels, and decreased MEL production.
The present study was undertaken to examine the effects of
a fluoride-free diet on the cellular morphology of the pineal
gland in aged (26-month-old) male rats. Animals previously
raised on standard fluoride-containing rat food and fluoridated
tap water served as controls. In an attempt to remove previ-
ously accumulated fluoride, animals were provided with
fluoride-free food and water for 4 or 8 weeks (fluoride flush).
Fluoride-Free Diet Stimulates Pineal Growth in Aged Male Rats
Our findings demonstrated that 4 weeks of fluoride-free
flush lead to an increase in interstitial supporting cell density
(number of cells per unit area), while 8 weeks of flush resulted
an increased density of both light and dark pinealocytes.
Although gland weights were not recorded, the initial increase
in supporting cells and later increase in pinealocyte numbers
suggest growth of the gland. A possible explanation for the
early increase in supporting cells may be growth of capillaries
as the pineal began to enlarge. Since pineal secretory activity
is directly proportional to parenchymal volume and
pinealocyte density [11–14], the later increase in pinealocyte
numbers suggests that longer-term fluoride-flush animals
would show a corresponding increase in MEL secretion.
Enlargement of the pineal in the aged animals used in this
study is an unusual finding, as previous researchers have re-
ported declines in MEL production [17–20], pineal size [15],
and pinealocyte numbers [16, 21–23] with increasing age.
One possible explanation for these reported declines is that
the human and animal pineal glands assayed in these previous
reports were regularly exposed to dietary fluoride.
The number of supporting cells per unit area was found to
be elevated after 4 weeks of flush but by 8 weeks, this number
had returned to the control level. Paradoxically, this observa-
tion provides additional evidence for our conclusion that the
fluoride flush promoted pineal growth. Since our observations
are reported as cellular density (the number of cells per unit
area), the decrease in supporting cells between 4 and 8 weeks
does not necessarily indicate a loss of cells. Instead, this
change is likely due to a stable population of supporting cells
during the last 4 weeks combined with a concurrent increase
in pinealocytes. As the population of pinealocytes expanded,
the surrounding supporting tissue occupied less space within
the gland, thereby decreasing the number of supporting cells
observed per unit area. The increase in pinealocytes thus
Bdiluted out^ supporting cell density.
The source of these new pinealocytes is unknown, but low
levels of mitotic activity have been observed in the adult pi-
neal [49, 50]. A recent study examining the pineal expression
of Pax 6, a homeobox transcription factor that is essential for
pineal gland formation, found that Pax6-positive cells differ-
entiated into both pinealocytes and astroglial supporting cells
[10]. Pax6-positive cells had the same morphological features
as previously described type II pinealocytes (dark cells) and
were found dispersed throughout the gland in mature rats.
This raises the interesting possibility that the small but signif-
icant increase in dark pinealocytes observed following 4
weeks of fluoride-free flush represents an expansion of the
Pax6 precursor cell population, which subsequently gives rise
to new supporting cells and pinealocytes. Since degenerating
or dying dark cells have been observed in the aged pineal
gland [1, 21–23], our findings suggest that the removal of
dietary fluoride may stimulate the growth and differentiation
of Pax6-positive cells.
To test the effects of dietary fluoride, additional animals
were placed on a fluoride-free diet for 4 weeks and then given
drinking water containing 1.2 ppm NaF for the last 4 weeks.
The change to fluoridated water completely reversed the pos-
tulated growth of the pineal gland induced by the fluoride-free
flush, with both supporting cell and pinealocyte numbers
returning to baseline levels. These effects of fluoride treatment
are similar to the decline in pinealocytes seen with aging [1,
16], disruptions to normal sleep patterns [51], or prolonged
constant illumination [28].
Dietary fluoride may have the potential to cause adverse
human health problems [40]. Previous animal studies have
found that dietary fluoride can induce oxidative stress and cell
death within the brain and other organs. Rats administered
moderate levels of fluoride in their drinking water (2 or
10 ppm NaF) showed neuronal losses within the hippocam-
pus, with remaining neurons exhibiting shrunken cell bodies,
reduced nuclear diameters, and increased levels of cathepsin
D, an intracellular protease that serves as a marker of cell
death [42]. Similarly, brain, heart, liver, and kidney tissues
of rats given drinking water with 150 ppm NaF showed in-
creased levels of malondialdehyde and elevated activities of
the antioxidant enzymes catalase, glutathione peroxidase, glu-
tathione reductase, and superoxide dismutase, all of which are
indicators of oxidative stress [52, 53].
Additional studies have found deleterious effects of fluo-
ride on reproductive functioning. Male gerbils administered a
high-fluoride diet (37 mg F/kg) showed significantly de-
creased MEL production prior to puberty and decreased testis
weight after puberty [30]. In female gerbils, the high-fluoride
diet did not decrease MEL production prior to puberty but it
was associated with a significant acceleration of puberty on-
set. These findings suggest adverse effects by fluoride prior to
and during puberty. Similarly, rats given drinking water with
high levels of fluoride (100–200 ppm) showed impaired sper-
matogenesis in males and decreased reproductive hormone
levels and fertility in females [43–45].
Our study found that an 8-week fluoride-free flush resulted
in an increased density of pinealocytes in the glands of elderly
rats (starting age 26 months). Since studies have reported
losses of pinealocytes with increasing age [16, 21, 22], our
finding that removal of dietary fluoride can stimulate an in-
crease in pinealocyte numbers raises the possibility that the
previously reported age-related loss of pinealocytes may not
be directly due to increasing age but instead could have result-
ed from the life-long consumption of fluoridated food and
water. Our results therefore suggest that fluoride may have
detrimental effects on the pineal gland, and these effects can
be reversed by the removal of dietary fluoride.
This study did not measure circulating MEL levels, but the
finding of pineal growth after removal of dietary fluoride sug-
gests the possibility of elevated MEL secretion [11–14].
Pineal calcification was also not examined. Since elevated

calcification has been linked to decreased MEL production
[29] and to the dementia of Alzheimer’s disease [13, 33, 34],
it would be of great interest to determine if a fluoride-free diet
was able to reduce pineal calcification and increase MEL pro-
duction in elderly individuals, a finding that could have im-
portant consequences for the treatment of neurodegenerative
diseases.
Acknowledgments Dr. Thomas P. Diggins (Department of Biological
Sciences, Youngstown State University) assisted with the statistical
analysis.
Author Contributions Mrvelj A and Womble MD designed and con-
ceived the study. Mrvelj A performed the experiments and data analysis.
Mrvelj A and Womble MD drafted and approved the manuscript.
Compliance with Ethical Standards
Conflict of Interest This study was supported by a Youngstown State
University Research Council grant to MDW and a Cushwa-Commericial
Shearing Graduate Fellowship to AM. The authors declare that there is no
conflict of interest associated with this manuscript.
Animal use adhered to the Guide for the Care and Use of Laboratory
Animals (8th edition, National Research Council, 2011) and was approved
by the Youngstown State University’s Institutional Animal Care and Use
Committee (protocol #04-16).
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