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 fT4 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 0.8-2.0 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 substantialMaybe use important in regulation of metabolism. Fluoride induced decreases in thyroid function could possibly make animals more susceptible to attainingmaybe developing 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 haphazardlyword not necessary 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 mgNaF (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 attainedSupplyed? 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 fT4concentrations (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).Personally I would work this part into the introduction, as this paragraph feels more like an introduction to the thyroid gland funtion. This is what you were studing after all.
The current study sought to determine water fluoridation’sinfluence 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 fluoride-a 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 correlationawkward phrase, 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.
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