The excessive fluoride (F) exposure is associated with damage to cellular processes of different tissue types, due to changes in enzymatic metabolism and breakdown of redox balance. However, few studies evaluate doses of F compatible with human consumption. Thus, this study evaluated the effects of chronic exposure to sodium fluoride (NaF) on peripheral blood of mice from the evaluation of biochemical parameters. The animals were divided into three groups (
Fluoride is a negatively charged nonmetallic halogen that can be naturally available in the soil, rocks, and water [
Water fluoridation initiated in the United States in 1945 and is currently practiced in approximately 25 countries around the world [
The fluoride can act as an enzyme inhibitor, due to its strong electronegativity. Thus, it forms ions in solution and the main toxic effect of fluoride derives from its interaction with enzymes [
After ingestion, fluoride is absorbed from the gastrointestinal tract, circulates in the organism and is taken up mainly by mineralized tissues and to a lower extent by soft tissues. The remaining amount is excreted mainly in the urine [
Although fluoride is absorbed largely by the mineralized extracellular matrix in calcified tissues, absorbed fluoride can lead to mitochondrial dysfunction, DNA damage, and lipid peroxidation in cells through the production of reactive oxygen species (ROS) [
The association between absorbed fluoride and changes in oxidative parameters is an important indicator of the toxic potential of fluoride on cellular mechanisms. The great importance of evaluating oxidative stress markers such as MDA in the peripheral blood is that this site is a useful source of biomarkers, as it is easily obtained and minimally invasive. Therefore, alterations in oxidative parameters can be detected in individuals exposed to compounds, even at low doses, and can distinguish them from individuals not exposed to these compounds or their metabolites. Accordingly, many studies show that biomarkers are preferentially quantified in accessible biological matrices (e.g., urine and blood). After fluoride reaches the systemic blood circulation, multiple organs are affected by exposure to the substance, but it is not clear yet by which mechanisms fluoride leads to systemic dysfunction. Thus, this study aimed at assessing the effect of fluoride exposure in levels similar to the ones found in areas of artificial water fluoridation and in areas of endemic fluorosis in blood oxidative processes, investigating that even small concentrations can trigger mechanisms that damage the body.
Thirty male Swiss albino mice (
Blood samples were collected in tubes containing 50
Fluoride concentrations in plasma were determined according to Whitford and Taves [
The NO was quantified as nitrate concentration based on the Griess method [
Lipid peroxidation (LPO) was measured by determining the thiobarbituric acid reactive substances (TBARS) as described by Kohn and Liversedge [
The method used to analyze TEAC levels is described by Ruffino et al. [
The CAT enzyme activity was determined according to the method described by Aebi [
The SOD activity was determined by following the modified method of McCord and Fridowich [
The GSH level measurements were determined by using a modified Ellman method [
Data were expressed as mean ± standard deviation for each fluoride levels and percentage of the control ± standard deviation for oxidative biochemistry assays. To calculate the standard data distribution, the normality Shapiro-Wilk test was performed. The data passed on normality and were analyzed by one-way ANOVA followed by Tukey’s test. The significance level adopted was
After 60-day exposure, the fluoride concentrations in the 10 mg/L NaF treatment (0.122 ± 0.0071) and 50 mg/L NaF treatment (0.142 ± 0.0127) were statistically higher when compared to control group (0.081
Analysis of plasma fluoride concentration. The graph shows the fluoride concentration in the plasma of mice in
As observed in Figure
Evaluation of oxidative biochemistry in blood. The graphs represent, as a percentage of the control, the results of oxidation biochemistry in the groups that received deionized water, 10 mg/L fluoride water and 50 mg/L fluoride water after the experimental period (60 days). (a) TEAC levels, (b) NO concentration, (c) SOD activity, (d) CAT activity, (e) GSH levels, and (f) TBARS concentration. One-way ANOVA followed by Tukey’s test,
In the present study, fluoride exposure significantly increased plasma fluoride concentrations. Moreover, chronic fluoride exposure induced biochemical alterations in the peripheral blood of mice, such as increased lipid peroxidation levels and decrease of the CAT activity and NO levels.
The fluoride exposure doses used in our study (10 and 50 mg/L) are often employed [
Once absorbed into the blood, fluoride is distributed rapidly throughout the body and is mainly retained in areas rich in calcium, such as bones and teeth (dentin and enamel). The fluorine with calcium forms calcium ionospheres that readily diffuse into the cell membrane [
In this regard, the organisms have a variety of antioxidant molecules and mechanisms that protect them against ROS, which include the enzymes SOD, CAT, and glutathione peroxidase (GSH-Px), and nonenzymatic antioxidants such as selenium and vitamins A, E, and C as well as compounds containing thiol groups [
In our study, the enzymatic assays showed that chronic fluoride treatment did not alter SOD activity when compared to control. Similar results were observed in human and rabbits exposed to 5 mg/L of fluoride in drinking water for 6 months [
The CAT activity was significantly decreased upon treatment with fluoride, regardless of the dose. However, only the highest fluoride dose significantly decreased GSH levels compared to control, denoting that fluoride can act as inhibitor of enzymatic antioxidants (CAT) or nonenzymatic antioxidants (GSH). Several studies report reduced enzymatic activity after fluoride exposure, followed by oxidative damage [
The total antioxidant capacity is commonly maintained by enzymatic and nonenzymatic systems, which reflect the compensatory capacity against external stimulus [
Studies show that treatment with antioxidants, such as ascorbic acid, tamarind seed coat extract, blackberry, and quercetin, prevented fluoride-induced changes such as increase of oxidant (reactive oxygen species generation, lipid peroxidation, protein carbonyl content, and NO) and inhibition of antioxidant (superoxide dismutase, catalase, glutathione peroxidase, and glutathione) parameters, suggesting that the major mode of action of fluoride is dependent of oxidative/nitrosative mechanism [
The oxidative stress, therefore, is characterized as an excess of ROS or decrease of antioxidant defenses that results in cellular macromolecule damage and changes cellular homeostasis [
Several studies reported fluoride as an inducer of oxidative stress and modulator of intracellular redox homeostasis, lipid peroxidation, and protein carbonyl content [
In normal conditions, the superoxide anion produced by vascular walls is detoxified by the enzyme SOD into hydrogen peroxide (H2O2) that may be converted into inactive forms by Fenton’s reaction or other enzymes, such as GSH-Px (before action on hydrogen peroxide) and CAT [
The importance of NO has been related to several biological processes, as inflammatory response, immunity, endothelial relaxation, and others [
The production of nitric oxide in our experiments decreased significantly after fluoride exposure. NO is a homeostasis regulator [
Changes in TBARS, NO, and TEAC, as well as CAT activity and GSH levels, especially in the group exposed to the highest dose of fluoride, indicate that this ion is a toxicant, inducing metabolic alterations in the blood and interacting with the antioxidant system in mice chronically exposed. Thus, exposure to excessive fluoride doses in the long term must be avoided.
The data used to support the findings of this study are available from the corresponding author upon request.
There are no conflicts of interest.
This work was supported by Pró-Reitoria de Pesquisa da UFPA (PROPESP, UFPA, Brazil), Brazilian National Council for Scientific and Technological Development (CNPq), Fundação de Amparo a Pesquisa do Estado do Pará (FAPESPA), and Brazilian Government/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Giza H. N. Miranda was a scholar supported by CAPES. Walessa A. B. Aragão was a scholar supported by FAPESPA and Leonardo O. Bittencourt, by CNPq. Rafael R. Lima is an investigator from CNPq (Edital MCTI/CNPQ/Universal 14/2014).