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Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats
Accepted Manuscript Title: Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats Author: Flavia Godoy Iano Maria Cec´ılia Ferreira Giovana Brino Quaggio Mileni Silva Fernandes Rodrigo Cardoso Oliveira Valdecir Farias Ximenes Mar´ılia Afonso Rabelo Buzalaf PII: DOI: Reference:
S0022-1139(14)00293-0 http://dx.doi.org/doi:10.1016/j.jfluchem.2014.09.029 FLUOR 8446
To appear in:
FLUOR
Received date: Revised date: Accepted date:
22-8-2014 25-9-2014 27-9-2014
Please cite this article as: F.G. Iano, M.C. Ferreira, G.B. Quaggio, M.S. Fernandes, R.C. Oliveira, V.F. Ximenes, M.A.R. Buzalaf, Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats, Journal of Fluorine Chemistry (2014), http://dx.doi.org/10.1016/j.jfluchem.2014.09.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats Short title: Chronic fluoride intake alters the antioxidant system of rats Flavia Godoy Iano*1, Maria Cecília Ferreira*2, Giovana Brino Quaggio**3, Mileni Silva Fernandes***4,
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Rodrigo Cardoso Oliveira*5, Valdecir Farias Ximenes**6, Marília Afonso Rabelo Buzalaf*7
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Brazil Al. Octávio P. Brisolla 9-75, 17012-901, Bauru-SP, Brazil
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*Department of Biological Sciences, Bauru Dental School, USP - University of São Paulo, Bauru, SP,
**Department of Chemistry Science School-UNESP, Bauru, SP, Brazil, Av. Engenheiro Luiz Edmundo
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Carrijo Coube, 14-01, 17033-360, Bauru, SP, Brazil
***Department of Genetics and Evolution Federal University of São Carlos- UFSCAR, São Carlos,
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Brazil Rodovia Washington Luís, km 235, 13565-905, São Carlos, SP, Brazil [email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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[email protected]
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To whom correspondence should be addressed at Department of Biological Sciences, Bauru Dental School, USP - University of São Paulo, Bauru, SP, Brazil Al. Octávio P. Brizolla 9-75, 17012-901, BauruSP, Brazil, E-mail: [email protected] phone: 55 14 32358346, fax: 55 14 32271486
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Abstract Excessive fluoride intake over a long period of time can lead to fluorosis, which may cause dental and skeletal manifestations. Metabolic, functional and structural damage caused by chronic fluorosis have been reported in many tissues, but the exact mechanisms modulated by fluoride remain
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unclear. The aim of this study was to evaluate the effect of fluoride administered in drinking water
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on the antioxidant defense system of rats. Four groups of Wistar rats were used for the study (n=10/group). The animals received drinking water containing 0 (control), 5, 15 or 50 mg/L of fluoride
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over 60 days. They were then euthanized, and their livers and kidneys were collected and homogenized. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced
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glutathione (GSH), antioxidants, thiobarbituric acid reactive substances (TBARS), lipid hydroperoxide
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(LH), and fluoride levels were analyzed. Data were analyzed by ANOVA and Tukey’s test or by the Kruskal-Wallis and Dunn’s tests (p<0.05). In the kidneys, the SOD, GPx, GSH and antioxidants levels
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significantly decreased, while the fluoride and LH levels significantly increased. In the liver, the CAT and TBARS levels significantly decreased, while the fluoride, SOD, LH and antioxidants levels
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significantly increased. In summary, these results show that chronic fluoride administration alters the antioxidant system of rats. Our data suggest that the conversion of the superoxide anion to water in
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the kidney upon exposure to high levels of fluoride occurs mainly through SOD and CAT and not through the glutathione system, in contrast to what occurs in the liver. Keywords: Fluoride, antioxidants, oxidative stress, liver, kidney.
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1. Introduction Excessive uptake of fluoride can cause fluorosis, a condition characterized by an altered appearance of the teeth, such as pitting or staining of the enamel, and
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skeletal manifestations at higher doses, such as bone deformities, osteoporosis and osteosclerosis. Endemic fluorosis is widely prevalent around the world and affects
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millions of people [1]
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Fluoride can affect cells in several ways, depending on the time of exposure, concentration and cell type. At micromolar levels, fluoride is an anabolic agent and
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promotes cell proliferation. However, millimolar concentrations inhibit several enzymes, including phosphatases, both in vivo and in vitro [2]. Stimulation of enzyme
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activity by fluoride has also been reported [3].
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Metabolic, structural and functional damage as a result of chronic fluorosis have been identified in different tissues, including renal, endothelial, gonadal and neuronal
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cells [4]. Oxidative stress following excessive fluoride exposure has been observed in several cell types in vitro and in soft tissues in vivo [5-7]. Fluoride affects the liver,
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spleen, brain, lungs and testicles of animals and humans living in endemic areas of fluorosis. Fluoride is reported to inhibit the activity of antioxidant enzymes such as SOD, GPx and CAT [5, 6, 8, 9]. In addition, fluoride can alter glutathione levels [912], causing excessive production of ROS at the mitochondrial level and damaging some cell components. In addition, fluoride can cause neuronal damage in rats treated with 600 ppm for one week due to increased oxidative stress in the brain [13]. While the effects of fluoride on the antioxidant system have been evaluated in many studies, the methodologies vary greatly, which impairs comparisons of the results obtained in different tissues. Furthermore, the doses of fluoride used in many 3 Page 3 of 21
cases are extremely high and do not have clinical significance [13, 14]. The aim of this study was to evaluate the effects of clinically significant doses of fluoride on the
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antioxidant systems of the liver and kidneys of rats.
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2. Results and Discussion
There were no significant differences in either the mean body weight or kidney
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weight between the groups (results not shown), which is consistent with the literature for the fluoride doses used [15]. The mean liver weight was higher in the
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experimental groups compared to the control group (p=0.0064). However, only the 15
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mg/L fluoride group (8.43±0.57 g) significantly differed from control (7.33±0.85 g); the increases observed in the 5 mg/L (8.03±0.55 g) and 50 mg/L fluoride groups
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(7.90±0.37 g) were not significant. However, when the ratio liver weight/body weight was considered, both the 5 and 15 mg/L fluoride groups presented ratios significantly
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higher than the one observed for the control group. It has been reported that
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exposure to higher fluoride doses (such as 100 mg/L) reduces the weight of the liver of rats [16].
After intake, fluoride is absorbed in the gastro-intestinal tract and reaches
systemic circulation [17]. Blood plasma is an important indicator of fluoride intake levels in cases of both acute [18-20] and chronic exposure [15, 18, 21-23]. An increase in plasma fluoride levels was observed as water fluoride concentrations were increased in the present study, which is consistent with the literature [15, 22, 23] (Table 1). Fluoride is distributed to the whole organism via plasma and is excreted in the urine [17]. In the present study, the group treated with 5 mg/L fluoride displayed plasma fluoride levels similar to those of the control group, consistent with 4 Page 4 of 21
previous studies using the same treatment protocol [15, 21-23], and could be partially explained by fluoride uptake in mineralized tissues [17]. Fluoride concentrations in the liver and kidney are shown in Table 1. In the
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kidney, a dose-dependent response was observed, with significant differences between all groups (p<0.0001). Significant differences in the liver fluoride
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concentrations were also observed among the groups (p<0.0001). The 50 mg/L fluoride group displayed the highest fluoride concentration, and both the 15 mg/L and
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50 mg/L groups had significantly increased fluoride levels compared to the control
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group, which displayed the lowest concentration. The kidneys are the main organs responsible for the reduction of plasma fluoride levels after fluoride intake;
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approximately 60% of the fluoride absorbed is excreted in the urine in healthy adults [24]. As the kidneys are the principal route for fluoride elimination from the organism,
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the fluoride levels in the kidneys displayed a dose-dependent effect in the kidneys in the present study: the fluoride levels increased upon exposure to higher
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concentrations of fluoride. In contrast, the fluoride levels in the liver (Table 1)
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decreased compared to those found in the plasma. This result was expected because intracellular fluoride levels are typically 10-50% lower than those found in plasma or interstitial fluid [17].
Table 1. Fluoride Concentration in the plasma (µg/ml) and the liver and kidney (µg/g tissue) of rats receiving water containing different fluoride concentrations
Plasma
Control
5 mg/L
15 mg/L
50 mg/L
0.032±0.011a
0.027±0.008a
0.043±0.010a
0.094±0.019b
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Liver
0.009±0.003a
0.010±0.002ad
0.014±0.004bd
0.023±0.008c
Kidney
0.045±0.012a
0.067±0.011b
0.112±0.020c
0.245±0.042d
Values are mean ±SD (n=10). In each line, different superscript letters indicate significant differences among the groups (p<0.05).
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Macroscopic analysis of the liver did not reveal any differences between the groups, and all groups displayed normal morphological characteristics, such as
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uniform appearance, intact hepatic lobules, intact portal area, defined hepatic vein, and intact sinusoids converging to the central lobule vein. However, the group treated
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with 50 mg/L fluoride (Figure 1B) displayed more disorganized hepatocytes
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compared to the control group (Figure 1A), and some hepatocytes were observed to have heterogeneous nuclear sizes, disorganized architecture and changes in the
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delimitation of the cytoplasm. Signs of toxicity include changes in organ weights and hematological and biochemical blood alterations [25]. We did not observe either
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macroscopic alterations or microscopic signs of toxicity. However, the group treated with 50 mg/L of fluoride displayed areas with altered hepatocytes; similar results
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were described by Bouaziza et al (2006) [26], who observed liver changes in mice
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intoxicated by fluoride.
Histological analysis of the kidneys did not reveal any differences between the
groups. Discrete vascular congestion was observed only in the group treated with 50 mg/L of fluoride (Figure 1C). This group also displayed a higher number of congested vessels compared to the control group, but over less than 25% of the tissue (Figure 1D). Vascular congestion has also been described by other authors. Kobayashi et al (2009) [22] also observed vascular congestion, but found no classic signs of nephropathy induced by fluoride. Hyperemia (vascular congestion) can be an early
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sign of the inflammatory response [27]. Other studies on the chronic toxicity of
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fluoride have also reported the presence of vascular congestion [8, 22, 28].
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Figure 1: Liver histopathology of control and fluoride-treated rats (HE stain). (1A) Section of liver tissue from control group rats showing S: Sinusoids, arrow: normal hepatocyte, Arrowhead: nucleus.
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(1B) Experimental group of rats treated with 50 mg/L of fluoride showing Arrowhead: nucleus, * Irregular hepatocytes. Kidney histopathology of control and fluoride-treated rats (HE stain). (1C) Experimental group of rats treated with 50 mg/L of fluoride showing V: vascular congestion, G: Kidney glomerulus. (1D) Section of kidney tissue from control group rats showing G: Kidney glomerulus. 40x
CAT activity in the kidneys (Table 2) was similar in all groups, and no significant
differences were observed (p=0.4157). However, CAT activity in the liver (table 3) was significantly different among the groups (p=0.0001). All the experimental groups displayed decreased CAT activity compared to the control group; the 15 mg/L
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fluoride group displayed the lowest value and was significantly different from both the control and the 5 mg/L fluoride groups. The SOD activity in the kidney was also significantly different between the
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groups (p=0.0012; Table 2). The 50 mg/L fluoride group displayed the lowest value (approximately a 50% decrease) and significantly differed from both the control and
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the 5 mg/L fluoride groups. No significant differences were observed between the control group and the 5 mg/L and 15 mg/L fluoride groups. SOD activity in the liver
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was also significantly different between the groups (p=0.0006): the 15 mg/L fluoride
and 5 mg/L fluoride groups (Table 3).
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group displayed the highest activity and significantly differed from both the control
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The GPx activity (Table 2) in the kidney was significantly different between the groups (p<0.0001). The 50 mg/L fluoride group displayed the lowest activity, while
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the 5 mg/L group displayed the highest activity. In the liver, the GPx activity
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increased in all the experimental groups compared to the control group, but no significant differences were detected (p=0.4964) (Table 3).
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The GSH levels in the kidney (Table 2) were significantly different between the
groups (p=0.0177). The concentration decreased in all experimental groups compared to the control group, but the decrease was only significant for the 50 mg/L fluoride group. In the liver (Table 3), all experimental groups displayed increased GSH concentrations, but no significant differences were detected (p=0.2996). The LH levels in the kidney and liver are shown in Tables 2 and 3, respectively. The LH levels in the kidney were significantly different between the groups (p=0.0075). All experimental groups displayed increased concentrations compared to the control group, but only the 15 mg/L fluoride group significantly differed from the 8 Page 8 of 21
control. The LH levels in the liver were also significantly different between the groups. The experimental groups displayed higher LH concentrations compared with control, but the difference was significant only for the 5 and 50 mg/L fluoride groups.
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No significant differences in the concentration of TBARS in the kidney were detected between the groups (table 2; p=0.0952). In the liver, however, significant
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differences in the TBARS level were observed (p=0.0478). The experimental groups displayed lower concentrations of TBARS compared to the control, but the difference
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was significant only for the 15 mg/L fluoride group (Table 3). The levels of LH, a
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substance related to lipid peroxidation, increased in both the liver and kidneys in the exposed groups compared to the control, which is consistent with the results of
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plasma fluoride levels, despite the fact that no dose-response was observed. However, TBARS levels remained unchanged in the kidneys and decreased in the
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liver of treated animals compared to the control. The concentration of MDA, a product of the termination phase of lipid peroxidation, has been reported to increase in the
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liver of rats that received water containing 100 mg/L fluoride for 4 months [6]. Similar
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results were obtained in the kidneys of rats that were treated for one week with 600 mg/L of fluoride [29]. In these cases, the increased dosage of fluoride and/or the different treatment length might have exhausted the antioxidant defenses, which does not seem to have occurred in the present study. Significant differences in antioxidant concentrations in the liver were observed
among the groups (Table 3; p<0.0001). Antioxidant concentrations in the 15 and 50 mg/L fluoride groups were similar, but significantly higher than those found in the control and 5 mg/L fluoride groups. Significant differences were also observed in the kidneys (Table 2; p<0.0001). An inverse dose-response effect was observed: 9 Page 9 of 21
antioxidant activity significantly decreased in all the experimental groups compared to the control. A significant reduction in antioxidant levels was observed in the groups treated with fluoride compared to the control, in contrast to what was observed for the liver. This can be explained by the higher fluoride concentrations found in the kidney
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compared to the liver, which may also have contributed to reducing the levels of
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antioxidant enzymes such as SOD and CAT.
The activity of SOD significantly increased in the liver of the 15 mg/L fluoride
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group, while the activity of CAT significantly decreased compared to that of the
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control. However, the activity of GPx and the GSH levels tended to be constant. In a recent study, all of these antioxidants were reduced in the liver of rats treated for 12
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weeks with 25 mg/L fluoride in drinking water [30]. The antioxidant profile observed in the kidneys, however, was notably different. The concentration of GPx decreased in
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the groups treated with 15 and 50 mg/L fluoride compared to the control. The levels of SOD and GSH also decreased, but only in the 50 mg/L fluoride group. Similar
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reductions were reported in the kidney of rats treated with 100 mg/L fluoride for one
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week [29]. However, CAT activity remained unaltered. When analyzed in conjunction, these findings suggest that the conversion of the superoxide anion to water in the kidney occurs mainly through the action of SOD and CAT rather than the glutathione system, in contrast to what occurs in liver.
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Table 2: Concentration of LH, TBARS, Antioxidants, CAT, SOD, GSH and GPx in the kidney of rats receiving water containing different fluoride concentrations Group of animals 5 mg/L
15 mg/L
50 mg/L
LH (µmol/g)
0.704±0.18a
0.961±0.47ab
1.229±0.29b
1.001±0.19ab
TBARS (µmol/g)
0.073±0.009
0.067±0.015
0.064±0.009
0.077±0.012
Antioxidants (mmol/g)
2.23±0.34a
1.51±0.41b
1.13±0.25b
1.03±0.18b
CAT(µmol/min*mg protein)
3.37±0.529
3.67±0.681
3.24±0.247
3.51±0.48
SOD (U/min*mg protein)
0.83±0.25a
0.91±0.41a
0.86±0.63ab
0.41±0.7b
GSH (nmol/mg)
3.29±0.52a
2.83±1.04ab
3.13±0.59ab
2.34±0.46b
GPx (µg/min*mg protein)
0.31±0.11ab
0.37±0.13a
0.25±0.05bc
0.17±0.03c
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Control
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Concentration or activity
Values are mean ±SD (n=10). In each line, different superscript letters indicate significant differences among the groups (p<0.05).
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Table 3: Concentration of LH, TBARS, Antioxidants, CAT, SOD, GSH and GPx in the
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liver of rats receiving water containing different fluoride concentrations Group of animals
Concentration or activity
5 mg/L
15 mg/L
50 mg/L
LH (µmol/g)
1.358±0.95a
2.425±0.69b
2.021±0.45ab
2.449±0.8b
TBARS (µmol/g)
0.067±0.018a
0.061±0.027ab
0.047±0.008b
0.05±0.004ab
Antioxidants (mmol/g)
1.32±0.11a
1.25±0.12a
2.52±0.15b
2.37±0.08b
CAT (µmol/min*mg protein)
11.37±2.57a
9.04±1.76b
6.74±0.7c
7.3±0.93c
SOD (U/min*mg protein)
19.76±4.78a
16.82±3.54a
25.17±3.36b
21.4±2.34ab
GSH (nmol/mg)
14.04±7.6
16.51±7.6
18.92±7.26
19.54±5.34
GPx (µg/min*mg protein)
0.093±0.026
0.112±0.046
0.112±0.052
0.132±0.07
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Control
Values are mean ±SD (n=10) In each line, different superscript letters indicate significant differences among the groups (p<0.05).
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3. Conclusions Exposure to the highest fluoride doses (15 and 50 mg/L) caused alterations in the antioxidant system of liver and kidney of rats. However, few changes in the parameters evaluated were observed upon treatment with 5 mg/L fluoride, the dose equivalent to 1 mg/L
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fluoride in drinking water for humans [31]. These findings reinforce the safety of controlled
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water fluoridation.
4. Materials and Methods
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4.1 Chemicals
Sodium fluoride is from Sigma Aldrich (St Louis, USA), HMDS hexamethyldisiloxane
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was purchased from Aldrich (St Louis, USA), Fluoride electrode (Cas #940907) Thermo Orion (USA), butylhydroxytoluene (BHT), thiobarbituric acid (TBA), trichloroacetic acid (TCA),
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nitroblue tetrazolium (NBT), 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), o-Phthalaldehyde (OPT), β-Nicotinamide adenine dinucleotide, reduced dipotassium salt (NADH), xanthine, oxidase,
glutathione
reduced
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xanthine
(GSH),
potassium
dichromate,
sodium
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pyrophosphate, phenazine methosulfate, meta-phosphoric acid and Antioxidant Assay Kit (Cas # CS0790) were purchased from Sigma Aldrich (St Louis, USA), hydrogen peroxide (H2O2), methanol, hydrochloric acid (HCl), Ethylenediaminetetraacetic acid (EDTA) and acetic acid were purchased from Merck (Germany).
4.2 Animals and treatment The study protocol was approved by the Committee on the Ethics of Animal Experiments of Bauru School of Dentistry, University of São Paulo (Proc. 001/2007). Male Wistar rats (21 days old) were divided into four groups containing ten animals each. The rats
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were housed in polypropylene cages and provided with ad libitum low-fluoride food (AIN-93) and water inside a climate-controlled room, which had a 12 h light/dark cycle. The experimental groups were treated for 60 days with three different concentrations of fluoride in drinking water (5, 15 or 50 mg/L fluoride, as NaF), while the control group received deionized
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water. These doses of fluoride were chosen because they are approximately equivalent to doses of 1, 3 or 10 mg/L fluoride in drinking water for humans, respectively [31]. These
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doses correspond to the fluoride concentration that is typically included in artificially fluoridated water and to fluoride concentrations naturally found in endemic regions of dental
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and skeletal fluorosis, respectively [1]. After the experimental period, the animals were
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anaesthetized with ketamine and xylazine and euthanized by decapitation. Liver and kidneys
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were collected and stored at -80ºC until analysis.
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4.3 Determination of fluoride concentration
The fluoride concentration in the kidneys and liver were determined after overnight
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HMDS-facilitated diffusion [18, 32] using an ion-specific electrode (Orion Research, Model 9409) and a miniature calomel electrode, (Accumet, #13-620-79) both of which were coupled
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to a potentiometer (Orion Research, Model EA 940, Cambridge, USA).
4.4 Sample preparation
To obtain the supernatant, the tissues (kidney and liver) were homogenized with a 10
mmol/L phosphate buffer (pH 7.0) and 1% BHT for 2 min (Marconi MA 102, Brazil) and centrifuged at 6400 g for 10 min. The supernatant was collected and used for analysis of TBARS, LH and antioxidant concentration.
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To analyze CAT, SOD and GPx activity, the kidney and liver tissues were homogenized in 10 mmol/L sodium phosphate buffer (pH 7.4) and centrifuged at 1500 g for
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10 min; the supernatant was collected and used for the determinations.
4.4.1 Determination of TBARS concentration
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Lipid peroxidation levels were measured by determination of the thiobarbituric acid
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reactive substances (TBARS) as described by Buege and Aust (1978) [33]. The supernatant (0.4 mL) was mixed with 0.8 mL thiobarbituric acid solution (0.375% TBA, 15% TCA and
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0.25% HCl), incubated in a boiling water bath for 15 min, placed in an ice water bath for 10 min and centrifuged at 13,900 g for 5 min. The absorbance of the supernatant was read at
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535 nm. The spectrophotometer was blanked with 0.4 mL phosphate buffer 10 mmol/L (pH 7.0) (Ultrospec 2000, Pharmacia Biotech, United Kingdom). The concentration of TBARS
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was calculated using its molar extinction coefficient (1.56×105 mol/L−1cm−1).
4.4.2 Determination of lipid hydroperoxide concentration
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The LH concentration in the kidney and liver was measured using the method
described by Jiang et al. (1971) [34], and calculated using a molar extinction coefficient of 4.3 ×104 (mol/L)−1cm−1.
4.4.3 Determination of antioxidant concentration Concentrations of antioxidants in the kidney and liver were obtained using the Antioxidant Assay kit (CS0790, Sigma–Aldrich (St Louis, USA).
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4.4.4 Determination of CAT activity The CAT activity was assayed as described previously by Sinha (1972) [35]. In brief, 0.1 mL of supernatant was incubated with 0.5 mL of 0.2 mol/L H2O2 at 37ºC for 90 s. The reaction was stopped by adding 5% potassium dichromate solution (5% potassium
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dichromate in acetic acid 1:3). Samples were incubated in a boiling water bath for 15 min, and the concentration of H2O2 consumed was determined by recording the absorbance at
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570 nm. The activity of CAT was expressed as µmol/min*mg protein.
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4.4.5 Determination of SOD activity
The SOD activity in the kidney was assessed as described by Kakkar et al. [36], with
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slight modifications. The supernatant was centrifuged at 10,000 g for 15 min, and 0.2 mL of 5% tissue homogenate was added to a reaction mixture containing 1.2 mL of sodium
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pyrophosphate buffer (0.052 mmol/L, pH 7.0), 0.1 mL of phenazine methosulfate (186 µmol/L), and 0.3 mL of NBT (300 µmol/L). The enzymatic reaction was initiated by adding 0.2
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mL of NADH (780 µmol/L) and was stopped precisely after 1 min by adding 1 mL of glacial
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acetic acid. The concentration of reduced NBT formed was measured by the absorbance at 560 nm. In the liver, SOD activity was measured as described previously by Beauchamp and Fridovich [37]. Samples (50 mg) were homogenized with 1 mL of sodium phosphate buffer (50 mmol/L pH 7.8), and after centrifugation at 10,621 g for 10 min, the supernatant was collected. The reaction mixture consisted of 0.96 mL of sodium and potassium phosphate buffer (50 mmol/L pH 7.8), 0.010 mL of xanthine (5 mmol/L), 0.010 mL of NBT (10 mmol/L), 0.010 mL of supernatant and 0.010 mL of EDTA (10 mmol/L). The reaction was initiated by adding 0.005 mL of xanthine oxidase (diluted 20 times), and the absorbance at 550 nm was recorded over a 3 min period. The Δabs/min was determined, and the activity of SOD was calculated and expressed as U/min*mg protein.
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4.4.6 Determination of glutathione peroxidase (GPx) activity The activity of GPx in the kidney and liver were measured as described by Flohe and Gunzler [38] and as modified by Modi and Flora [39]. The supernatant was centrifuged at 10,000 g for 30 min, and the reaction was initiated by adding 0.3 mL of a phosphate buffer
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(10 mmol/L pH 7.4), 0.2 mL of GSH (2 mmol/L), 0.1 mL of H2O2 (1 mmol/L) and 0.3 mL of supernatant. The reaction was incubated in a 37ºC water bath for 15 min and then stopped
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by adding 0.5 mL TCA (5%). The samples were centrifuged at 1500 g for 5 min, the
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supernatant was collected, and 0.1 mL of the supernatant was mixed with 0.2 mL phosphate buffer (0.1 mol/L, pH 7.4) and 0.7 mL of DTNB (0.4 mg/mL). The absorbance was read at
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420 nm with a spectrophotometer (Spectra Max M2, Molecular Devices, Sunnyvale, USA).
4.4.7 Determination of reduced glutathione (GSH) activity
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The GSH activity in the kidney and liver were assayed as described by Hissin and Hilf [40], with slight modifications. The tissues were mixed with phosphate-EDTA buffer (0.1
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mol/L) and meta-phosphoric acid (25%) and centrifuged at 20,817 g for 10 min. Then 1.8 mL
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of a phosphate-EDTA buffer, 0.1 mL of supernatant and 0.1 mL of OPT solution (1 mg of OPT diluted in 1 mL of methanol) were combined, and the reactions were incubated for 15 min. The fluorescence was determined (ex 350 nm/em 420 nm) (Spectra Max M2, Molecular Devices, Sunnyvale, USA), and the concentration was calculated using a calibration curve for GSH and expressed as nmol/mg tissue.
4.8 Histopathological analysis A portion of the kidney and liver tissues were fixed in 10% neutral buffered formalin solution for histological analysis. After fixation, the tissues were embedded in paraffin, and 5 µm sections were sliced and stained with hematoxylin and eosin (HE) using standard
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techniques. The sections were examined under light microscope (Carl Zeiss, German) and photomicrographs were taken.
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4.9 Statistical analysis All results are expressed as means ± SD (standard derivation). GraphPad InStat
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version 3.0 for Windows was used for the statistical analysis (GraphPad Software Inc., La
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Jolla, USA). The data were checked for normality (Kolmogorov and Smirnov test) and homogeneity (Bartlett test); when these criteria were satisfied, the data were analyzed by
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ANOVA and Tukey’s tests. Otherwise, they were analyzed by the Kruskal-Wallis and Dunn’s
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tests. In all cases, the significance level was set to 5%.
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Acknowledgements
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
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no conflict of interest.
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(FAPESP), grant numbers 2007/03723-2, 2007/01788-0, 2009/01996-7. The authors have
References
[1] J. Fawell, K. Bailey, J. Chilton, E. Dahi, L. Fewtrell,Y. Magara, Fluoride in Drinking-water, ed., World Health Organization (WHO), 2006. [2]E. Adamek, K. Pawlowska-Goral,K. Bober, Ann Acad Med Stetin, 51 (2005) 69-85. [3] A. Mendoza-Schulz, C. Solano-Agama, L. Arreola-Mendoza, B. Reyes-Marquez, O. Barbier, L.M. Del Razo,M.E. Mendoza-Garrido, Toxicol Lett, 190 (2009) 179-186. [4] W. Committee on Fluoride in Drinking, C. National Research, Fluoride in Drinking Water: A Scientific Review of EPA's Standards, ed., The National Academies Press, 2006. 17 Page 17 of 21
[5] E.A. Garcia-Montalvo, H. Reyes-Perez, L.M. Del Razo, Toxicology, 263 (2009) 75-83. [6] J.A. Izquierdo-Vega, M. Sanchez-Gutierrez, L.M. Del Razo, Toxicol Appl Pharmacol, 230 (2008) 352-357. [7] C.D. Nobes,A. Hall, Cell, 81 (1995) 53-62.
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Gopalakrishna,R.S.
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[13] S.M. Nabavi, A. Sureda, S.F. Nabavi, A.M. Latifi, A.H. Moghaddam,C. Hellio, J Fluorine Chem, 142 (2012) 79 - 82.
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[14] S.F. Nabavi, S.M. Nabavi, S. Habtemariam, A.H. Moghaddam, A. Sureda,M. Mirzaei, Cell Mol Neurobiol, 33 (2013) 261-267.
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[15] J.G. Carvalho, A.L. Leite, D. Yan, E.T. Everett, G.M. Whitford,M.A. Buzalaf, J Dent Res, 88 (2009) 1054-1058.
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[16] R.A. Vasant,A.V. Narasimhacharya, Environ Health Prev Med, 17 (2012) 484493. [17] M.A. Buzalaf,G.M. Whitford, Monogr Oral Sci, 22 (2011) 20-36. [18] G.M. Whitford, Monogr Oral Sci, 16 Rev 2 (1996) 1-153. [19] M.A. Buzalaf, E.E. Caroselli, J.G. de Carvalho, R.C. de Oliveira, V.E. da Silva Cardoso,G.M. Whitford, J Anal Toxicol, 29 (2005) 810-813. [20] M.A. Buzalaf, E.E. Caroselli, R. Cardoso de Oliveira, J.M. Granjeiro,G.M. Whitford, J Anal Toxicol, 28 (2004) 249-252. [21] A.L. Leite, E.E. Caroselli, A.G. Maria, M.S. Fernandes,M.A. Buzalaf, Methods Find Exp Clin Pharmacol, 30 (2008) 209-214. [22] C.A. Kobayashi, A.L. Leite, T.L. Silva, L.D. Santos, F.C. Nogueira, R.C. Oliveira, M.S. Palma, G.B. Domont,M.A. Buzalaf, Chem Biol Interact, 180 (2009) 305-311.
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[23] J.G. de Carvalho, T.M. Cestari, R.C. de Oliveira,M.A. Buzalaf, Methods Find Exp Clin Pharmacol, 30 (2008) 287-294. [24] A. Villa, M. Anabalon, V. Zohouri, A. Maguire, A.M. Franco,A. Rugg-Gunn, Caries Res, 44 (2010) 60-68.
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[25] F.H.D. González,S.C. Silva, Introdução à Bioquímica Clínica Veterinária., ed., UFRGS, Porto Alegre, 2003.
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[26] H. Bouaziza, S. Ketatab, K. Jammoussic, T. Boudawarad, F. Ayedic, F. Ellouzec,N. Zeghala, Pesticide Biochemistry and Physiology, 86 November 2006 (2006) 124 - 130.
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[27] A.L. Ogilvie, J Dent Res, 32 (1953) 386-397.
[28] E. Karaoz, M. Oncu, K. Gulle, M. Kanter, F. Gultekin, S. Karaoz,E. Mumcu, Biol Trace Elem Res, 102 (2004) 199-208.
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[29] S.M. Nabavi, S.F. Nabavi, S. Habtemariam, A.H. Moghaddam,A.M. Latifi, Ren Fail, 34 (2012) 901-906.
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[30] L. Panneerselvam, K. Subbiah, A. Arumugam,J.G. Senapathy, Biol Trace Elem Res, 151 (2013) 85-91.
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[31] A.J. Dunipace, E.J. Brizendine, W. Zhang, M.E. Wilson, L.L. Miller, B.P. Katz, J.M. Warrick,G.K. Stookey, J Dent Res, 74 (1995) 358-368. [32] D.R. Taves, Talanta, 15 (1968) 1015-1023.
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[33] J.A. Buege,S.D. Aust, Methods Enzymol, 52 (1978) 302-310.
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[34] Z.Y. Jiang, A.C. Woollard,S.P. Wolff, Lipids, 26 (1991) 853-856. [35] A.K. Sinha, Anal Biochem, 47 (1972) 389-394. [36] P. Kakkar, B. Das,P.N. Viswanathan, Indian J Biochem Biophys, 21 (1984) 130132. [37] C. Beauchamp,I. Fridovich, Anal Biochem, 44 (1971) 276-287. [38] L. Flohe,W.A. Gunzler, Methods Enzymol, 105 (1984) 114-121. [39] M. Modi,S.J. Flora, Cell Biol Toxicol, 23 (2007) 429-443. [40] P.J. Hissin,R.A. Hilf, Anal Biochem, 74 (1973) 214-226.
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*Graphical Abstract - Pictogram
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*Highlights (for review)
Highlights
Chronic exposure to high doses of fluoride alters the antioxidant system of rats;
Doses equivalent to those found in artificially fluoridated water do not provoke any alterations; The effects of fluoride in the antioxidant systems of the liver and kidneys are
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remarkably different;
Upon exposure to high fluoride levels, the kidneys convert the superoxide anion to
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H2O mainly through SOD and CAT.
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S0022-1139(14)00293-0 http://dx.doi.org/doi:10.1016/j.jfluchem.2014.09.029 FLUOR 8446
To appear in:
FLUOR
Received date: Revised date: Accepted date:
22-8-2014 25-9-2014 27-9-2014
Please cite this article as: F.G. Iano, M.C. Ferreira, G.B. Quaggio, M.S. Fernandes, R.C. Oliveira, V.F. Ximenes, M.A.R. Buzalaf, Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats, Journal of Fluorine Chemistry (2014), http://dx.doi.org/10.1016/j.jfluchem.2014.09.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats Short title: Chronic fluoride intake alters the antioxidant system of rats Flavia Godoy Iano*1, Maria Cecília Ferreira*2, Giovana Brino Quaggio**3, Mileni Silva Fernandes***4,
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Rodrigo Cardoso Oliveira*5, Valdecir Farias Ximenes**6, Marília Afonso Rabelo Buzalaf*7
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Brazil Al. Octávio P. Brisolla 9-75, 17012-901, Bauru-SP, Brazil
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*Department of Biological Sciences, Bauru Dental School, USP - University of São Paulo, Bauru, SP,
**Department of Chemistry Science School-UNESP, Bauru, SP, Brazil, Av. Engenheiro Luiz Edmundo
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Carrijo Coube, 14-01, 17033-360, Bauru, SP, Brazil
***Department of Genetics and Evolution Federal University of São Carlos- UFSCAR, São Carlos,
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Brazil Rodovia Washington Luís, km 235, 13565-905, São Carlos, SP, Brazil [email protected]
2
[email protected]
3
[email protected]
4
[email protected]
5
[email protected]
6
[email protected]
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1
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To whom correspondence should be addressed at Department of Biological Sciences, Bauru Dental School, USP - University of São Paulo, Bauru, SP, Brazil Al. Octávio P. Brizolla 9-75, 17012-901, BauruSP, Brazil, E-mail: [email protected] phone: 55 14 32358346, fax: 55 14 32271486
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Abstract Excessive fluoride intake over a long period of time can lead to fluorosis, which may cause dental and skeletal manifestations. Metabolic, functional and structural damage caused by chronic fluorosis have been reported in many tissues, but the exact mechanisms modulated by fluoride remain
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unclear. The aim of this study was to evaluate the effect of fluoride administered in drinking water
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on the antioxidant defense system of rats. Four groups of Wistar rats were used for the study (n=10/group). The animals received drinking water containing 0 (control), 5, 15 or 50 mg/L of fluoride
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over 60 days. They were then euthanized, and their livers and kidneys were collected and homogenized. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced
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glutathione (GSH), antioxidants, thiobarbituric acid reactive substances (TBARS), lipid hydroperoxide
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(LH), and fluoride levels were analyzed. Data were analyzed by ANOVA and Tukey’s test or by the Kruskal-Wallis and Dunn’s tests (p<0.05). In the kidneys, the SOD, GPx, GSH and antioxidants levels
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significantly decreased, while the fluoride and LH levels significantly increased. In the liver, the CAT and TBARS levels significantly decreased, while the fluoride, SOD, LH and antioxidants levels
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significantly increased. In summary, these results show that chronic fluoride administration alters the antioxidant system of rats. Our data suggest that the conversion of the superoxide anion to water in
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the kidney upon exposure to high levels of fluoride occurs mainly through SOD and CAT and not through the glutathione system, in contrast to what occurs in the liver. Keywords: Fluoride, antioxidants, oxidative stress, liver, kidney.
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1. Introduction Excessive uptake of fluoride can cause fluorosis, a condition characterized by an altered appearance of the teeth, such as pitting or staining of the enamel, and
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skeletal manifestations at higher doses, such as bone deformities, osteoporosis and osteosclerosis. Endemic fluorosis is widely prevalent around the world and affects
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millions of people [1]
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Fluoride can affect cells in several ways, depending on the time of exposure, concentration and cell type. At micromolar levels, fluoride is an anabolic agent and
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promotes cell proliferation. However, millimolar concentrations inhibit several enzymes, including phosphatases, both in vivo and in vitro [2]. Stimulation of enzyme
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activity by fluoride has also been reported [3].
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Metabolic, structural and functional damage as a result of chronic fluorosis have been identified in different tissues, including renal, endothelial, gonadal and neuronal
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cells [4]. Oxidative stress following excessive fluoride exposure has been observed in several cell types in vitro and in soft tissues in vivo [5-7]. Fluoride affects the liver,
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spleen, brain, lungs and testicles of animals and humans living in endemic areas of fluorosis. Fluoride is reported to inhibit the activity of antioxidant enzymes such as SOD, GPx and CAT [5, 6, 8, 9]. In addition, fluoride can alter glutathione levels [912], causing excessive production of ROS at the mitochondrial level and damaging some cell components. In addition, fluoride can cause neuronal damage in rats treated with 600 ppm for one week due to increased oxidative stress in the brain [13]. While the effects of fluoride on the antioxidant system have been evaluated in many studies, the methodologies vary greatly, which impairs comparisons of the results obtained in different tissues. Furthermore, the doses of fluoride used in many 3 Page 3 of 21
cases are extremely high and do not have clinical significance [13, 14]. The aim of this study was to evaluate the effects of clinically significant doses of fluoride on the
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antioxidant systems of the liver and kidneys of rats.
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2. Results and Discussion
There were no significant differences in either the mean body weight or kidney
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weight between the groups (results not shown), which is consistent with the literature for the fluoride doses used [15]. The mean liver weight was higher in the
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experimental groups compared to the control group (p=0.0064). However, only the 15
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mg/L fluoride group (8.43±0.57 g) significantly differed from control (7.33±0.85 g); the increases observed in the 5 mg/L (8.03±0.55 g) and 50 mg/L fluoride groups
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(7.90±0.37 g) were not significant. However, when the ratio liver weight/body weight was considered, both the 5 and 15 mg/L fluoride groups presented ratios significantly
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higher than the one observed for the control group. It has been reported that
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exposure to higher fluoride doses (such as 100 mg/L) reduces the weight of the liver of rats [16].
After intake, fluoride is absorbed in the gastro-intestinal tract and reaches
systemic circulation [17]. Blood plasma is an important indicator of fluoride intake levels in cases of both acute [18-20] and chronic exposure [15, 18, 21-23]. An increase in plasma fluoride levels was observed as water fluoride concentrations were increased in the present study, which is consistent with the literature [15, 22, 23] (Table 1). Fluoride is distributed to the whole organism via plasma and is excreted in the urine [17]. In the present study, the group treated with 5 mg/L fluoride displayed plasma fluoride levels similar to those of the control group, consistent with 4 Page 4 of 21
previous studies using the same treatment protocol [15, 21-23], and could be partially explained by fluoride uptake in mineralized tissues [17]. Fluoride concentrations in the liver and kidney are shown in Table 1. In the
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kidney, a dose-dependent response was observed, with significant differences between all groups (p<0.0001). Significant differences in the liver fluoride
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concentrations were also observed among the groups (p<0.0001). The 50 mg/L fluoride group displayed the highest fluoride concentration, and both the 15 mg/L and
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50 mg/L groups had significantly increased fluoride levels compared to the control
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group, which displayed the lowest concentration. The kidneys are the main organs responsible for the reduction of plasma fluoride levels after fluoride intake;
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approximately 60% of the fluoride absorbed is excreted in the urine in healthy adults [24]. As the kidneys are the principal route for fluoride elimination from the organism,
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the fluoride levels in the kidneys displayed a dose-dependent effect in the kidneys in the present study: the fluoride levels increased upon exposure to higher
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concentrations of fluoride. In contrast, the fluoride levels in the liver (Table 1)
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decreased compared to those found in the plasma. This result was expected because intracellular fluoride levels are typically 10-50% lower than those found in plasma or interstitial fluid [17].
Table 1. Fluoride Concentration in the plasma (µg/ml) and the liver and kidney (µg/g tissue) of rats receiving water containing different fluoride concentrations
Plasma
Control
5 mg/L
15 mg/L
50 mg/L
0.032±0.011a
0.027±0.008a
0.043±0.010a
0.094±0.019b
5 Page 5 of 21
Liver
0.009±0.003a
0.010±0.002ad
0.014±0.004bd
0.023±0.008c
Kidney
0.045±0.012a
0.067±0.011b
0.112±0.020c
0.245±0.042d
Values are mean ±SD (n=10). In each line, different superscript letters indicate significant differences among the groups (p<0.05).
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Macroscopic analysis of the liver did not reveal any differences between the groups, and all groups displayed normal morphological characteristics, such as
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uniform appearance, intact hepatic lobules, intact portal area, defined hepatic vein, and intact sinusoids converging to the central lobule vein. However, the group treated
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with 50 mg/L fluoride (Figure 1B) displayed more disorganized hepatocytes
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compared to the control group (Figure 1A), and some hepatocytes were observed to have heterogeneous nuclear sizes, disorganized architecture and changes in the
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delimitation of the cytoplasm. Signs of toxicity include changes in organ weights and hematological and biochemical blood alterations [25]. We did not observe either
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macroscopic alterations or microscopic signs of toxicity. However, the group treated with 50 mg/L of fluoride displayed areas with altered hepatocytes; similar results
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were described by Bouaziza et al (2006) [26], who observed liver changes in mice
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intoxicated by fluoride.
Histological analysis of the kidneys did not reveal any differences between the
groups. Discrete vascular congestion was observed only in the group treated with 50 mg/L of fluoride (Figure 1C). This group also displayed a higher number of congested vessels compared to the control group, but over less than 25% of the tissue (Figure 1D). Vascular congestion has also been described by other authors. Kobayashi et al (2009) [22] also observed vascular congestion, but found no classic signs of nephropathy induced by fluoride. Hyperemia (vascular congestion) can be an early
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sign of the inflammatory response [27]. Other studies on the chronic toxicity of
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fluoride have also reported the presence of vascular congestion [8, 22, 28].
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Figure 1: Liver histopathology of control and fluoride-treated rats (HE stain). (1A) Section of liver tissue from control group rats showing S: Sinusoids, arrow: normal hepatocyte, Arrowhead: nucleus.
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(1B) Experimental group of rats treated with 50 mg/L of fluoride showing Arrowhead: nucleus, * Irregular hepatocytes. Kidney histopathology of control and fluoride-treated rats (HE stain). (1C) Experimental group of rats treated with 50 mg/L of fluoride showing V: vascular congestion, G: Kidney glomerulus. (1D) Section of kidney tissue from control group rats showing G: Kidney glomerulus. 40x
CAT activity in the kidneys (Table 2) was similar in all groups, and no significant
differences were observed (p=0.4157). However, CAT activity in the liver (table 3) was significantly different among the groups (p=0.0001). All the experimental groups displayed decreased CAT activity compared to the control group; the 15 mg/L
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fluoride group displayed the lowest value and was significantly different from both the control and the 5 mg/L fluoride groups. The SOD activity in the kidney was also significantly different between the
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groups (p=0.0012; Table 2). The 50 mg/L fluoride group displayed the lowest value (approximately a 50% decrease) and significantly differed from both the control and
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the 5 mg/L fluoride groups. No significant differences were observed between the control group and the 5 mg/L and 15 mg/L fluoride groups. SOD activity in the liver
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was also significantly different between the groups (p=0.0006): the 15 mg/L fluoride
and 5 mg/L fluoride groups (Table 3).
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group displayed the highest activity and significantly differed from both the control
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The GPx activity (Table 2) in the kidney was significantly different between the groups (p<0.0001). The 50 mg/L fluoride group displayed the lowest activity, while
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the 5 mg/L group displayed the highest activity. In the liver, the GPx activity
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increased in all the experimental groups compared to the control group, but no significant differences were detected (p=0.4964) (Table 3).
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The GSH levels in the kidney (Table 2) were significantly different between the
groups (p=0.0177). The concentration decreased in all experimental groups compared to the control group, but the decrease was only significant for the 50 mg/L fluoride group. In the liver (Table 3), all experimental groups displayed increased GSH concentrations, but no significant differences were detected (p=0.2996). The LH levels in the kidney and liver are shown in Tables 2 and 3, respectively. The LH levels in the kidney were significantly different between the groups (p=0.0075). All experimental groups displayed increased concentrations compared to the control group, but only the 15 mg/L fluoride group significantly differed from the 8 Page 8 of 21
control. The LH levels in the liver were also significantly different between the groups. The experimental groups displayed higher LH concentrations compared with control, but the difference was significant only for the 5 and 50 mg/L fluoride groups.
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No significant differences in the concentration of TBARS in the kidney were detected between the groups (table 2; p=0.0952). In the liver, however, significant
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differences in the TBARS level were observed (p=0.0478). The experimental groups displayed lower concentrations of TBARS compared to the control, but the difference
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was significant only for the 15 mg/L fluoride group (Table 3). The levels of LH, a
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substance related to lipid peroxidation, increased in both the liver and kidneys in the exposed groups compared to the control, which is consistent with the results of
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plasma fluoride levels, despite the fact that no dose-response was observed. However, TBARS levels remained unchanged in the kidneys and decreased in the
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liver of treated animals compared to the control. The concentration of MDA, a product of the termination phase of lipid peroxidation, has been reported to increase in the
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liver of rats that received water containing 100 mg/L fluoride for 4 months [6]. Similar
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results were obtained in the kidneys of rats that were treated for one week with 600 mg/L of fluoride [29]. In these cases, the increased dosage of fluoride and/or the different treatment length might have exhausted the antioxidant defenses, which does not seem to have occurred in the present study. Significant differences in antioxidant concentrations in the liver were observed
among the groups (Table 3; p<0.0001). Antioxidant concentrations in the 15 and 50 mg/L fluoride groups were similar, but significantly higher than those found in the control and 5 mg/L fluoride groups. Significant differences were also observed in the kidneys (Table 2; p<0.0001). An inverse dose-response effect was observed: 9 Page 9 of 21
antioxidant activity significantly decreased in all the experimental groups compared to the control. A significant reduction in antioxidant levels was observed in the groups treated with fluoride compared to the control, in contrast to what was observed for the liver. This can be explained by the higher fluoride concentrations found in the kidney
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compared to the liver, which may also have contributed to reducing the levels of
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antioxidant enzymes such as SOD and CAT.
The activity of SOD significantly increased in the liver of the 15 mg/L fluoride
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group, while the activity of CAT significantly decreased compared to that of the
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control. However, the activity of GPx and the GSH levels tended to be constant. In a recent study, all of these antioxidants were reduced in the liver of rats treated for 12
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weeks with 25 mg/L fluoride in drinking water [30]. The antioxidant profile observed in the kidneys, however, was notably different. The concentration of GPx decreased in
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the groups treated with 15 and 50 mg/L fluoride compared to the control. The levels of SOD and GSH also decreased, but only in the 50 mg/L fluoride group. Similar
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reductions were reported in the kidney of rats treated with 100 mg/L fluoride for one
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week [29]. However, CAT activity remained unaltered. When analyzed in conjunction, these findings suggest that the conversion of the superoxide anion to water in the kidney occurs mainly through the action of SOD and CAT rather than the glutathione system, in contrast to what occurs in liver.
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Table 2: Concentration of LH, TBARS, Antioxidants, CAT, SOD, GSH and GPx in the kidney of rats receiving water containing different fluoride concentrations Group of animals 5 mg/L
15 mg/L
50 mg/L
LH (µmol/g)
0.704±0.18a
0.961±0.47ab
1.229±0.29b
1.001±0.19ab
TBARS (µmol/g)
0.073±0.009
0.067±0.015
0.064±0.009
0.077±0.012
Antioxidants (mmol/g)
2.23±0.34a
1.51±0.41b
1.13±0.25b
1.03±0.18b
CAT(µmol/min*mg protein)
3.37±0.529
3.67±0.681
3.24±0.247
3.51±0.48
SOD (U/min*mg protein)
0.83±0.25a
0.91±0.41a
0.86±0.63ab
0.41±0.7b
GSH (nmol/mg)
3.29±0.52a
2.83±1.04ab
3.13±0.59ab
2.34±0.46b
GPx (µg/min*mg protein)
0.31±0.11ab
0.37±0.13a
0.25±0.05bc
0.17±0.03c
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Control
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Concentration or activity
Values are mean ±SD (n=10). In each line, different superscript letters indicate significant differences among the groups (p<0.05).
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Table 3: Concentration of LH, TBARS, Antioxidants, CAT, SOD, GSH and GPx in the
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liver of rats receiving water containing different fluoride concentrations Group of animals
Concentration or activity
5 mg/L
15 mg/L
50 mg/L
LH (µmol/g)
1.358±0.95a
2.425±0.69b
2.021±0.45ab
2.449±0.8b
TBARS (µmol/g)
0.067±0.018a
0.061±0.027ab
0.047±0.008b
0.05±0.004ab
Antioxidants (mmol/g)
1.32±0.11a
1.25±0.12a
2.52±0.15b
2.37±0.08b
CAT (µmol/min*mg protein)
11.37±2.57a
9.04±1.76b
6.74±0.7c
7.3±0.93c
SOD (U/min*mg protein)
19.76±4.78a
16.82±3.54a
25.17±3.36b
21.4±2.34ab
GSH (nmol/mg)
14.04±7.6
16.51±7.6
18.92±7.26
19.54±5.34
GPx (µg/min*mg protein)
0.093±0.026
0.112±0.046
0.112±0.052
0.132±0.07
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Control
Values are mean ±SD (n=10) In each line, different superscript letters indicate significant differences among the groups (p<0.05).
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3. Conclusions Exposure to the highest fluoride doses (15 and 50 mg/L) caused alterations in the antioxidant system of liver and kidney of rats. However, few changes in the parameters evaluated were observed upon treatment with 5 mg/L fluoride, the dose equivalent to 1 mg/L
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fluoride in drinking water for humans [31]. These findings reinforce the safety of controlled
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water fluoridation.
4. Materials and Methods
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4.1 Chemicals
Sodium fluoride is from Sigma Aldrich (St Louis, USA), HMDS hexamethyldisiloxane
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was purchased from Aldrich (St Louis, USA), Fluoride electrode (Cas #940907) Thermo Orion (USA), butylhydroxytoluene (BHT), thiobarbituric acid (TBA), trichloroacetic acid (TCA),
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nitroblue tetrazolium (NBT), 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), o-Phthalaldehyde (OPT), β-Nicotinamide adenine dinucleotide, reduced dipotassium salt (NADH), xanthine, oxidase,
glutathione
reduced
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xanthine
(GSH),
potassium
dichromate,
sodium
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pyrophosphate, phenazine methosulfate, meta-phosphoric acid and Antioxidant Assay Kit (Cas # CS0790) were purchased from Sigma Aldrich (St Louis, USA), hydrogen peroxide (H2O2), methanol, hydrochloric acid (HCl), Ethylenediaminetetraacetic acid (EDTA) and acetic acid were purchased from Merck (Germany).
4.2 Animals and treatment The study protocol was approved by the Committee on the Ethics of Animal Experiments of Bauru School of Dentistry, University of São Paulo (Proc. 001/2007). Male Wistar rats (21 days old) were divided into four groups containing ten animals each. The rats
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were housed in polypropylene cages and provided with ad libitum low-fluoride food (AIN-93) and water inside a climate-controlled room, which had a 12 h light/dark cycle. The experimental groups were treated for 60 days with three different concentrations of fluoride in drinking water (5, 15 or 50 mg/L fluoride, as NaF), while the control group received deionized
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water. These doses of fluoride were chosen because they are approximately equivalent to doses of 1, 3 or 10 mg/L fluoride in drinking water for humans, respectively [31]. These
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doses correspond to the fluoride concentration that is typically included in artificially fluoridated water and to fluoride concentrations naturally found in endemic regions of dental
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and skeletal fluorosis, respectively [1]. After the experimental period, the animals were
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anaesthetized with ketamine and xylazine and euthanized by decapitation. Liver and kidneys
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were collected and stored at -80ºC until analysis.
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4.3 Determination of fluoride concentration
The fluoride concentration in the kidneys and liver were determined after overnight
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HMDS-facilitated diffusion [18, 32] using an ion-specific electrode (Orion Research, Model 9409) and a miniature calomel electrode, (Accumet, #13-620-79) both of which were coupled
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to a potentiometer (Orion Research, Model EA 940, Cambridge, USA).
4.4 Sample preparation
To obtain the supernatant, the tissues (kidney and liver) were homogenized with a 10
mmol/L phosphate buffer (pH 7.0) and 1% BHT for 2 min (Marconi MA 102, Brazil) and centrifuged at 6400 g for 10 min. The supernatant was collected and used for analysis of TBARS, LH and antioxidant concentration.
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To analyze CAT, SOD and GPx activity, the kidney and liver tissues were homogenized in 10 mmol/L sodium phosphate buffer (pH 7.4) and centrifuged at 1500 g for
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10 min; the supernatant was collected and used for the determinations.
4.4.1 Determination of TBARS concentration
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Lipid peroxidation levels were measured by determination of the thiobarbituric acid
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reactive substances (TBARS) as described by Buege and Aust (1978) [33]. The supernatant (0.4 mL) was mixed with 0.8 mL thiobarbituric acid solution (0.375% TBA, 15% TCA and
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0.25% HCl), incubated in a boiling water bath for 15 min, placed in an ice water bath for 10 min and centrifuged at 13,900 g for 5 min. The absorbance of the supernatant was read at
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535 nm. The spectrophotometer was blanked with 0.4 mL phosphate buffer 10 mmol/L (pH 7.0) (Ultrospec 2000, Pharmacia Biotech, United Kingdom). The concentration of TBARS
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was calculated using its molar extinction coefficient (1.56×105 mol/L−1cm−1).
4.4.2 Determination of lipid hydroperoxide concentration
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The LH concentration in the kidney and liver was measured using the method
described by Jiang et al. (1971) [34], and calculated using a molar extinction coefficient of 4.3 ×104 (mol/L)−1cm−1.
4.4.3 Determination of antioxidant concentration Concentrations of antioxidants in the kidney and liver were obtained using the Antioxidant Assay kit (CS0790, Sigma–Aldrich (St Louis, USA).
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4.4.4 Determination of CAT activity The CAT activity was assayed as described previously by Sinha (1972) [35]. In brief, 0.1 mL of supernatant was incubated with 0.5 mL of 0.2 mol/L H2O2 at 37ºC for 90 s. The reaction was stopped by adding 5% potassium dichromate solution (5% potassium
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dichromate in acetic acid 1:3). Samples were incubated in a boiling water bath for 15 min, and the concentration of H2O2 consumed was determined by recording the absorbance at
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570 nm. The activity of CAT was expressed as µmol/min*mg protein.
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4.4.5 Determination of SOD activity
The SOD activity in the kidney was assessed as described by Kakkar et al. [36], with
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slight modifications. The supernatant was centrifuged at 10,000 g for 15 min, and 0.2 mL of 5% tissue homogenate was added to a reaction mixture containing 1.2 mL of sodium
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pyrophosphate buffer (0.052 mmol/L, pH 7.0), 0.1 mL of phenazine methosulfate (186 µmol/L), and 0.3 mL of NBT (300 µmol/L). The enzymatic reaction was initiated by adding 0.2
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mL of NADH (780 µmol/L) and was stopped precisely after 1 min by adding 1 mL of glacial
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acetic acid. The concentration of reduced NBT formed was measured by the absorbance at 560 nm. In the liver, SOD activity was measured as described previously by Beauchamp and Fridovich [37]. Samples (50 mg) were homogenized with 1 mL of sodium phosphate buffer (50 mmol/L pH 7.8), and after centrifugation at 10,621 g for 10 min, the supernatant was collected. The reaction mixture consisted of 0.96 mL of sodium and potassium phosphate buffer (50 mmol/L pH 7.8), 0.010 mL of xanthine (5 mmol/L), 0.010 mL of NBT (10 mmol/L), 0.010 mL of supernatant and 0.010 mL of EDTA (10 mmol/L). The reaction was initiated by adding 0.005 mL of xanthine oxidase (diluted 20 times), and the absorbance at 550 nm was recorded over a 3 min period. The Δabs/min was determined, and the activity of SOD was calculated and expressed as U/min*mg protein.
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4.4.6 Determination of glutathione peroxidase (GPx) activity The activity of GPx in the kidney and liver were measured as described by Flohe and Gunzler [38] and as modified by Modi and Flora [39]. The supernatant was centrifuged at 10,000 g for 30 min, and the reaction was initiated by adding 0.3 mL of a phosphate buffer
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(10 mmol/L pH 7.4), 0.2 mL of GSH (2 mmol/L), 0.1 mL of H2O2 (1 mmol/L) and 0.3 mL of supernatant. The reaction was incubated in a 37ºC water bath for 15 min and then stopped
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by adding 0.5 mL TCA (5%). The samples were centrifuged at 1500 g for 5 min, the
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supernatant was collected, and 0.1 mL of the supernatant was mixed with 0.2 mL phosphate buffer (0.1 mol/L, pH 7.4) and 0.7 mL of DTNB (0.4 mg/mL). The absorbance was read at
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420 nm with a spectrophotometer (Spectra Max M2, Molecular Devices, Sunnyvale, USA).
4.4.7 Determination of reduced glutathione (GSH) activity
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The GSH activity in the kidney and liver were assayed as described by Hissin and Hilf [40], with slight modifications. The tissues were mixed with phosphate-EDTA buffer (0.1
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mol/L) and meta-phosphoric acid (25%) and centrifuged at 20,817 g for 10 min. Then 1.8 mL
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of a phosphate-EDTA buffer, 0.1 mL of supernatant and 0.1 mL of OPT solution (1 mg of OPT diluted in 1 mL of methanol) were combined, and the reactions were incubated for 15 min. The fluorescence was determined (ex 350 nm/em 420 nm) (Spectra Max M2, Molecular Devices, Sunnyvale, USA), and the concentration was calculated using a calibration curve for GSH and expressed as nmol/mg tissue.
4.8 Histopathological analysis A portion of the kidney and liver tissues were fixed in 10% neutral buffered formalin solution for histological analysis. After fixation, the tissues were embedded in paraffin, and 5 µm sections were sliced and stained with hematoxylin and eosin (HE) using standard
16 Page 16 of 21
techniques. The sections were examined under light microscope (Carl Zeiss, German) and photomicrographs were taken.
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4.9 Statistical analysis All results are expressed as means ± SD (standard derivation). GraphPad InStat
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version 3.0 for Windows was used for the statistical analysis (GraphPad Software Inc., La
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Jolla, USA). The data were checked for normality (Kolmogorov and Smirnov test) and homogeneity (Bartlett test); when these criteria were satisfied, the data were analyzed by
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ANOVA and Tukey’s tests. Otherwise, they were analyzed by the Kruskal-Wallis and Dunn’s
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tests. In all cases, the significance level was set to 5%.
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Acknowledgements
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
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no conflict of interest.
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(FAPESP), grant numbers 2007/03723-2, 2007/01788-0, 2009/01996-7. The authors have
References
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*Graphical Abstract - Pictogram
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*Highlights (for review)
Highlights
Chronic exposure to high doses of fluoride alters the antioxidant system of rats;
Doses equivalent to those found in artificially fluoridated water do not provoke any alterations; The effects of fluoride in the antioxidant systems of the liver and kidneys are
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remarkably different;
Upon exposure to high fluoride levels, the kidneys convert the superoxide anion to
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H2O mainly through SOD and CAT.
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