Download Lesneski and Szlanta

The Effect of Water Fluoridation on Thyroid Function in Rattus norvegicus
and its Relevance to Hypothyroidism
Amy L. Lesneski and Christina Szlanta
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Fluoride, a highly electronegative ion, is a common additive in water supplies due to its
ability to prevent tooth decay through enamel and dentin strengthening. However, in high
concentrations it may interfere with thyroid function, causing the thyroid to produce lowered
amounts of thyroid hormones, thyroxine (T4) and triiodothyronine (T3). The current study
sought to determine whether decreases in free thyroxine (fT4) resulted from fluoride
administration. The effect of fluoride on the hormone level fT4 was studied by orally
administering calculated amounts of sodium fluoride (NaF) to fifteen male fancy rats. The
control group (0 ppm) did not receive any NaF. Group 1 was given 4.42 x 10-2 mg NaF
(equivalent to 2 ppm) and group 2 was given 2.21 mg NaF (equivalent to 100 ppm). The water
with the appropriate amount of NaF was administered on a daily basis at a dose of 0.3 ml per
rat. The hormone levels in the serum were measured with an fT 4 enzyme immunoassay (EIA).
The fT4 levels within the control group increased by 0.004586 ± 0.004255 ɳg·dL-1·g-1 (Mean ±
95% C.L., n=15). Group 1 had a slight increase in fT4 levels with a hormone concentration of
0.0001979 ± 0.003396 ɳg·dL-1·g-1 (Mean ± 95% C.L., n=15). Hormone levels in group 2 had
insignificant decreases of 0.006503 ± 0.001294 ɳg·dL-1·g-1 (Mean ± C.L., n=15). A single-factor
ANOVA revealed there to be no significant differences between hormone levels in any of the
three groups (n= 15, p = 0.08850). Although differences were not significant, extreme fluorosis
(100 ppm) induced slight thyroid dysfunction, resulting in minor decreases of fT4 production
by the thyroid gland. These results could have a correlation to ATP halting due to inhibition of
glycolysis, competition of iodide and fluoride through the sodium/iodide symporter (NIS), and
fluoride induced DNA damage, resulting in alterations in protein synthesis.
Introduction
Water fluoridation is prevalent in many countries, including the United States (Peterson,
2003). As an added material to public water systems, fluoride’s role in water encompasses the
improvement of dental enamel and dentin, and the prevention of tooth decay (Rolla et al., 1993;
Yoon et al., 1959). Fluoride is also used for treatment of osteoporosis (El-Khoury et al., 1982),
stimulating osteoblast activity and resulting in increased bone density. Amounts deemed
beneficial are determined by various governmental agencies. According to the California
Department of Public Health, adequate fluoride concentrations in water supplies range from 0.82.0 mgL-1 (CDPH website, 2011). Although human exposure to small doses of sodium fluoride
can be beneficial for teeth and bone, little regard is taken into consideration to the possible
effects on small and large mammals, whom, unknowingly, are exposed to fluoridated water.
Minor and chronic fluoride exposure can result in physiological alterations within
mammalian bodies. For instance, increased fluoride intake has been shown to cause dental
fluorosis, muscular and neurological effects, and decreased brain and muscle enzyme activity,
which may alter growth and development (Bixler and Muhler, 1956; Lakshmi et al., 2000;
Trabelsi et al., 2001). Most importantly, research illustrates fluoride to be detrimental on thyroid
function, which is substantial in regulation of metabolism. Fluoride induced decreases in thyroid
function could possibly make animals more susceptible to attaining hypothyroidism- a condition
in which an underactive thyroid permanently produces lowered amounts of thyroid hormones. A
study administered by Hillman and colleagues (1979) directly correlated prevalence in fluoride
ingestion to increased frequency of hypothyroidism and anemia throughout dairy cattle. The
dysfunction in thyroid activity observed with fluoride intake is a direct response to decreases in
thyroid hormone production, thyroxine (T4) and triiodothyronine (T3), synthesized by the thyroid
gland (Bouaziz et al., 2005; Cinar et al., 2005; Wang et al., 2009). Although high quantities
clearly have deleterious effects on thyroid activity, amounts as low as 5.0 ppm have been
recognized to cause suppression in thyroid hormone production (Cinar et al., 2005).
The purpose of the current study was to investigate the effects of water fluoridation on free
thyroxine (fT4) production in rats administered legal California levels of fluoride (2 ppm), and
amounts equivalent to excessive fluorosis (100 ppm).
Methods and Materials
Animals and Housing
Fifteen 40 day old male fancy rats were purchased from Sand Bar Pet shop in Mission
Viejo and transported to Lake Elsinore, California. Upon arrival, rats were haphazardly separated
into three groups such that each group contained two albino rats, two bi-colored fancy rats, and
one solid colored rat. Groups were housed within separate wire cages containing wood shavings
in a room with proper ventilation, standard hygienic conditions, and overall ambient
temperatures of 20-25°C. Rats had free access to water purified through reverse osmosis. Food
containing nutrient quantities of 18% protein and 0.9% calcium (Wang et al., 2009) was given to
each group twice a day. Food amounts were increased accordingly from 8-14 tablespoons per
group per day, as to compensate for growth of the rats over the experimental period. In addition,
the rats were acclimatized to the new environment, food requirements, water, and temperature
conditions for six days prior to experimentation. All procedures listed below were approved by
Professor Teh at Saddleback College, Mission Viejo.
Procedures
Prior to experimentation, the rats were labeled and weighted, and hair was removed from
the ventral portion of their hind legs. Blood was withdrawn from the saphenous vein according to
Hem (1998), and captured in non-heparinized twist cap centrifuge tubes. Blood samples were
then centrifuged for four minutes at 6000 rpm with a Spectrafuge 16M centrifuge, and then
immediately stored at -20°C. Afterward, the rats were orally administered calculated
concentrations of sodium fluoride daily for 16 days. Each individual rat within the two
experimental groups was given the following daily amounts of sodium fluoride dissolved within
0.3 mL of water: experimental group 1: 4.42 x 10-2 mg NaF (equivalent to 2.00 ppm F-);
experimental group 2: 2.21 mg NaF (equivalent to 100 ppm F-); rats in the control group were
given no sodium fluoride. After 16 days, rats were again weighed and final blood samples were
extracted.
All frozen blood samples were thawed, centrifuged, and blood serum was separated from
the remaining blood cells. Free thyroxine (fT4) levels were determined using an enzyme
emmunoassay (EIA) produced from MP Biomedicals Incorporated. Samples were processed
through the required protocols within the attained kit. The microplate reader (Fischer Scientific
Multiskan MCC 340), set at 405 ɳm, was used to determine absorbencies of the samples. These
values were later used to calculate fT4 concentrations.
A standard curve was created from calibrated samples at an optical density reading of
405 nm. Absorbencies were obtained and plotted against fT4 reference standard concentrations,
and the equation of the best fit curve was determined to be y=0.607e-0.246x (Figure 1).
Figure 1. Standard curve for the free thyroxine (fT4) EIA kit. Absorbance for standards at 405 ɳm versus free T4
concentrations (ɳg·dL-1).
The equation was utilized to calculate the fT4 concentrations (ɳg·dL-1) for each rat. The differing
weights of each rat was then factored out through calculation of mass specific concentrations by
division of their weights, producing the actual fT4 concentrations (ɳg·dL-1·g-1). The difference
between before and after concentrations was then used to analyze hormone changes after sodium
fluoride administration. A single-factor ANOVA was performed to determine differences
between groups.
Results
Masses of each rat were determined before and after experimentation. All groups
experienced weight gains; however, gains within the experimental groups were less than the
control group. Average weight gain for the control group was 90.44 g, whereas, the 2 ppm and
100 ppm experimental groups had average weight gains of 83.91g and 72.29 g. A single-factor
ANOVA revealed there to be no significant differences in weight gains between the groups
(n=15; p= 0.1710).
Free T4 concentrations were determined and calculated through enzyme immunoassay
(EIA). The current study revealed that free T4 levels increased for the rats within the control
group (0 ppm); moreover, average increases were 0.004586 ±0.004255 ɳg·dL-1·g-1 (Mean ± 95%
C.L., n=15). Hormone levels increased slightly for the experimental group 1 (2 ppm), revealing
concentrations of 0.0001979 ± 0.003396 ɳg·dL-1·g-1 (Mean ± 95% C.L., n=15). Experimental
group 2 (100 ppm) revealed decreases of 0.006503 ± 0.001294 ɳg·dL-1·g-1 in hormone levels
(Mean ± C.L., n=15). Using the calculated changes in fT4 concentrations, a single-factor
ANOVA statistical test revealed there to be no significant difference between hormone levels in
any of the three groups (p= 0.08850; Figure 2).
Figure 2. Changes in free thyroxine (fT4) concentrations over the 16 day experimental period for each
of the three tested groups (n=15, p >0.050, single-factor ANOVA). Water fluoridation with
concentrations of 0-2 ppm did not significantly alter fT4 concentrations. Fluoride concentrations of 100
ppm resulted in insignificant decreases in thyroid hormone production (p=0.0885, n=15). Error bars are
mean ± 95% Confidence Levels.
Discussion
The thyroid gland is instrumental in maintaining metabolic homeostasis and various other
physiological functions; moreover, its effects are due to the creation of thyroid hormones, which,
themselves, are created through a hormone cascade pathway involving the hypothalamus and the
pituitary gland. In response to a sensory neuron received signal and reaction from the
hypothalamus, the pituitary gland releases Thyroid-Stimulating Hormone (TSH), which travels
through the blood stream to the thyroid gland, stimulating the production of thyroid hormones:
thyroxine (T4) and triiodothyronine (T3) (Reece et al., 2011). These hormones are later released
into the blood stream to perform various functions including protein synthesis, growth, bone
development, and neurological maturation (Lakshmi et. al., 2000; Scow, 1959; Trabelsi et al.,
2001).
The current study sought to determine water fluoridation’s influence on the thyroid, and
thus, the production of thyroid hormones, specifically thyroxine (T4). In ascertaining thyroid
status, free thyroxine (fT4) is ideal to study. According to MP Biomedical protocols (2011) about
80% of thyroxine is bound to Thyroxine-Binding Globulin (TBG) in the blood stream; only
0.03% of thyroxine is free and active to influence cellular activity. As a result, fT4 is considered
a better indicator for thyroid activity. This was the influencing factor in choosing the enzyme
immunoassay (EIA) for this particular study.
Although a single-factor ANOVA revealed there to be no significant differences in
thyroid hormone production in rats (p= 0.0885), trends in serum levels were observed. Increases
in fT4 levels occurred in both the control group (0 ppm) and experimental group 1 (2 ppm). This
increase in thyroid hormone levels was to be expected for the control group. As young growing
rats, increases in metabolic activity would be necessary for growth and development; thus, more
thyroxine would be required to synthesize new proteins and aid in their growth. The
experimental group 1 resulted in diminutive increases in fT4 compared to the control group; in
fact, average thyroid hormone concentrations after the testing were nearly constant with those
measured before fluoride administration. Perhaps 2 ppm of fluoride in water was enough to
interfere with thyroxine production, resulting in smaller raises. The experimental group 2 (100
ppm) experienced overall decreases in plasma free thyroxine levels. This observation is
consistent with previous studies on rats, mice, and cattle, which also observed decreases in
hormone levels in geographical areas with chronic fluorosis (Bouaziz et al., 2005; Cinar et al.,
2005; Wang et al., 2009; Trabelsi et al., 2001).
Several explanations are considered for these hormone decreases. One such explanation
is that fluoride may compete with iodide for transport into the thyroid follicle cell through
sodium/iodide symporters (NIS) - membrane glycoproteins responsible for ion movement into
the cell. Speculation suggests NIS to have an affinity for highly charged ions with similar forms
such as perchlorate and iodide (BEST, 2005). Thus, NIS may have a greater affinity for fluoridea small, highly electronegative ion- than for iodide, resulting is lowered amounts of iodide
available for hormone production.
An additional explanation resides in fluoride’s effects on intracellular activity. Dickens
and Simer (1929) discerned decreases in tissue glycolysis of rat sarcoma, brain, and testicular
tissue when exposed to fluoride. In correlation, Bouaziz and researchers (2005) discovered
smaller colloid diameters in thyroid follicles of mice exposed to fluoride concentrations of 226
ppm. Although the current study avoided dissection of the thyroid glands from the rats,
correlations can be derived from previous studies for justification in observations of the lowered
fT4 levels in experimental group 2. Glycolysis is an important anaerobic intracellular mechanism
responsible for production of pyruvate and ATP (adenosine triphosphate), serving as a precursor
for the ignition of mitochondrial ATP production through aerobic cellular respiration. If
glycolysis is inhibited through fluoride’s binding to an important molecule, as predicted by
Dickens and Simer (1929), ATP production would consequently be inhibited as well. Through
decreases in ATP production, thyroid membrane ion pumps, specifically ATPase, would have
lessened energy available to transport sodium ions against the concentration gradient out of the
cell and potassium ions into the cell. The slowed transport of sodium out of the cell may affect
the concentration gradient, slowing the facilitation of iodide and sodium ions through NIS. This
would ultimately slow the production of thyroid hormones.
Furthermore, the decreased production of ATP may alter the rate of thyroglobulin
production within the cell. Iodide undergoes iodination to thyroglobulin- a protein manufactured
in the rough endoplasmic reticulum of the thyroid cell and used in the production of thyroid
hormone. Enzymes such as aminoacyl-tRNA synthetase require ATP to attach specified amino
acids to the polypeptide building block of these proteins during translation (Reece et al., 2011).
With lessened quantities of ATP available, thyroglobulin production may be inhibited and,
ultimately, result in decreases in thyroid hormones.
Recent studies have also found fluoride to induce rupturing of DNA strands in thyroid
cells of rats exposed to 45 ppm fluoride and low iodine (Ge et al., 2005). Perhaps this damage in
cell structure and DNA altered DNA transcription and, overall, protein synthesis (Lakshmi et al.,
2000). Inhibition of protein synthesis will influence the outcome of thyroglobulin production. If
lowered quantities of thyroglobulin resulted, hormone production and secretion would
subsequently be inhibited. Bouaziz and others (2005) described an important observation
supporting this supposition. Upon excision of rat thyroid glands, they discovered decreases in
follicular colloid mass in fluoridated rats. Studies suggest that colloids serve as reservoirs for
storage of iodinated thyroglobulin and iodide (Knight, 1982). Thus, the smaller colloid sizes
indicate either inhibition of thyroglobulin production or iodide facilitation, which influence
thyroid hormone production. This supports the trends observed of declines in thyroxine levels
with increased amounts of fluoride in water during the current study. However, further
examination would be needed to confirm the above explanations in correlation to the current
experiment. For instance, it would be interesting to investigate changes in iodine metabolism
within the thyroid. Measuring changes in plasma iodine-131 levels before and after fluoride
administration may provide insight into the activity of NIS in the thyroid gland.
Another important quantitative observation during the study was the difference in
weight gains experienced between the three groups before and after the 16 day experimental
period. Despite a single factor ANOVA rendering no significant differences in weight gains
between the groups (p= 0.1710), weight gain trends were observed. More fluoride administered
through the water resulted in smaller weight gains. The control group gained an average
estimated 6.53g more than experimental group 1 (2 ppm), and 18.29g more than experimental
group 2 (100 ppm). This is consistent with previous studies on mice and rats that also observed
lesser weight gains to increases in fluoride intake (Trabelsi et al., 2001; Wang et al., 2009). This
observation is against what was expected, though. In an instance with thyroid dysfunction, it was
predicted that weight would increase in rats experiencing larger quantities of NaF, especially
since hypothyroidism typically results in weight gains. However, Robert Scow (1959) discovered
thyroidectomized- hypophysectomized rats experienced accelerated growth in muscle and bone
lengths when treated with 2.5 µg/day of thyroxine as opposed to growth hormone. The current
results reveal that increases in fluoride resulted in slight inhibition of thyroxine production.
Perhaps the decrease in weight gains of the young fluoridated rats within the experimental
groups is due to the lowered thyroxine, resulting in slowed bone and body development and
lower weights.
Overall it appears that with either legal California water fluoridation amounts (2 ppm), or
chronic fluorosis (100 ppm), there is no significant decrease in serum fT4 levels. Emphasis must
be provided that despite no significant differences, experimental group free thyroxine levels did
decrease in comparison with the control group. Moreover, the results of the current study
indicate that fluoride administration in water is not enough to induce clinical primary
hypothyroidism in small mammals.
References
Bixler, D., and Muhler, J.C. 1957, The relation of systemic fluoride and thyroid gland activity to
the incidence of dental caries in the rat. Journal of Dental Research, 36: 304.
Bouaziz, H., Soussia, L., Guermazi, F., and Zeghal, N. 2005, Fluoride-induced thyroid
proliferative changes and their reversal in female mice and their pups. Fluoride, 38, no.
3: 185-192.
California Department of Public Health. 2011. Fluoride and poison. http://www.cdph.ca.gov/
programs/Pages/FluorideandPoison.aspx accessed 4 SEPT. 2011.
Cinar, A., and Selcuk, M. 2005, Effects of chronic fluorosis on thyroxine, triiodothyronine, and
protein-bound iodine in cows. Fluoride, 38, no.1: 65-68.
Board of Environmental Studies and Toxicology (BEST), National Research Council,
2005. Health Implications of Perchlorate Ingestion. Washington, D.C.: National
Academies Press. pp. 36-50.
Diagnostics Division. 2011, Enzyme immunoassay for the quantitative determination of free
thyroxine (fT4) concentration in human serum. Solon, Ohio: MP Biomedicals, pp. 1-4.
Dickens, F., and Simer F. 1929, Observations on tissue glycolysis: the effect of fluoride and
some other substances. Biochem J, 23, no. 5: 936-958.
El-Khoury G. Y., Moore T.E., Albright J.P., Huang H.K., and Martin R.K. 1982, Sodium
fluoride treatment of osteoporosis: radiologic findings. American Journal of
Roentgenology, 139:39-43.
Ge Y., Ning H., Wang S.Wang, Shanxi, J. 2005, DNA damage in thyroid gland cells of rats
exposed to long-term intake of high fluoride and low iodine. Fluoride, 38, no. 4: 318323.
Hem, A., Smith, A.J., and Solberg, P. 1998, Saphenous vein puncture for blood sampling of the
mouse, rat, hamster, gerbil, guinea pig, ferret, and mink. Laboratory Animals, 32: 364368.
Hillman, D., Bolenbaugh, D.L., Convey, E. 1979, Hypothyroidism and anemia related to fluoride
in dairy cattle. Journal of Dairy Science, 62, no. 3: 416-423.
Knight, G.D. 1982, Resolution and reconstruction of the NADPH-dependent tyrosyl-peptide
iodinating activity from porcine thyroid tissue. U of Texas: Austin, 36-40.
Lakshmi, V.M., K Pratap R. 2000, Effects of fluoride accumulation on some enzymes
of brain and gastrocnemius of mice. Fluoride, 33, no. 1: 17-26.
Peterson, P.E. 2003, Continuous improvement of oral health in the 21st century- the approach of
the WHO global oral health program. Community Dentistry and Oral Epidemiology,
31: 3-24.
Reece, J., Urry, L., Cain, M., Wasserman, S., Minorsky, P., Jackson, R. 2011. Campbell Biology.
San Francisco: Pearson Education, Inc. pp. 986-989.
Rolla, G., Osard, B., Almelda, R.C. 2005, Topical application of fluorides on teeth. Journal of
Clinical Peridontology, 20, no. 2: 105-108.
Scow, R.O. 1959. Effect of growth hormone and thyroxine on growth and chemical composition
of muscle, bone and other tissues in thyroidectomized-hypophysectomized rats.
American Journal of Physiology, 196, no. 4: 859-865.
Trabelsi, M., Guermazi, F., and Zeghal, N. 2001, Effect of fluoride on thyroid function and
cerebellar development in mice. Fluoride, 34, no. 3: 165-173.
Wang, H., Yang Z., Zhou B., Gao H., Yan X., and Wang J. 2009, Fluoride-induced thyroid
dysfunction in rats: roles of dietary protein and calcium level. Toxicology and
Industrial Health, 25: 49-57.
Yoon, S.H, Brudevold, F., Gardner, D.E., and Smith F. A. 1960, Distribution of fluoride in teeth
from areas with different levels of fluoride in the water supply. Journal of Dental
Research, 39: 845.
Acknowledgment
We would like to give special thanks to Professor Teh for all of his help, patience,
and support throughout our experiment, especially with assisting with blood withdrawal
from the rats and supplying and giving practical insight into the workings of the EIA kit.
We also thank Professor Huntley for assisting with preparation of the Fischer Scientific
Multiskan MCC 340 microplate reader and supplying access to the student research
room. In addition, we would like to acknowledge Dr. Joan Parent Thatcher D.V.M. and
her assistance in helping us to better understand thyroid activity. Finally, we are indebted
to Saddleback College for providing many of the necessary materials for successful
execution of our experiment.