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SCIENCE SECTION

Westendorf 1

I. Introduction

The element fluorine was discovered in ivory at the beginning of the 19th century by Morodini, a student of Gay-Lussac's, and first purified in 1886 by Henri Moissan. Fluorine is, as has only been known for a few years, a so called essential element, without which at least vertebrates can not live. Knappwost(1) demonstrated this in humans after F analysis of urine through the use of the well known curve for the dependence of the frequency of caries formation on fluoride content of the drinking water. McClendon(2) proved that rats with F-free nutrition do not thrive well. Hayek et al(3) found that under physiological conditions traces of F are necessary to precipitate hydroxyapatite (Abbr. HA).

Research of the effects of small physiological F-doses was significantly spurred by the observation that a regular uptake of about 1mg F per day served as an effective protection against tooth-decay.(4) This protection is strongest when F administration is begun before the teeth decay. This effect is due to an improvement of the mineralization density of the tooth enamel. An evenly and densely mineralized enamel offers a visibly higher protection against the corrosive influences of cariogenic microorganisms. The deciding factor is therefore not formation of F-HA but rather the mineralization density. In vivo the concentration of F-HA only reaches a level of a few ppm, at which, according to measurement by Knappwost and Raju(5), only a few meaningless reductions in solubility are achieved.

However, a reduction in decay can even be achieved in teeth that have already begun to decay, as long as a daily intake of at least 1 mg of fluoride is maintained. According to Knappwost(6?), the reduction in decay relies for the most part not on the presence of fluoride in the saliva, but rather on an influence on the saliva quality and quantity. According to McClure(7), the F-content of saliva, independent of the intake level, should never exceed 0.1 mg/l.

More recent experiments by K.Yao and P. Groen(8), which were carried out with the help of an F-specific electrode, confirm these results. The F-content of the saliva secreted by the parotid gland, which for test subjects drinking water with <0.1ppm of F was 0.007ppm, rose to 0.009ppm upon transition to concentrations of 1ppm of F in the water.

Knappwost developed a model as part of his "resistance theory"(9) that describes a correlation between cariogenic effects and the viscosity of the saliva. The pH-value of the saliva also takes on an important role in this model. The physiologically efficient saliva can thereby be viewed as a supersaturated solution of HA that has the task of stabilizing initial corrosive defects on the enamel surface through remineralization.

The rate of the remineralization is, at a given supersaturation of HA ions, limited by the level of diffusion across a boundary layer attached to the surface of the tooth. The relationship between the remineralization rate vR and the viscosity h of the saliva can be expressed through the following equation:

Where n>0 and depends on specific conditions like, for example, the flow velocity of the saliva.

The pH-level has an effect on the solubility of the HA, and therefore also on the level of supersaturation. A correlation must therefore exist between oral uptake of fluoride and the viscosity of the saliva. Experimental findings support this conclusion (10,11)). Of all the factors that might explain a possible effect of fluoride on the caries, disregarding the effect on the mineralization density, Knappwost's theory is least questionable.

As has already been stated, the amount of fluoride that is incorporated into the tooth enamel under physiological conditions is too small to cause a significant reduction in the solubility of the HA. An antifermentative effect of F- on the glycolysis of bacteria in the mouth only appears at F- concentrations above 0.5 ppm(12). The concentration of 0.0033 ppm(8) of F that is reached when the F concentration in drinking water is 1 mg F/l, a level that was recognized as sufficient for tooth decay prophylaxis, however lies well below the concentration necessary to achieve this effect.

Knappwost's theory assumes an effect of fluoride on the salivary glands in the form of an elevated secretion rate, a decrease in the viscosity, and an increase in the pH. These symptoms can always be observed with large intakes of fluoride, both orally as well as parenterally (11,13). Upon transition to physiological concentrations the effect is difficult to observe because of a number of other variables that influence saliva secretion. In addition, it is difficult to turn off inductive effects that arise in conjunction with this outcome. For this reason, one must attempt to find an influence of the fluoride on the fundamental biochemical and biophysical processes involved in saliva secretion.

The salivary glands are innervated by the autonomic nervous system (sympathetic and parasympathetic), with the parasympathetic being of greater importance. The stimulation occurs as a nervous reflex, the control center of which lies in the nucleus salivatorius of the medulla oblangata. The composition of the saliva depends on the type of stimulus, which creates a unique stimulatory pattern through the smell and taste receptors. This stimulatory mechanism allows, depending on demand, for either a more serous secretion (stimulation of the parotis) or a more viscous mucin rich secretion (stimulation of the sublingual glands) to be created.

For the resistance of the tooth's surface, however, the so called "resting-saliva" is of great importance. Like all autonomic organs, the salivary glands have a basal level of activity, which in this case serves to moisten the mouth and throat regions. This moisture is important for maintenance of muculmembranes and the surfaces of teeth (by way of remineralization). A general increase in the tone of the parasympathetic system has an effect on the composition of the resting saliva in accordance with Knappwost's resistance theory, that is to say towards an increased release of a watery and possibly also more alkaline saliva.

According to recent experiments, the mechanism of saliva secretion is the following(14): After stimulation by the neurotransmitter acetylcholine (ACh), active transport of Cl- from the interstice into the cell occurs as a result of hyperpolarization. Passive transport of Na+ follows the Cl- and is in turn followed by water, which results in an increase in osmotic pressure within the gland-cell. As a result of the rising pressure, cellular fluid penetrates the membrane bordering the lumen. Na+ is actively reabsorbed at the lumen wall and is followed by Cl- by way of passive transport. Water penetrates the lumen wall slowly, which is the reason for the hypotonia of the saliva. As a result of the delay in Cl- migration with respect to that of Na+, the lumen wall becomes negatively charged on the inside, which causes a flow of K+ from the inside of the cell into the lumen. The abnormally high potassium excess in the saliva results from this influx. (The K+/Na+ ratio of the saliva is 1.3, compared to 0.05 in the serum.)

Figure 1. Schematic Representation of Saliva Secretion

A possible effect of fluoride on the secretion of saliva could lie either in an influence on the cholinergic system, or in a direct influence, perhaps on the membrane permeability for cations and anions.

1. Fluoride and the Cholinergic System

Stimulus conduction takes place by way of the "complete ACh system" at the synapses of the motor endplate as well as those of the target organs of the parasympathetic system. The ACh is synthesized from "activated" acetic acid (in acetyl-CoA, the acetate is bound to the coenzyme by a high energy thioester bond) and choline and is collected in small storage bubbles (vesicles), from which it is released upon stimulation. The excitation is passed on by way of depolarizing the bordering cell, which is the result of a change in Na+ permeability caused by the ACh. The released ACh is quickly inactivated (saponified) by the enzyme acetylcholinesterase (AChE). Inactivation is necessary for the reestablishment of excitability.

Drugs that inhibit AChE (Physostigmine, Neostigmine, Diisopropylfluorophosphate, E-605, among others) cause the ACh, which is constantly released in small amounts, to collect in the tissues. The build up of ACh leads to the appearance of parasympathetic stimulation (activation of the intestinal tract, increased levels of glandular secretion, decreased blood pressure and heart rate). In the progressed state a constant depolarization of the cholinergic membrane, and thereby an un-excitability, is established. The effected organism dies as a consequence of this depolarization.

These symptoms, which are typical of drugs influencing the parasympathetic nervous system, are also observed when toxic amounts of fluoride are administered. MIYAZAKI(15) found overabundant salivation when toxic amounts of NaF were given to rats. In 1872 RABOUTEAU(16) determined that ingesting 0.25g of NaF resulted in an increase in his salivation. The increase began after 4.5 hours and lasted 1.5 hours. He could make the same observation with dogs and rabbits. WEDDEL(17) induced diarrhea as well as increased salivation in a dog by administering 0.5g of NaF. The salivation could not be inhibited by atropine. This last finding suggests that in this case the fluoride must be influencing the salivary glands directly. Otherwise, atropine would have inhibited the salivation by displacing the ACh, collecting due to AChE, from its receptors. An anti-cholinesterase effect of fluoride at lower concentrations than those applied here is thereby however not out of the question.

Inhibition of AChE by fluoride has been described often. E. HEILBRONN(18) and R.M. KRUPKA(19) completed detailed studies. The authors describe the inhibition of AChE by NaF, as well as the pH dependence of the inhibition. This dependence is, however, not traced back to the un-dissociated HF molecule, in contrast to which we will, over the course of this report, show that the inhibition of AChE by fluoride occurs in proportion to the concentration of HF.

It follows from Heilbronn and Krupka's experiments that an inhibition of AChE by fluoride only arises at concentrations that are acutely toxic and even lethal in vivo. If one shifts to physiological concentrations (0.1-1 ppm) the inhibitions become so small that they lie below the threshold for accurate measurement.

The inhibition of AChE by fluoride can be drawn into the discussion of a vagotonic fluoride influence if effects are found that, in vivo, can lead to an increase of fluoride's normal inhibitory influence. The inhibitory effect of fluoride was assigned to F- in all previous investigations of the inhibition of AChE by fluoride. In our opinion, the inhibition does not necessarily appear only in this form in the organism. For example, if one dissolves magnesium hexafluorosilicate (MgSiF6) or cryolite (Na3AlF6) in a buffer at pH of 7.4, which corresponds to that of human blood, only partial hydrolysis occurs, as we will show over the course of this work. The residual complexes, at least in the case of (SiF6)2-, inhibit AChE more strongly than fluoride. Therefore, if one postulates the existence of such complexes in the organism, the range in which inhibition still appears shifts towards physiological F concentrations.

Due to the constant contact of natural waters with silicates as well as Fe and Al compounds, one must expect that these compounds and silicates will form complexes with the fluoride contained in the water. These complexes can then, by way of drinking water, enter the body, where they persist and carry out their influences. New, and as of yet unreleased, experiments by Knappwost and Rastaedter, suggest that fluoride is present in several mineral springs as a Si complex. Taking such compounds into account one can easily imagine that fluoride causes an AChE inhibition in vivo, which makes itself noticeable as a slight vagotonia.

2. Effect of Fluoride on Membrane Permeability

As Weddel describes(17), a strong saliva flow developed at high fluoride concentrations. Since this flow could not be inhibited by atropine we assume a direct effect of fluoride on the salivary glands in this case. In studying the influence of toxic F doses on the nervous system and muscles, TAPPEINER(13) found that depression of the central nervous system and stimulation of the motor endplate appeared initially. Uncontrolled fibrillary twitches, which were removed by Curare, arose as well. It is true that these observations suggest a cholinergic effect of F, since Curare blocks ACh from binding the receptor of the effected membrane. This does not, however, necessarily contradict Weddel's observations of the salivary glands, since the stimulatory processes of gland cells differ from those of the skeletal musculature (Stimulation at the motor endplate is preceded by a depolarization; at the gland cells a hyperpolarization precedes stimulation).

After long term exposure, high doses of F eventually led to a blockage of all stimulus conduction, which suggests a constant depolarization of nerve and muscle cells.It has long been known that fluoride can affect (Na+-K+) distribution at cell membranes.(20) This influence was first observed in red blood cells, which have a high intra-cellular K+ concentration together with a low Na+­ concentration. In serum, on the other hand, the relationship is reversed.

The unequal distribution of these ions, which is found in all bodily cells, can only be maintained with constant energy use, as expressed by the "Gibbs-Helmholtz" equation:

Since the membrane is permeable to the cations, the T•D S term is positive when the distribution is uneven. In the case of a quasi stationary equilibrium the change in free energy (D G) must equal 0. This means that T•D S must be counteracted by an equal, but opposite, D H, in this case the enthalpy change (D H = -7 Cal./Mol.) that results from the splitting of ATP in the membrane.

Fluoride can affect the (Na+-K+) distribution at cell membranes and, thereby for nerve cells, also the resting potential, in three ways:

  1. By inhibiting the enzymatic degradation of glucose, and therefore also ATP synthesis.
  2. By suppressing the splitting of ATP at the membrane, which normally provides the energy for the active transport of cations, by inhibiting the membrane bound (Na+- K+) activated ATPase.
  3. By fluoride directly affecting the permeability of the membrane for the aforementioned ions. This effect perhaps involves a reciprocal action by fluoride with the membrane proteins by changing their spatial conformation.

There is evidence to support all of these possibilities. O. WARBURG(21) has already reported on the inhibition of glycolysis by F-. He suggests that fluoride's effect is caused by an inhibition of enolase by a Mg-fluorophosphate complex. We discovered a decline in ATP formation in our own experiments at F- concentrations > 10-3 M.

L.J. OPIT(22) reports of an inhibition of the (Na+-K+) activated ATPase of kidney cells (guinea pig). According to OPIT, 4 x 10-3 M NaF inhibits the enzyme up to 50%. S. LEPKE and H. PASSOW(23) could determine a direct effect on the membrane in that they discovered K+ efflux in so called "Erythrocyte Ghosts" after action of 4 x 10-2 M NaF.

Which of these effects dominates in vivo has not been determined. The concentrations used here all lie above the physiological concentration. We must still investigate if similar effects can be observed at smaller F concentrations. We must also determine which effects become effective at which concentrations, when F- acts on the entire system. The possibilities for a fluoride effect, possibly also a selective effect on the salivary glands, are very complex. The possibilities can be divided into:

  1. an effect on the cholinergic system, that is, on synthesis, storage, release, and inactivation of ACh.
  2. an effect on the (Na+-K+) distribution and thereby on the resting potential of nerve cells, whereby the parasympathetically stimulated cells should be most sensitive.
  3. a direct effect on the processes at the salivary gland, perhaps through activation of the active ion transport, or through independent enlargement of the hyperpolarization during the stimulatory phase.

II. Presentation of the Problem

According to a theory of KNAPPWOST's(9), watery vagotonic saliva causes an increase in the rate of the natural processes that maintain the surface of teeth, known as remineralization. The saliva functions as a supersaturated solution of HA in this process. The level of supersaturation rises with the pH level, so that a watery and slightly alkaline saliva possesses the best reparative properties.

Numerous findings show that vagotonic symptoms can be observed after administration of fluoride.(10,11,13,15,17) We undertook the task of looking for a possible influence of fluoride ions on the tone of the vagus nerve by measuring the inhibition of AChE, and at the same time of studying the kinetics of this inhibition. We also felt it necessary to study the effect of fluoride complexes on the ACh-AChE system, due to their frequent occurrence in nature.

Since it could be assumed that fluoride can also trigger vagotonic effects indirectly, we were also interested in the effect of fluoride on the transport of ions and molecules through the cell membrane. Furthermore, radioactive tracing methods, with the help of which biochemical reactions and even entire chain reactions can be studied in a single procedure, were to be applied for this experiment. The pathway of ions and molecules in the body and at cell membranes can also be followed using this technique.


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