FLUORIDE 31(2), 1998, pp 96-99	International Society for Fluoride Research	Table of Contents

Objective
The questions being addressed involved the effects of aluminum fluoride and sodium fluoride on the brain. Until the etiology of Alzheimer’s dementia was better understood it was considered appropriate to investigate the possible contribution of aluminum to neurotoxicity. Following earlier experiments, a study was made of the effect of aluminum fluoride on the brain. After considering the results of this work a further experiment was carried out in which the effect on the brain of aluminum fluoride was again studied and, in addition, that of sodium fluoride.

Method
In the first experiment aluminum fluoride, AlF₃, was given in the drinking water, for 45 weeks, to three groups of rats in concentrations of 0.5 ppm, 5 ppm and 50 ppm (as Al³⁺) starting at about 4 months of age. A fourth control group received only distilled water for drinking. In the second experiment, in addition to having a control group, two groups of rats received 0.5 ppm aluminum fluoride or 2.1 ppm of sodium fluoride. Both the aluminum fluoride and sodium fluoride gave a fluoride level of about 1 ppm. Aluminum fluoride was of special interest due to its lipid solubility and ability to pass through hydrophobic membranes. After sacrifice, the brain aluminum content was measured and histological examination made including study of the cortex, hippocampus and brain blood vessels. Tissue staining was done by the Morin fluorescent stain method, for aluminum, hematoxylin and eosin staining, cresyl-violet staining and by using the Bielchowsky silver stain. An immunohistochemical method was used to test for the immunoglobulin IgM. Vascular inclusions were studied by X-ray pulsation diffraction patterns and scanning electronmicroscopy. The kidneys were examined including histological examination and measurement of aluminum content.

Results
In the first experiment a high mortality rate occurred in the animals receiving the lowest dose of aluminum fluoride: 0.5 ppm with 1 ppm of fluoride. Eighty percent of this group died before the end of the experiment at 45 weeks. Only a few animals died in other groups and the increased mortality in the 0.5 ppm aluminum fluoride group was significant. They also looked poorly prior to their deaths with thinner hair. Their exposed skin was bronzed, mottled or flaky. Their teeth and toe nails were excessively dark. The same high mortality in this group also occurred in the second experiment. When autopsies were performed on the bodies of the animals in the second experiment, a number of disease-related changes were found in the animals receiving aluminum fluoride or sodium fluoride including splenomegaly and mycoplasma pulmonis lung infections. The respiratory infection, which is common in rodents, was always much more virulent in the animals receiving the AlF₃ or the NaF. There appeared to be a general impairment in the immune capacities of the treated animals. The animals treated with aluminum fluoride were most affected.

In both experiments deaths occurred in the 0.5 ppm AlF₃ dose groups when the additional stress of a behavioral training regime was imposed. It was as if an already diminished immune capacity was unable to tolerate the stress induced by the behavioral manipulations.

In the first experiment, all the AlF₃-exposed groups had increased brain levels of aluminum. These were about double those found in the control animals. No dose-response relationship occurred. Despite a 100-fold difference in dose all three groups had the same level of aluminum in the brain.

In the second experiment it was found that the prolonged administration of low levels of sodium fluoride, 2.1 ppm, corresponding to 1 ppm of fluoride, also resulted in an elevated brain level of aluminum. The aluminum was considered to have originated in the food provided to the animals. In the first experiment, for the animals receiving AlF₃, histologically, there was a reduction in the number of cells in the hippocampus in areas CA1 and CA3 together with a disorganization of the hippocampal pyramidal cells. Changes were observed in both the phosphorylated and nonphosphorylated neurofilaments in the neocortex which are usually considered to be related to cell dysfunction. Aluminum was found in brain blood vessels frequently in large amounts. It was particularly prominent in areas where blood vessels were most dense such as the median eminence. It was largely confined to the interior of blood vessels both large and small. The obvious presence of these inclusions in the blood vessels was seen to cause reductions in cerebral blood flow and most certainly a reduction in aerobic metabolism.

In the second study the same Al-positive inclusions were found in the group receiving 0.5 ppm of AlF₃. A few of these were also found in the group receiving 2.1 ppm sodium fluoride together with other nonaluminum types of inclusions that reacted positively for the immunoglobulin IgM. The inclusions were seen have the effect of reducing the amount of blood which could flow through the vessels together with producing a turbulence in blood flow. The turbulence was considered likely to reduce the transport of oxygen, nutrients, and waste products across the vessel walls.

On scanning electronmicroscopy, the vascular inclusions, which were presumed from the staining to contain aluminum, had a rather spiky crystalline configuration resembling an inorganic crystal. The presumed IgM inclusions had a more complex shape typical of an organic crystal. On X-ray diffraction spectrum examination, the presumed Al inclusions showed a peak corresponding to aluminum. For the presumed IgM inclusions the diffraction spectrum showed a peak corresponding to a high level of sulfur. This was seen to be consistent with the paired sulfur bonds in the immunoglobulins. These analyses were seen to confirm what had been assumed on the basis of histological and immunohistologic procedures.

In both studies the low dose of AlF₃ produced the highest mortality rates, the greatest signs of poor health, and substantial neural aberrations. Higher doses produced fewer deaths, healthier animals, and few neurological faults. Since all the doses of the AlF₃ produced the same levels of Al in the brains, it was wondered if the difference in toxicity might be related to the difference in the amount of fluoride in the different dose levels.

In the second study, when rats were given the same amount of fluoride (2.1 ppm of sodium fluoride or 1 ppm of fluoride) in the form of sodium fluoride as was in the low dose of AlF₃ (0.5 ppm ionic aluminum with 1 ppm of fluoride) in the first study, no increase in mortality was found. The data were considered to indicate that as the doses of AlF₃ increased, the increasing amounts of fluoride must have acted to offset the devastating effects of the aluminum. It was presumed that a protective level of fluoride was achieved at the 5 and 50 ppm Al dose levels.

In the second study, the AlF₃-treated animals had more cortical cell loss and more cells with clear-cut structural anomalies than did the NaF-treated animals. The AlF₃-treated animals had cortical aberrancies in the upper layers, 2 and 3, and in the lower layers, 5 and 6. Cells in layer 4 were unaffected in all groups.

Neuronal abnormalities were observed in the sodium fluoride treated animals, however, especially in the deeper cell layers. Hemispheric differences were found in both the AlF₃ and NaF groups which were consistent in the groups. Cell losses were found in the hippocampal CA3 area and in the molecular layers of the dentate gyrus in the AlF₃-treated animals who also had plentiful deformations of the neurones of the CA1 and CA4 areas.

Both the AlF- and NaF-treated animals had substantial numbers of argentophilic cells on Bielchowsky staining and these cells showed condensed Nissl substance with hematoxylin and eosin staining. The NaF treatment also produced distortions of cells and, in some rats, cell losses could be demonstrated in particular brain regions. Both AlF₃ and NaF induced vascular inclusions, although of a different character: the AlF₃ produced the Al-based particles and the NaF produced the IgM inclusions. AlF₃ and NaF were seen to produce different reactions within the vascular tissue and the results of the first experiment were interpreted as being an interaction between aluminum and fluoride.

Pathological changes were found in the kidneys of animals in both the AlF₃ and NaF groups. Aluminum-containing deposits were found in the kidney blood vessels, and the renal aluminum content was doubled when the rats drank the AlF₃ water. The kidneys from rats drinking the NaF-treated water exhibited glomerular hypercellularity and mesangial proliferation together with patchy focal nephritis.

Discussion and Conclusions
It was unclear why the greatest impairments and mortality rates were found in the animals with the lowest level of added AlF₃, 0.5 ppm as Al³⁺. Why higher levels of AlF₃ produced less impairment, fewer deaths and generally healthier animals than the low levels remained a mystery. It is possible, based primarily on epidemiological reports, that fluoride, at certain low levels, may exert a protective effect against the aluminum when given at a certain absolute level. The general aluminum levels of brain tissue was the same in the treated groups, in both experiments, at a level about double that of the control groups. What differed in the higher AlF₃ groups was the amount of fluoride in the complexes. If the amount in the lowest level groups was insufficient to provide protection but above a protection threshold in the higher dose groups, then this could account for the findings.

The immune deficiencies found in the animals support the general thesis of dementia as a microvascular-immune disease beginning with attacks on the integrity of the capillary linings in the brain and other organs. This could be due to the mechanisms involved in the formation of the inclusions found in the vessels. Damage to the vessel linings could provide the locations for the deposits of aluminum-containing materials, immunoglobulin, cholesterol, albumin, collagen, or amyloid. The brain’s metabolic activity would be reduced hand in hand with the initiation of the innate immune response to foreign matter and the subsequent cascade of immunological responses. The combination of reduced aerobic metabolism and the immune reactions would act to enhance each other with the result being a progressive disease. A number of possible protective measures were seen to be suggested by the data:

1.  Protecting the blood vessel linings from attack. If free radicals were involved prevention might be possible with scavengers, vitamin E and drugs like allopurinol.

2.  Protection of the immune system from toxins and events that tend to reduce its efficiency. More attention might be paid towards discovering the interactions between the environmental factors and overall immune competencies including the effects of aluminum and other reactive metals.

3.  Reducing any further impediments to blood flow. Since the effect of AlF₃ is largely to reduce the effectiveness of transport of oxygen and nutrients across the capillary walls, as well as the efficient removal of waste materials, every effort should be made to reduce any further impediment to blood flow. Treatments that reduce hypertension which impairs blood flow would be helpful especially those active at the blood vessels themselves.

4.  Maintaining an effective cardiovascular system. Exercise and overall fitness might be encouraged.

5.  Understanding the reciprocal relationship between the use of nonsteroidal antiinflammatory drugs, other than aspirin, and dementia. Medication may have a role in more than just the early phase of the disease-related immune response in the brain.

6.  Understanding the role of fluoride in providing protection from certain types of toxins. Based on knowledge of this element’s own toxicity, it cannot be used without understanding the risks it imposes at various doses to different organs. The possible benefits from its use must be balanced with its potential harm. Nevertheless, there is evidence supporting a neuroprotective effect of fluoride in water supplies in regard to dementia from epidemiological studies whether or not aluminum is also in the water. If the protective mechanisms are understood, it may be possible to find other agents to provide them with less harmful side effects.

Key words: Aluminum-fluoride; Amyloid; Brain; Cerebrovasculature; Hippocampus; Neurotoxicity; Rat; Sodium fluoride.
Reprints: Robert L Isaacson, Department of Psychology,
Binghamton University, Binghamton, New York 13902-6000, USA. Fax: +1-607-777-4890.
E-mail: isaacson@binghamton.edu

FLUORIDE 31(2), 1998, pp 96-99	International Society for Fluoride Research
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