FLUORIDE 31(1)
 1998 pp 26-32		 International Society for Fluoride Research Back to Table of Contents


TOXIC EFFECTS OF FLUORIDE ON BEATING
MYOCARDIAL CELLS CULTURED IN VITRO

FuYuan Wang,a DeXin Zhang,a and RuiMian Wangb
Wuhan, China


SUMMARY:
Objective: To study the effect of fluoride on myocardial cells.
Method: Three days after seeding in vitro, five groups of neonatal rat myocardial cells were cultured for one day in media containing fluoride at concentrations of 0, 3.23, 6.46, 12.91, and 25.81 ppm respectively.
Results: Higher fluoride levels were associated with an increased beat arrest rate, media lactate dehydrogenase (LDH) activity and media nitric oxide (NO) level, and a decreased cell glycogen level and cell succinate dehydrogenase (SDH) activity. Scanning electron microscope study of the beat-arrested cells showed them to be rounder and smaller than the beating cells with the loss of microvilli, the shortening or loss of cell processes, and the appearance of pits and blebs on the cell surfaces.
Conclusion: Fluoride can be toxic to myocardial cells with possible mechanisms for this toxicity being impaired energy metabolism, cell membrane damage, increased NO production causing cell relaxation, and dissociation of the cytoskeleton. A threshold for fluoride toxicity was not established as significant toxicity was present at the lowest level studied, of 3.23 ppm of fluoride in the media.

Key words: Beat arrest rate; Fluoride toxicity; Myocardial cells; Rat.

aDepartment of Hygiene. bDepartment of Histology and Embryology, Hubei Medical University, Wuhan, 430071 People's Republic of China.

INTRODUCTION

There are differing reports of the effect of fluoride on cardiovascular disease. A more rapid decline in cardiovascular mortality has been reported in association with fluoridation.1 Another report, from Antigo, Wisconsin, USA, found a seven-fold increase in the annual cardiac death rate from 5 deaths per 100,000 in 1949, to 35 deaths per 100,000 population in 1960, when fluoridation was stopped.2 When an adjustment was made for age the cardiac death rate increased ten-fold from 1.2 deaths per 100,000 in the decade prior to fluoridation to 12 deaths per 100,000 after the introduction of fluoridation. The increase in the annual cardiac death was interrupted when fluoridation was discontinued for five years but resumed again two years after the reintroduction of fluoridation. In China, significantly more cardiovascular disease3 and more electrocardiogram abnormalities 4 have been found in an area with endemic fluorosis compared to an area without endemic fluorosis. Foetal myocardial cells have been found to be abnormal in an endemic fluorosis area.5 Experiments in vitro have shown that fluoride may have a positive or negative effect on the contractility of muscle.6,7

Because of the potential for confounding factors to affect an epidemiological study, the difficulties involved in detecting toxic effects of fluoride on myocardial cell in vivo, and the only limited time for which myocardial cells can be maintained in a viable and stable state for in vitro experimentation, it was elected to use cultured neonatal rat myocardial cells in vitro to study the effect of fluoride on myocardial cells, concentration-response effects, and the mechanisms by which toxicity occurred.

MATERIALS AND METHODS

1. Primary culture of beating myocardial cells
The hearts of neonatal Sprague-Dawley rats were excised, washed with phosphate-buffered saline (PBS) at pH 7.2 and minced.8 The cells were isolated with 0.125% trypsin and purified with 6% bovine serum albumin (BSA) and cytosine-1b -D-arabinofuranoside (ARA-C), the final concentration of which in the medium was 10 m M. The muscle cells were seeded at a density of 105 cells/mL in plastic culture dishes which had 24 wells with cover-slips. The muscle cells were maintained in medium 199 (Gibco) supplemented with 15% foetal bovine serum (Gibco) and antibiotics. The cultures grew at 37°C in a humidified incubator gassed with 95% air/5% carbon dioxide.

2. Fluoride exposure
Seventy-two hours after seeding the cultures were divided into five groups each containing seven cultures. Medium 199 was added to the medium of the control group and sodium fluoride to the other four groups to give final concentrations of fluoride for Group A of 3.23 parts per million (ppm) (mg/L), Group B 6.46 ppm, Group C 12.91 ppm, and Group D 25.81 ppm. The culture was then continued for 24 hours.

3. Beat arrest rate of myocardial cells
The number of beating cell clusters was counted under an inverted microscope at 72 hours after seeding before exposure to fluoride and 96 hours after seeding following 24 hours of exposure to fluoride. The beat arrest rate was calculated from the formula:
Beat arrest rate (%) =A-B
A		x 100 (%)
A: number of beating clusters at 72 hours, before exposure to fluoride
B: number of beating clusters at 96 hours, 24 hours after exposure to fluoride

4. Cytochemical reactions and image analysis
Twenty-four hours after exposure to fluoride, the Periodic Acid Schiff Reaction (PAS) and the Nachlass Nitro-blue Tetrazolium Reaction (Nitro-BT) were carried out on the cultures to detect glycogen and SDH activity in the myocardial cells.9 The optical density (OD) values of the reactions were determined with image analysis equipment (Kontron IBAS). Thirty microscopic visual fields in 7 cultures (4 or 5 visual fields in one culture) of each group were determined.

5. Analysis of media LDH and NO
Media from each group were collected at 72 hours and 96 hours to determine LDH activity and the NO level.10 The NO level was determined by measuring absorption at 490 nm and calculating using a curve calibrated from sodium nitrite standards.

6. Scanning electron microscopy (SEM)
At 96 hrs the control culture and Groups B and D were prepared with routing for SEM and observed under a scanning electron microscope (Hitachi S-450).

7. Statistical analysis
Statistical analysis was carried out using the Statistical Analysis System V6.03. Analysis of variance (F value) was used to test for significance of the experimental values obtained for the groups. The Student-Newman-Keuls test (q test) was used to test for significant differences between groups.

RESULTS

1. Beat arrest rate of myocardial cells
When examined under the inverted microscope 24 hours after seeding the myocardial cells were found to have developed into cylindrical or polygonal cells with some processes but the majority of them were not beating. At 72 hours the myocardial cells had increased in size and cell clusters had formed through active proliferation. As the cells came in contact with one another they connected through the long processes on the cells and most began to beat in synchrony. At 96 hours, after exposure to fluoride for 24 hours, the beat lost its regularity and beat arrest appeared. The beat arrest rate increased with increasing concentrations of fluoride in the media (Table 1). The median beat arrest concentration (BAC50) for fluoride, using the modified Karber method of calculation, was 8.05 ppm, 95% confidence interval 7.23-8.91 ppm.


TABLE 1. Beat arrest rate of myocardial cells and media fluoride concentration
  No. of beating clusters  
Group Fluoride in
media (ppm)		 No. of
Samples		 72 hours
(pre-exposure)		 96 hours
(post-exposure)		 Beat arrest rate (%)
(Mean ± SD)
Control 0 7 69 69 0 ± 0
A 3.23 7 93 77 17.2 ± 0.77
B 6.46 7 94 68 27.7 ± 2.01
C 12.91 7 103 27 73.8 ± 2.71
D 25.81 7 87 1 98.9 ± 0.58
  Comparing the media fluoride level and the beat arrest rate
  r = 0.9688 y = 5.855+3.9005x


2. Cytochemical reaction and image analysis
In the control group of beating cells, the presence in the cell bodies and processes, of glycogen was indicated by pink granules or coarse clumps (Figure 1), and SDH activity by purplish-blue granules (Figure 3). In the groups with fluoride, A - D, there was a progressive reduction in the intensity of the pink (Figure 2) and purplish-blue colouration (Figure 4), and optical density values. A significant negative correlation was present between the fluoride concentration and the glycogen content and the SDH activity (Table 2).


TABLE 2. Optical density values of glycogen content and succinate
dehydrogenase activity after exposure to fluoride for 24 hours
Group Fluoride in
media (ppm)		 No. of
samples		 Glycogen contenta
(mean ± SD)		 Succinate dehydrogenase
activityb (mean ± SD)
Control 0 30 0.3066 ± 0.0281 0.4569 ± 0.0563
A 3.23 30 0.3017 ± 0.0254 0.4486 ± 0.0449
B 6.46 30 0.2656 ± 0.0141* 0.4048 ± 0.0330
C 12.91 30 0.2277 ± 0.0101* 0.2828 ± 0.0124*
D 25.81 30 0.2164 ± 0.0093* 0.2252 ± 0.0165*
  aF = 140.33 p < 0.0001 y = 0.2996-0.004x
  bF = 242.75 p < 0.001 y = 0.4585-0.0010x
  *compared to the control p < 0.05

Figures 1-6. Myocardial cells from rats cultured for 96 h [NOTE by webmaster: images not displayed here. Please see membership page for instructions on how to subscribe to the FLUORIDE Journal]


3. Lactate dehydrogenase activity and nitric oxide content in the media
After 24 hours of exposure to fluoride, the LDH activity in the media was higher in groups B,C and D compared to the control, and increased, in all groups, as the fluoride concentration in the media increased (Tables 3 and 4). Similarly after 24 hours of exposure to fluoride the nitric oxide content was higher in group D compared to the control (Table 3).


TABLE 3. Changes in lactate dehydrogenase activity and nitric oxide content of the media,
between 72 hours and 96 hours after exposure to fluoride for 24 hours
Group Fluoride in
media (ppm)		 No. of
Samples		 Change in Lactate dehydrogenase
activitya (activity units) between
72 and 96 h (mean ± S.D.)		 Change in Nitric oxide content
(nitrite N mg/L) between
72 and 96 h (mean ± S.D.)
Control 07 23.3 ± 22.2 0.0003 ± 0.0035
A 3.23 7 104.9 ± 49.1 0.0002 ± 0.0015
B 6.46 7 282.6 ± 56.0** 0.0002 ± 0.0090
C 12.91 7 272.4 ± 54.3** 0.0008 ± 0.0223
D 25.81 7 470.1 ± 62.0** 0.0328 ± 0.0105*
  aF=9.94 p<0.0001 r=0.9730
  *compared to the control p<0.05
  **compared to the control p<0.01


TABLE 4. Lactate dehydrogenase activity of the media, at 96 hours
after seeding, after exposure to fluoride for 24 hours
Group Fluoride in
Media (ppm)		 No. of
samples		 Lactate dehydrogenase activitya
(activity units) at 96 h (mean ± S.D.
Control 0 7 483.86 ± 28.15
A 3.23 7 551.71 ± 74.35
B 6.46 7 713.86 ± 77.12*
C 12.91 7 782.29 ± 51.36*
D 25.81 7 982.57 ± 68.20*
  aF=9.94 p<0.0001 r=0.97 y = 520.79 - 18.80x
  *compared to the control p<0.05


4. Damage to the cell surface ultrastructure
In the control group the beating myocardial cells had a cylindrical or polygonal shape with microvilli distributed evenly on the cell surfaces along with slender processes growing from the cell bodies connecting with the processes or cell bodies of nearby cells (Figure 5). In groups B and D the beat-arrested cells became smaller and rounder than the beating cells with the microvilli disappearing, the processes becoming shortened or absent, and the appearance of many pits or blebs on the cell surfaces (Figure 6).

DISCUSSION

Two types of myocardial cells were present in the cultures. One was pacemaker cells from the conductive system which contracted rhythmically with diastolic depolarization. The other type was the normal atrial and ventricular myocardial cells which were not able to contract or beat until able to make contact with the pacemaker cells or other beating cells.11 When exposed to fluoride the myocardial cells may stop beating. In the present study a positive correlation and concentration-response relationship were found to exist between the concentration of fluoride and the beat arrest rate. Fluoride was found to be toxic or poisonous to the myocardial cells.

The myocardial cells in culture absorb glucose from the medium to produce energy for contracting and other metabolic functions or the synthesis of glycogen. The glycogen content reflects not only the availability of an energy store but the adenosine triphosphate (ATP) content in the cells. In measuring the activity of SDH, one of the dehydrogenases in the citric acid (tricarboxylic acid, Kreb's) cycle an indication is obtained of the ability to produce ATP. This study showed that a possible mechanism by which fluoride produced beat arrest in myocardial cells was by reducing the glycogen content and SDH activity in the cells indicating a reduced availability of energy in the form of ATP.

Lactate dehydrogenase is an enzyme in the cell cytoplasm. The raised levels of LDH in the media after the addition of fluoride indicates that the cell membrane is damaged by fluoride resulting in an increase in the permeability of the cell membrane and the escape of the enzyme from the cytosol. As the cell membrane of the myocardial cell is closely involved with excitation and contraction, damage to it may be another factor contributing to beat arrest.

Nitric oxide is formed from L-arginine by the enzyme NO synthase,12 several isoforms of which have been purified and characterized from different cell types.13 In the myocardial cell a calcium-independent NO synthase isoform (iNOS) predominates.14 When iNOS is activated it produces NO which exerts a negative inotropic effect by increasing the level of the cyclic guanosine monophosphate (cGMP). In the present study the NO content in the medium increased when the fluoride content in the medium reached 25.81 ppm. In association with this a large number of myocardial cells stopped beating suggesting that the NO increase resulting from iNOS activated by fluoride may be another mechanism for beat arrest.

The marked alteration in cellular morphology associated with fluoride reflects damage to the cytoskeleton. In the course of plating, an adhesion plaque, made of vinculin and talin, appears at the inside of the cell membrane, which induces actin to assemble, producing fibres which support the cell and allow it to plate, spread, and extrude processes. A prerequisite for the assembly of actin is the presence of ATP15 which is reduced in availability by fluoride. The deficiency of ATP produced by fluoride may result in the dissociation of the skeleton of actin filaments bringing about injurious alterations in cell form and cell surface ultrastructure with finally cell death and shedding from the cover slip.

In conclusion, fluoride may be directly toxic or poisonous to myocardial cells. While there may be debate as to whether or not fluoride in small amounts may have a beneficial effect on the presence of dental caries, it is clear that in increasing amounts it is increasingly toxic to the heart. Exposure to excessive amounts of fluoride should be avoided. A threshold for fluoride toxicity was not established, as significant toxicity was present at the lowest level studied, of 3.23 ppm of fluoride in the media.

REFERENCES

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  12. Lowenstein CJ, Dinerman JL, Snyder HS. Nitric oxide: a physiologic messenger. Annals of Internal Medicine 120 227-237 1994.
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  15. Carlier MF, Pantaloni D. Nucleotide hydrolysis in actin and tubulin polymerization: liberation of Pi is the crucial step in the regulation of dynamic of microtubles and actin filaments. In: Rousset BAF (Ed). Structure and Functions of the Cytoskeleton: Biological and Physiopathological Aspects. John Libbey, London 1988 pp 33-41.

FLUORIDE 31(1)
1998, pp 26 - 32		 International Society for Fluoride Research
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