Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
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EFFECT OF SODIUM FLUORIDE ON SOME PHYSIOLOGICAL
PARAMETERS AND HISTOPATHOLOGY IN ADRENAL AND THYROID
GLANDS IN ADULT MALE RATS
Marwa Adel Hameed*, Bader Khatlaan Hameed* , Khalid Ahmed Hadi**,
Enaam Anad Gabori***, Ahmed Abdalaali Aziz****, Wassan Sarhan Obid**,
Dakheel Hussein Hadree**,
*Deparment of Anatomy and Histology,Vet. Medicine faculty, Tikrit University, Tikrit, Iraq
**Department of physiology, pharmacology and biochemistry, Vet. Medicine faculty, Tikrit
University, Tikrit, Iraq
*** Department of Medical laboratory technique, Al-noor University college, Mosul, Iraq.
****Departmentof physiology, pharmacology and biochemistry, Vet. Medicine faculty,
Kirkuk University, Kirkuk, Iraq
Corresponding Author: [email protected]
Key words : sodium fluoride, adrenal gland, thyroid gland
ABSTRACT
This study was carried out to investigate the harmful effect of water fluoridation on both
(thyroid and adrenal gland) in adult male rats its weight about (400-300gm) exposed to sodium
fluoride(NaF) in the drinking water. We are used in this study (٣٠) adult male rats (albino) were
distributed randomly and divided into two equal groups (15 animals per group) . The first group
was given normal water and considered as a control group (control group), while the second group
were given drinking water with 100 ppm of sodium fluoride(NaF) (treated group). this study carried
out in the animal house in the college of Vet. Med.\ Tikrit University. blood was drawn through the
eye pupil for periods (0,30,60 days) in order to measure the following parameters: Measurement of
Glutathione(GSH) concentration, total cholesterol and blood glucose concentration . In addition
taking tissue sections of the thyroid and adrenal glands.The results of this study showed that
exposure of animals to sodium fluoride at a concentration of 100 ppm in drinking water for (60)
days cause adrenal and thyroid gland dysfunction, represented by a significant decrease in the level
of glutathione in blood serum on days 30 and 60 of the experiment and a significant increase in total
cholesterol and blood glucose concentrations.The results of the histological examination of the
thyroid gland of the treated group showed hyperplasia of the epithelial cell layer lining the acini
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College
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vesicles, and severe lipid changes were seen in the function of Reticularis in the histological
sections of the thyroids gland of the same group animals. The results of this study confirmed the
harmful effect of sodium fluoride(NaF) on thyroid and adrenal functions in addition to its effect on
some biochemical parameters that are indicative of the occurrence of harmful effects of some
chemical compounds .
INTRODUCTION
The terms “fluorine” and “fluoride” are using interchangeably as a generic term. The term
“fluorine gas” is often used as an emphasized reference to the elemental form of fluorine rather than
a combined form (1). Fluoride is the light element of Group 17 (VIIA) of the periodic table. This
group, also includes chloride, bromine, and iodine. As with the other halogens, fluorine happened as
a diatomic molecule, F2, in its elemental form. It has one stable isotope and its valence in all
compounds is -1. Fluorine is the most reactive of all the elements, which may be because of its large
electronegativity (estimated standard potential +2.85 V). It reacts at normal room temperature or
elevated temperatures with all elements other than nitrogen, oxygen, and the lighter gases. Fluorine
is also notable for its small size; large numbers of fluorine atom fit around atom of other element.
This, along with its electronegativity, allows the formation of many simple and complex fluorides in
which the other elements is in its highest oxidation date (2). Sodium fluoride is an in-organic
chemical compounds with the formula NaF. A colorless solid, it is a source of the fluoride ions in
diverse applications. Sodium fluoride is less expensive and less hygroscopic than the related salt
fluoride. Sodium fluoride is an ionic compound, dissolving to give separated Na+ and F− ions (1).
Sodium fluoride (NaF) is using as a flux for deoxidizing rimmed steel, as a component of laundry
sours (removal of iron stains), in casein glues and heat-treating salts, and in the re-melting of
aluminum, manufacture of vitreous enamels, pickling of stainless steel, and manufacture of coated
papers (3). Sodium fluoride is also used in various pesticide formulations, including insecticides
and woods preservations (4). Sources of exposure The major sources of fluoride in the world are
food, drinking water (5), beverages, and fluoride-containing dental products (6). The atmosphere
carries some fluoride, but it supplies a small fractions of the daily exposure except in heavily
polluted areas (7).
MATERIALS AND METHODS
١. Animals: We are used in this study thirty male albino rats (400-300 g) were used in this
investigation. Animals were housed in iron cages in (22-25 °C) in the animal house at the College
of Veterinary Medicine -University of Tikrit.
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2. Preparation of NaF:
Sodium fluoride (NaF) was used as described by (8).
3.Experimental Design:
30 adult male were divided into 2 groups (15 rats/ group) and treated daily for (60) days as
follows:
1. Control Group: all animals in this group were submitted to natural conditions.
2. Treated Group: all animals in this group were submitted to dose of 100 ppm of sodium fluoride
with water (8).
4.Blood Sampling:
Blood samples were collected from each animal groups after periods 30 days. blood was drawn
from eye vein by a capillary tube containing heparin implanted in the inner corner of the eyeball. The
blood was allowed to flow into a dry and clean test tube. The blood was then clotted and the tubes were
placed in the centrifuge at 3000(rpm) for 15 minutes to separate the serum and kept it in refrigerator at -
22 C (9).
5. Studied Parameters:
A. biochemical parameters include:
1- Concentration of Glutathione (GSH).
The level of glutathione was estimated by using the modified method used by the researcher (10).
2- Concentration Total Cholesterol (TC) (mg/dl):
The level of cholesterol was estimated by using the method used by the researcher (11).
3- Concentration of serum glucose:
it was measured enzymatically by using glucose kit (Biomaghreb).
6. Histopathologic study:
For histological examinations, rats were killed after completing period of study. Immediately,
after killing, tissue pieces from thyroid and adrenal glands were preserved in 10% neutral buffer
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formalin till the preparation of histological sections. Several tissue sections were prepared and
stained according to (12).
Statistical Analysis:
Data were analyzed using two way analysis of variance (ANOVA) then followed with post
hoc test (Least Square Deviation). The probability level (P≤ ٠.٠٥).
RESULTS AND DISCUSSION
1.Level of glutathione (GSH):
The exposure to 100 ppm NaF (T1) on the mean value of serum (GSH) concentrations of adult
male rats were clarified in table (1). A statistical analysis indicated that the mean values of serum
(GSH) were non significantly different (P ≤ 0.05) in all experimental groups at the zero time when
compared to each other. During treatment (After 30 days) a significant decreasing in (GSH) level
with mean value of (16.8±0.15) was detected in NaF Treated Group in comparing with control
group (22.4±0.15). and after 60 days there is significant increase in the level of glutathione in the
infected group.
Table (1): level of glutathione concentration (μmol/l) of adult male albino rats treated with (NaF) in
drinking water for 30 and 60 days.
- Values represent Mean ± standard error.
- No. of animals in each group = 15.
- The different letters within the columns and rows mean a significant difference at a level of probability
(p≤0.05).
Groups Control G. Treated G.
Day
23.3±0.17
Aa
22.1±0.15
Zero Aa
16.8±0.15
Cb
22.4±0.15
30 Ba
11.1±0.14
Cc
22.8±0.73
60 Ba
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The mechanisms of fluoride effects have not been determined yet. However, oxidative stress
with lipid peroxidation has been suggested as one of the important factor of toxic effects of fluoride
(13). When an imbalance in the oxidant-antioxidant system occurs, the potentiality for tissue
damage increases, with disturbances of the antioxidant system (14) including GSH concentration
(15). it may be suggested that fluoride inhibits glucose-6-phosphates dehydrogenase (G6PD) due to
an oxidative stress, and the decrease of Pentose phosphate pathway flow could make the cell unable
to maintain the normal GSH/GSSG ratio, which is lowered due to fluoride. Increased consumption
of Glutathione(GSH) and decreased capability to regenerate the reduced cofactor could, in turn,
trigger a vicious circle, allowing a further oxidative damage of G-6-PD, lowering GSH
concentration and cell injury (16).
2.Total serum cholesterol (TC) Concentration.
Table (2) showed the mean of TC concentration in the two groups along the experimental
period. It can be seen that serum TC concentration was close in all groups (P≤ 0.05) in the
pretreated period. After 30 days of treatment, a significant increasing (P≤ 0.05) in serum total
cholesterol level was observed in NaF, (181.8±6.66) as compared to control groups (109.8±3.15).
Further significant elevation in the (TC) parameter were observed at the end of experiment in NaF
treated group comparing to control. The mean values of this parameter in treated group was
(181.8±6.66) and (25٦±3.14), for the same previous treated periods (30 and 60 days respectively).
Table (2): level of Total serum cholesterol level (mg/dl) of adult male rats treated with (NaF) in
drinking water for 30 and 60 days.
- Values represent Mean ± standard error.
- No. of animals in each group = 15.
- The different letters within the columns and rows mean a significant difference at a level of probability
(p≤0.05).
-
Groups Control G. Treated G.
Day
118±3.22
A a
115.4±2.92
Zero A a
181.8±6.66
A b
109.8±3.15
30 C a
256±3.14
A c
120.4±0.67
60 C a
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3.Serum Glucose Concentration.
Table (3) illustrated the values of serum Glucose level in male rats after oral intubation of 100
ppm NaF for two months. Data pointed there is no significant differences (P≤ 0.05) between groups
at zero time. During treated period (after 30 days) a significant increasing in glucose level with
mean value of (137.4 ± 4.47) was detected in NaF treated Group in-comparing with other group
(92.20±2.59).while at 60 days there is increase in the level of glucose in the treated group.
Table (3): level of glucose (mg/dl) of adult male rats treated with (NaF) in drinking water for 30
and 60 days.
- Values represent Mean ± standard error.
- No. of animals in each group = 15.
- The different letters within the columns and rows mean a significant difference at a level of probability
(p≤0.05).
The effects of fluoride as mentioned by (17) who reported impaired glucose tolerance in 10 of
25 residents of an area with endemic fluorosis. A significant increase (17%) was observed in serum
glucose in rats given F− in drinking water at 100mg/L for 6 months (18). Additionally, (19)
reported decreased insulin, increased plasma glucose and disturbance of the glucose tolerance test in
rats after an oral dose of 40μM NaF per 100 g body weight. In vitro study showed that exposure of
Langerhans islets isolated from rat to NaF, caused depression in insulin secretion in a dose
dependent manner (20) with a subsequent elevation in glucose concentration.
Inhibition of enzymes involved in cellular metabolism is one of the mechanical toxic effects of
fluoride on enzymes involved in the glycolytic pathway, such as hexokinase, enolase, and pyruvate
kinase, these are all subject to F− inhibition. Antioxidant enzymes such as superoxide dismutase
(SOD). Na+/K+-ATPases are also inhibited by fluoride, leading not only to ATP depletion, but to
Groups Control G. Treated G.
Day
92.6 ± 11.53
A a
90.2 ± 12.13
Zero A a
137.4 ± 14.47
A b
92.2 ± 12.59
30 C a
196.0 ± 17.92
A c
93.2 ± 14.61
60 C a
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disturbances in the cell’s membrane potential (21), with subsequent stimulation of glucagon
dependent pathways like gluconeognesis, glycogenolysis leading to an elevation of serum glucose.
Interestingly, mitochondria considers the main target of fluoride toxicity (22), because of
increasing production of O2–(23). There is no evidence that explain linking between fluoride
exposure and insulin expression. However, there was an evidence that fluoride exposure may be
influence the transcription of many gens (24). Also its effects on expression of insulin mRNA
production cells (25).
Apoptosis plays a rode role in fluoride toxicity in all cells (26), like pancreatic cells (27),
possibly because its role in oxidative stress (28).which is greatly accompanied with a
hyperglycemia (29).
Histological examination
Adrenal gland: Figure (1) pointed to histological section of adrenal gland in animals treated with
100 ppm of NaF, where sever fatty changes which appeared as large clusters cystic structure in
internal layer comparing to normal (Fig 2). There are some changes were observed in Thyroid and
adrenal glands supported the (Functional) changes in all experimental groups.
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Figure (1) Histological section in adrenal of rats treated with 100 ppm of NaF
for 60 days, reveals severe fatty change which appear large cluster cyctic
structure in internal layer ( ) (H and E 40X)
Figure (2) Histological section in control rats, showed normal structure of
adrenal gland.(H and E 40X)
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Thyroid gland
Histological sections in the thyroid gland post 60 day of treatment with 100 ppm NaF showed
irregular colloid substances with fluctuation, together with hyperplasia of epithelial lining cells of
acinied (Fig 3) comparing to histological section of normal thyroid gland (Fig 4). Besides, uniform
colloid substance .
Figure (3) Histological section in the thyroid gland of rats treated with 100 ppm of
NaF for 60 days, showed acini field with irregular colloid substance with vaculation
together with hyperplasia of epithelial lining cells of acinied ( ).(H and E 40X).
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