Boyd Haley PhD
Abstract: Mercury(II) or Hg2+, is neurotoxic and when exposed to normal brain tissue homogenates, is capable of causing many of the same biochemical aberrancies found in Alzheimer's diseased (AD) brain. Also, rats exposed to mercury vapor show some of these same aberrancies in their brain tissue. Specifically, the rapid inactivation of the brain thiol-sensitive enzymes tubulin, creatine kinase and glutamine synthetase occurs on the addition of low micromolar levels of Hg2+ or exposure to mercury vapor, and these same enzymes are significantly inhibited in AD brain. Further, extended Hg2+ exposure to neurons in culture has been shown to produce three of the widely accepted pathological diagnostic hallmarks of AD. These are elevated amyloid protein, hyper-phosphorylation of Tau, and formation of neurofibillary tangles. The hypothesis is that mercury and other blood-brain permeable toxicants that have enhanced specificity for thiol-sensitive enzymes are the etiological source of AD. Included in this category are other heavy metals such as lead and cadmium that act synergistically to enhance to toxicity of mercury and organic-mercury compounds, like thimerosal that is found in vaccines and other medicines. This hypothesis is also able to explain the genetic susceptibility to AD that is expressed through the APO-E gene family. Specifically, a reduction of APO-E gene types carrying cysteines decreases the ability to remove mercury and other thiol-reactive toxicants from the cerebrospinal fluid. This increases brain exposure to thiol-reactive toxicants and the risk of AD.
AD is a disease of unknown etiology. However, it is widely accepted that most AD is not directly genetically inherited and that some external vector, such as a toxicant exposure or an infection, must be involved for the disease to progress into a clinically observable condition. In the USA the rate of AD is very similar for rural versus urban peoples and it does not vary appreciably from state to state. Therefore, if a toxicant is involved then this toxicant must be of a very personal nature, like what we eat or what is placed into our bodies through other sources such as dental fillings, vaccines, etc.
The involvement of infectious agents such as bacteria, virus or yeasts; while possible at this time, seems not to be directly involved. This is based on the huge amount of National Institutes of Health (USA) and other world-wide funds spent on AD to identify the causal factors and they have not detected a consistent microbial vector. If an infectious agent were involved (like in AIDS and polio) it seems as if it would have been identified by now. However, focal infections caused by microbes in the oral cavity must still be considered as these microbes are known to produce toxicants such as hydrogen sulfide, methyl-mercaptan, gliatoxin and other compounds that inhibit thiol-sensitive enzymes.
For any toxicant, or class of toxicants, to be proposed as involved in the etiology of AD they must be available equally to individuals living in markedly different locations. The toxicant proposed must explain the genetic susceptibility concept of AD. Further, under experimental conditions the toxicants must be able to cause the exacerbation of the many biochemical aberrancies found in AD brain. Based on our research and a literature review, mercury and mercury containing compounds from dental amalgams, vaccines, other medicinals and preservatives used in paints, seed grains, etc. represent a class of compounds that fill this requirement.
Mercury and organic mercurials are neurotoxicants. Further, the enzyme inhibitory effects of mercury are synergistically enhanced by exposures to other toxicants such as lead and cadmium (smokers). Even the simultaneous presence of EDTA (ethylene-diamine-tetraacetic acid, a common food additive) or metal binding antibiotics such as tetracycline can enhance mercury toxicity. Therefore, any determination of a safe level of mercury exposure using rats in a cage being feed carefully monitored food and water is not reliable for determination of a "safe level of exposure to mercury" for humans. The fact is that science does not know what the combined toxic effects of many toxicants or enhancers of toxicity would be if present with mercury and therefore cannot identify a safe level of exposure.
Therefore, thiol-reactive toxicants such as mercury, cadmium, lead and certain organics are rational suggestions as being exacerbating factors for AD, or possibly even causal. However, mercury is the one toxicant that has been shown to reproduce many of the biochemical aberrancies and diagnostic hallmarks of AD. Also, mercury exposure is readily available to most humans. It is reasonable to propose that exposure to mercury is one of the major toxic factors involved in early onset AD. Further, that simultaneous exposures to other toxicants or factors enhance the toxicity of mercury and hasten the onset of AD, especially in those individuals who are genetically susceptible.
Enzyme Inhibition and Protein Partitioning Results.
Research regarding Alzheimer's disease (AD) done in our laboratory in the late 1980s was directed towards detecting aberrancies in the nucleotide binding proteins of AD post-mortem brain tissue versus age-matched, non-demented control brain samples. Basic to all of our findings was the following observation. Two very important brain nucleotide binding proteins, tubulin and creatine kinase (CK), showed greatly diminished activity and nucleotide binding ability. Further, they were abnormally partitioned into the particulate fraction versus the soluble fraction of AD brain tissue by simple centrifugation (1,2).
Both tubulin and CK are proteins that bind the nucleotides GTP (guanosine-5'-triphosphate) and ATP (adenosine-5'-triphosphate), respectively. We use a "photoaffinity labeling" technology to determine the availability of these binding sites before and after addition of mercury or other toxicants (21). This technology is explained in detail at www.altcorp.com for those interested in the detailed chemistry. Using this technology our laboratory has demonstrated that both tubulin and CK had diminished biological activity in AD brain compared to age-matched controls. Since AD is not directly a genetically inherited disease we searched for possible toxicants that might mimic the specific findings observed in AD brain.
Our first finding was simple and straight-forward. After testing numerous heavy metals we observed that only Hg2+ could mimic the AD effect in homogenates of normal brain at concentrations that might be expected to be found in brain (3,4). The observation was that Hg2+ at very low micromolar levels (@ 1 micromolar) could rapidly and selectively abolish the GTP binding activity of tubulin (Mr = 55,000 daltons) without any noticeable effect on the other GTP binding proteins protein(s) observed at an Mr of about 42,000 daltons, that are present in both control and AD brain at approximately equal levels. Therefore, concerning heavy metals the addition of only mercury at low micromolar levels to control brain homogenates gave a GTP binding profile that was identical to that observed in AD brain and that chelation of Hg2+ by EDTA did not prevent but enhanced this effect (4,5,6). Further, additional results have shown that the addition of Hg2+ to control brain homogenates not only caused the decrease in nucleotide interaction but could also support the abnormal partitioning of tubulin into the particulate fraction as observed in AD brain (7). This was especially effective in the presence of other divalent metals, such as zinc, which is elevated in AD brain. The recent video demonstrating Hg2+ specific stripping the tubulin from the neurofibrils shows the tubulin abnormally aggregating at the base of the neuron, supporting the partitioning we observed in brain homogenates (http://movies.commons.ucalgary.ca/mercury).
It is critical to understand that both tubulin and CK in normal brain are found primarily in the soluble fraction of a homogenate. Yet, both proteins appear of normal size and unmodified on reducing polyacrylamide gel electrophoretic analysis (PAGE). This indicates that both intact tubulin and CK have formed crosslinks with other proteins that are insoluble under physiological conditions. Yet, these crosslinks are readily disrupted by the common dithiolthreitol (DTT) reduction procedure used before PAGE. What tubulin and CK have in common is that both have a very reactive sulfhydryl in their nucleotide binding sites that, if modified, inhibits their biological activity (14, 15).
Mercury has a very high affinity for sulfhydryls and has been proven to be a potent inhibitor of the biological activity of both of these proteins. Also, mercury is divalent and can form crosslinks between soluble proteins like tubulin and CK and is known to cause protein aggregation. A generalized single step reaction would be as given in reaction 1.
1: Protein-A-SH + Protein-B-SH + Hg2+ Þ Protein-A-S-Hg-S-Protein-B + 2 H+
This chemistry would allow the formation of aggregates that would abnormally appear in the particulate fraction. Due to its dithiol structure DTT is an excellent chelator of mercury. The massive amounts of DTT used in reducing gels could chelate and remove mercury from the proteins resulting in their becoming soluble again and migrating as unmodified on gel electrophoresis as observed as shown in reaction 2.
2. Protein-A-S-Hg-S-Protein-B + DTT Þ Protein-A-SH + Protein-B-SH + DTT-Hg
The correct criticism of any homogenate test is that it may not occur in a living animal. Therefore, experiments were done to determine if mercury vapor, the primary form that escapes from dental amalgams, could mimic the effect in rats exposed to such vapor for various periods of time (5). Rats are different from humans in that they can synthesize vitamin C whereas humans have to ingest vitamin C. Vitamin C is thought to be somewhat protective against heavy metal toxicity and other oxidative stresses. However, we observed that the tubulin in the brains of rats exposed to mercury vapor lost between 41 and 75 percent of the nucleotide binding capability demonstrating a similarity to the aberrancy observed in AD brain and confirming the homogenate results (5).
There is also an "excito-toxic" amino acid hypothesis for the cause of AD wherein excito-toxic amino acid glutmate builds up in brain tissue causing neuronal death. This is a reasonable hypothesis and could co-exist with the thiol-sensitive enzyme/mercury hypothesis. The activity of Hg2+ sensitive glutamine synthetase (GS) was measured in AD brain and the amount of GS in the cerebrospinal fluid of AD versus control patients was determined. GS was found it to be inhibited in AD brain and copies of GS were elevated in the cerebrospinal fluid (12, 22). It has also been predicted by two groups that the elevation of GS in the cerebrospinal fluid of AD patients has potential as a diagnostic aid for AD (12,16). However, it is reasonable to conclude that brain GS would be rapidly inhibited by Hg2+ produced by oxidation of mercury vapor. This inhibition would cause a rise in glutamate based excito-toxicity and could cause neuron death. Further, glutamate is transported by molecular motors down the microtubules that are destroyed by Hg2+. Therefore, both the metabolism and transport of glutamate would be immediately affected by exposure to mercury. The measurement of GS in cerebrospinal fluid is most likely a measure of glial cell toxicity and death as would be expected in several central nervous system diseases, including AD.
Illnesses that lower our metabolic energy levels also lower our ability to synthesize the reducing equivalents that allow our body to bind and dispose of excess mercury. Hg2+ is known to inhibit the metabolic processes in mitochondria that produce ATP and NADH by inhibiting the enzymes of the citric acid cycle and the electron transport system. These nucleotides are absolutely required for both the synthesis of reduced glutathione (GSH) and to reduce glutathione after it is oxidized. GSH in the reduced state is the major biomolecule involved in the natural removal of mercury from the body. Therefore, as mercury slowly accumulates in the body it weakens the body's natural defense against all forms of other heavy metal toxicities and increases the overall oxidative stress expressed by reactive oxygen species formation. It is well known that AD brain tissue suffers from greater oxidative stress in all cellular components versus similar tissues from control subjects. This would be expected and it is well documented that mercury increases oxidative stress in biological tissues. Further, Hg2+ is well known to inhibit numerous other enzymes important to neurological function, including the Na/K ATPase that is necessary for recovery from a nerve-action potential. Therefore, the many numerous aberrancies observed in AD brain would be expected within a hypothesis that proposes exposure to Hg2+ is a major contributor to this disease.
Mercury from Dental Amalgams;
The fact that mercury has inhibitory effects on tubulin, CK and GS and that these proteins are proven to be aberrantly inhibited in AD does not alone conclusively prove that mercury exposure causes AD. However, it definitely proves that chronic, daily exposure to mercury would at least exacerbate the clinical conditions of AD. Is such an exposure to mercury likely? The answer is yes, and this makes mercury involvement in AD plausible.
First, the question must be addressed if there is enough mercury in an amalgam filling to continue a low chronic level exposure for years? The answer is yes. For example, if a single large amalgam filling contained 1 gram of mercury (1 million micrograms) and lost a significantly toxic 10 micrograms per day there would be enough mercury for 100,000 days or about 274 years of exposure. A small tenth of a gram mercury filling would last 27 years. So enough mercury is within amalgam fillings to provide a consistent chronic toxic exposure for the life of most fillings.
Second, does mercury emit from amalgams at a rate that should cause concern? The answer is yes. Dental amalgams, or "silver fillings" as organized dentistry calls them, are approximately 50% mercury by weight and it is quite easy to demonstrate that mercury vapors readily emit from these fillings. The actual amount can only be determined with the amalgam in a closed container and the amount of mercury released being determined using solid, time proven chemical techniques and instrumentation. The accurate level of mercury released cannot be accomplished on amalgams in the mouth. In a carefully designed study in a sealed container Chew et al. tested the "long term dissolution of mercury from a non-mercury-releasing amalgam (trade name Composil)" (9). Their results demonstrated "that the overall mean release of mercury was 43.5 +/-3.2 micrograms/cm2/24hr, and the amount of mercury released remained fairly constant during the duration of the experiment (2 years)".
In my opinion, this 43.5 micrograms/cm2/day is not an insignificant amount of mercury exposure if one considers the number of years a 70 year old individual living today may have been exposed to chronic mercury levels from his amalgams. Additionally, 43.5 micrograms/cm2/day is the level released without galvanism, excess heat, or pressure from chewing, all factors that increase mercury release from amalgams in the mouth (26).
Some may disagree with the figure presented above and indeed, amalgams of different manufacture may release more or less. However, the pro-amalgam supporters have not published any carefully controlled study similar to the one above repudiating the finds of this research group. They definitely have all of the scientific laboratory expertise needed to do this. Instead, they utilize "estimates" of release based on urine and blood levels that are widely known to vary dramatically with time and not to be reliable. In judging science one looks for what is not published that obviously should have been.
There have been numerous published reports of increased tissue mercury levels in subjects and the relationship to increased number of amalgams fillings (see 10, 11, 25 and references therein). Also, the World Health Organization Scientific Panel found ranges of mercury exposures from 3 to 70 micrograms/day with the bulk being from amalgam fillings (31). Data relevant to this question was addressed by a recent NIH study using 1,127 military personnel (20). Soldiers in this study had an average of 20 amalgam surfaces with ranges from 0 to 66 surfaces. Each 10 surfaces increased the urine mercury level 1microgram/liter or an average of 4.5 micrograms/day. This study indicated that individuals with an average number of amalgam fillings had about 4.5 times the urine mercury levels as controls without amalgams. Those soldiers with over 49 surfaces averaged over 8 times the urine level observed in the non-amalgam controls. Further, the blood and urine mercury levels corresponded well with the number of amalgam fillings (20). The results above are consistent with an earlier study where urinary mercury levels dropped by a factor of 5 after the removal of several amalgam fillings. The conclusion of the authors was that mercury from dental amalgams exceeds that from all forms of food, air and fluids (23). All of the data on urine or blood mercury levels must be considered with the knowledge that approximately 80% of inhaled mercury vapor is retained in the body. Mercury typifies a "retention" toxicity and much of the mercury taken into the body is absorbed by the solid tissues. The amount in urine represents mercury being excreted. However, the main question is how much is being retained in the different body tissues.
In contrast to other reports there was published in the J. American Dental Association research that measured mercury levels in brain and other neurological tissues and concluded "Our results do not support the hypothesis that dental amalgam is a major contributor to brain Hg levels. They also do not support the hypothesis that Hg is a pathogenetic factor in AD (25)." I can't explain how amalgams can increase blood mercury levels and not increase brain mercury levels. However, these researchers presented data showing no significant increase in Hg level in several brain regions between control and AD subjects. They surprisingly included data showing that the Hg levels in control olfactory region was more than double that of the corresponding AD olfactory tissue. This olfactory mercury increase in control subjects could have several explanations.
One explanation could be they were not precise in estimating the amount of mercury exposures of their subjects and the controls they selected were much more exposed to mercury than the AD subjects selected. The olfactory region is outside the blood-brain barrier and should be a consistent internal standard for mercury exposure in the air breathed in by the subjects.
Another explanation would be that the controls, even though exposed to more than double the mercury levels of the AD subjects, as evidenced by the olfactory region Hg levels, had a mechanism that protected their brain tissues from also having double the mercury levels. If this were true, then dividing the brain mercury levels by the olfactory mercury levels would give results that clearly show a significant ability of the controls to have a mechanism that protects brain tissue from mercury that is lacking in the AD subjects. This mechanism could be the presence of the protective APO-E protein genotypes (see below) and other predisposition factors not yet known.
The debate continues on whether or not human mercury exposures reach levels in the brain and other tissues that could be considered toxic or harmful (24,25). There are several reasons why the brain levels of mercury would not directly correlate to the damage being done. The level of selenium in the diet, which could bind with mercury rendering it less toxic, is the most straight-forward example. Also, the determination of the levels of mercury toxicity that could cause neurological disease has been done using animals, such as rats and monkeys, under tightly controlled laboratory conditions where the diet is carefully monitored to exclude other toxicants. Further, any test animal that becomes ill or infected by microbial sources is removed from the study. However, humans do not live under such restricted conditions. For example, we are exposed to numerous infections and additional heavy metal imbalances in AD brains have been reported numerous times. Cigarette smokers are exposed to excess cadmium (Cd2+) and lead (Pb2+) toxicity is not that uncommon in the inter-city environment or for those exposed to leaded gasoline fumes for many years. This means that the synergistic toxicities of combined heavy metals must be considered for humans.
It is also questionable whether or not brain mercury levels should be expected to remain high in AD brain. A report by Hock et al. (27) stated that in early onset AD the blood levels of mercury were almost three fold higher than the control groups and that these increases were unrelated to the patients' dental status. The concluded that the explanation of increased mercury in AD would include yet unidentified environmental sources or release from the brain tissue with the advance in neuronal death. The AD brain loses 25% of its average weight by time of death making the latter explanation reasonable. It is a well-known biochemical event that cells or tissues rid themselves of denatured, unusable protein.
The inhibition and break down of neuronal tissue may also explain another observation related to AD. It is documented that AD patients have elevated olfactory thresholds and impaired odor identification. It is further suggested that in patients with mild cognitive impairment, olfactory problems may have clinical value as an early diagnostic predictor for diagnosis of AD(28, 29, 30). Mercury in the oral cavity must interact with the olfactory bulb. Due to the neurotoxicity of mercury, this could impair olfactory sensitivity. Also, based on our hypothesis impaired olfactory response would almost have to occur.
Our laboratory has shown that one can add various metals to human brain homogenates to levels that alone do not affect nucleotide binding to tubulin, yet the very presence of these metals synergistically increases the toxicity of Hg2+. That is, the presence of Pb2+, Zn2+ and Cd2+, at non-toxic levels, decrease the amount of Hg2+ required for 50% inhibition of tubulin or creatine kinase viability. It is important to remember the "Periodic Chart of the Elements" which places Zn, Cd and Hg in the same IIB category and all have high affinity for thiol groups. In other words, mercury is much more toxic in the presence of other metals that compete with mercury for the binding sites on protective biomolecules (e.g., APO-E2 & E3, glutathione or GSH, metallo-thionine, etc.).
It is also important to note that the "test tube levels" of mercury are not representative of what would happen in a dynamic system where a constant level of mercury is being supplied by the amalgams. Since mercury toxicity is a "retention toxicity" all mercury pulled from the system, or retained by the tissue, is replaced by more mercury being constantly released from the amalgams and the Hg2+ level and toxicity in solution remains constant. In the test tube as the mercury is pulled out of solution the free Hg2+ concentration in solution drops making the soluble aspect less toxic with time.
To propose deleterious effects of amalgams while in the mouth the amalgams must be able to produce toxic effects outside of the mouth. Wataha et al. reported that extracts of the amalgam material (trade name, Dispersalloy) "was severely cytotoxic when Zn release was greatest, but less toxic between 48 and 72 hours as Zn release decreased" (8). Zn is a trace material in dental amalgams and a needed supplement for living neurons. Therefore, it did not seem likely that the toxicity was due to Zn emitting from the amalgams. When we compare the toxicity of Hg2+ in brain homogenates as described above (refs. 3 & 4), the addition of 0, 10 and 20 micromolar Zn2+ increased the inhibition of GTP binding to tubulin from 4% to 50% and 76%, respectively (7,13). This supports the concept that the Zn correlation to increased toxicity was due to the synergistically enhanced toxicity of the mercury released from the amalgam. Further,other studies in our laboratory have shown that soaking of amalgams in distilled water for less than one hour created a solution that also caused rapid inhibition of brain tubulin and creatine kinase similar to that observed on adding Hg2+ solutions. Therefore, it appears that the toxicity of solutions in which amalgams were soaked is not caused by direct Zn2+ toxic effects. Rather, enhanced toxicity is due to the Zn2+ or other amalgam heavy metals stimulating the toxicity of mercury by occupying biomolecule chelation sites. This would result in a higher concentration of free Hg2+ capable of inhibiting the activity of critical nucleotide binding proteins such as tubulin and CK.
The observed synergistic toxicity of other heavy metals with Hg2+ has been supported in animal models. Combining an LD-1 solution of Pb2+ with an LD-1 solution of Hg2+ gave a solution with an LD of 100, instead of an LD-2, when injected into rats (19). The bottom line is that mercury toxicity is enhanced by the presence of other heavy metals. Therefore, when one considers the toxicity of a certain body level of mercury it is somewhat meaningless unless the body level of other heavy metals is also considered.
With the complexity of our environment and the confounding factors involving neurological diseases, and without major government supported epidemiological studies proving safety, it is impossible to state with assurance, as many amalgams supporters do, that this exposure does not place the individuals at greater health risk. The "lack of proof of damage" from mercury exposure seems unwarranted to be used as "proving the safety of any material" that unnecessarily exposes individuals daily to several micrograms of mercury.
Any hypothesis of the etiology of AD must consider information on genetic susceptibility. The best known genetic risk factor for AD is the correlation of APO-E genotypes to the age of onset of AD (24a,b). Individuals can inherit any combination of the alleles APO-E2, E3 or E4. Individuals inheriting APO-E2 or combinations of APO-E2 and E3 are much less likely to get early onset AD than are individuals who have inherited APO-E4 genes. Also, APO-E2 appears to be more protective than APO-E3 against early onset AD. Therefore, it is necessary that the mechanism of mercury toxicity contain an explainable relationship for the APO-E genetic susceptibility. This is accomplished in a straight-forward manner by considering the basic structural difference between these three alleles. Simply put, the protective APO-E2 has two sulfhydryls (cysteines) that can bind mercury or other heavy metals that APO-E4 lacks. For example, in APO-E3, one of APO-E2 cysteines is replaced by an arginine and in APO-E4, both of the APO-E2 cysteines are replaced by arginines (32). Therefore, lack of protection against early onset AD was proposed to follow the loss of mercury binding sulfhydryls from APO-E proteins (6).
The protection provided by APO-E2 is reasonable when considering the nature and biochemical assignment of APO-E proteins. APO-E proteins are involved in cholesterol transport and all three alleles do this reasonably well. However, APO-E is classified as a "housekeeping protein". That is, in contrast to tubulin, GS and CK, which are meant to stay inside of cells where they are synthesized, APO-E is meant to leave the brain cells carrying damaged cholesterol through the cerebrol spinal fluid (CSF), across the blood-brain barrier into the blood where it is removed by the liver. It fits into the hypothesis that while APO-E2 or E3 are leaving the brain cells and traversing the CSF they likely bind and remove mercury, other heavy metals or other sulfhydryl reactive toxins that may have made it into the central nervous system thereby protecting the brain neurons (6). APO-E4 cannot as effectively bind mercury and therefore does not provide the protective parameters that APO-E2 and E3 have. It is interesting to note that the second highest level of APO-E protein in the body is in the CSF that bathes and protects the brain.
Many recent literature and popular press reports state that the presence of periodontal disease raises the risk factor or exacerbates the condition of several other seemingly unrelated diseases such as stroke, low birth weight babies, cardiovascular disease (See October 1996 issue of Periodontology). The anerobic bacteria of periodontal disease produce hydrogen sulfide (H2S) and methyl thiol (CH3SH) from cysteine and methionine, respectively. This accounts for the "bad breath" many individuals have.
However, in a mouth that produces H2S, CH3SH (from periodontal disease) and Hgo (from amalgam fillings) the very likely production of their reaction products, HgS (mercury sulfide), CH3S-Hg-Cl (methyl-thiol mercury chloride) and CH3S-Hg-S-CH3 (Dimethylthiol mercury) has to occur. This is simple, straight-forward chemistry whose occurrence is supported by easily observable "amalgam tattoos". These tattoos are purple gum tissue surrounding certain teeth where the gum and tooth meet and primarily caused by HgS as determined by elemental analysis of such tissue.
HgS is one of the most stable forms of mercury compounds and is the mineral form found in ore, called cinnabar, from which mercury is mined from the earth. All of these oral site produced compounds are classified as extremely toxic and the latter compound, dimethylthiol-mercury is very hydrophobic and its solubility would be similar to dimethyl-mercury (CH3-Hg-CH3). Dimethyl-mercury was the compound that was made famous in the press where only a small amount spilled on the latex gloves of a Dartmouth University chemistry professor caused severe neurological problems and finally death 10 months later. In my opinion, the extreme lethality of CH3-Hg-CH3 compared to other forms of mercury is due to its ability to collect in hydrophobic regions of the body, like the central nervous system. CH3-Hg-CH3 is similar to CH3-S-Hg-S-CH3 in its hydrophobic characteristics.
Logic implies that anyone with periodontal disease, anaerobic bacterial infected teeth and mercury containing fillings would be exposed daily to these very toxic compounds. In our laboratory we synthesized the two methylthiol-mercury compounds and tested them. They are extremely cytotoxic at 1 micromolar or less levels and are potent, irreversible inhibitors of a number of important mammalian enzymes, including tubulin and CK.
A recent report stated that the tissues of individuals who died of Idiopathic Dilated Cardiomyopathy (IDCM) had mercury levels of 178,400 ng/g tissue or 22,000 times more than their controls who died of other forms of heart disease. IDCM is a disease where young athletes drop dead during strenuous exercise. It seems impossible for a tissue to bind this much mercury on protein without early notice of injury through pain and lack of bioenergy. However, if this mercury were to combine with H2S produced by a local anerobic infection the mercury could precipitate out in the tissue as HgS as it does in "amalgam tattoos" causing a buildup without killing the tissue immediately. However, one has to ask where does this excess mercury come from. Many times this occurs to young intercity athletes who are not on a high seafood diet. My opinion is that dental amalgam is the source of this mercury. Also, if HgS is being made in the heart tissue the very cytotoxic CH3-S-HgX and CH3-S-Hg-S-CH3 are also being made.
To determine if toxic teeth could have an effect on the enzymes/proteins of human brain we have done the following study. Several very toxic teeth were incubated for 1 hour in distilled water. Aliquots of these solutions were then added to control human brain homogenates and the resulting samples tested for tubulin viability and partitioning. The results showed that about 40% inhibited the viability of tubulin and caused partitioning. Therefore, depending on the type of anerobic microbial infection existing in avital teeth it is possible to have a toxicant production that would exacerbate the condition classified as AD. It is also probable that many of these teeth were extracted from mouths containing amalgam and the toxins in these teeth may also consist partially of extremely organic-mercury compounds as described above.
Based on the potential clearance represented by elevated blood levels of mercury in early onset AD patients, the synergistic effects of other heavy metals, the fluctuating GSH levels during illness and aging, and dietary factors (e.g. selenium levels) there is no reason to believe that the adverse effects of mercury from amalgams would be dose dependent in any straight-forward manner in post-mortem AD brain. To expect this would fly in the face of published data and scientific logic. Further, to eliminate mercury as a factor in AD based on statistically insignificant increases above normal in post-mortem brain samples is not warranted. Also, involvement of genetic factors likely plays a key role.
A recent publication supports our contention that mercury from dental amalgams poses a major threat to the exacerbation of AD. Olivieri et al. demonstrated that exposure of neuroblastoma cells to sub-lethal doses (36 X 10-9 molar) of Hg2+ caused a rapid drop in GSH, an increased secretion of b–amyloid protein and an increased phosphorylation of the microtubulin protein Tau (17). The latter two of these biochemical changes are uniquely observed in AD brain tissues and are widely considered to be diagnostic, pathological markers of the disease. b-amyloid protein makes up the 'amyloid plaques' that was one of the first diagnostic markers reported for AD brain pathology. A very strong component of AD researchers believe that amyloid protein is the cause of AD. Therefore, mercury exposure at nanomolar levels causes neuroblastoma cells to produce a protein that is believed to be involved directly in AD. This lead the authors of this paper to conclude that mercury would have to be consider as causal for AD (17).
Further, the recent report of the response of neurons in culture rapidly forming neurofibillary tangles on exposure to extremely low levels of mercury, by a process involving loss of microtublin structure, completes the picture that mercury is capable of causing the formation of three widely accepted major pathological diagnostic hallmarks of AD in neuronal cultures (18). An impressive video accompanying this publication and available at http://movies.commons.ucalgary.ca/mercury shows the addition of 2 microliters of 10-7M mercury to a 2 milliliter solution bathing neurons caused a rapid stripping of the tubulin from the neurofibrils leaving them bare. This would be predictable from our earlier data showing mercury interfering with normal tubulin-GTP interactions and the abnormal partitioning of tubulin into the particulate fraction of brain tissue(3,4,6). The bare neurofibrils then aggregate forming neurofibrillary tangles (NFTs) similar to those observed in AD brain. The final mercury concentration of 10-10M in these experiments is roughly 100 to 1000 times lower than the 10-7M levels normally found in human brain of individuals with amalgam fillings. The majority of the mercury in brain is likely bound by protective compounds like GSH or selenium and not free to cause neuronal damage. However, it is not unreasonable to consider that some of this mercury is present as free Hg2+ some fraction of the time, especially when illness or other toxicities lower the GSH levels.
However, these two recent publications supports the initial contention that mercury first rapidly inhibits thiol-sensitive enzymes like tubulin, creatine kinase and glutamine synthetase and dramatically affects metabolism and membrane structure. The stripping of tubulin leads to the formation of NFTs and the exposing Tau for hyper-phosphorylation. This is followed by elevated production of b –amyloid protein that can aggregate into senile plaques. all diagnostic markers for AD. It is consistent with the mercury toxicity hypothesis for AD that neurofibillary tangles, hyper-phosphorylated Tau, amyloid plaques and increased oxidative stress observations are the result of neuronal toxicity and death in AD, they are not the cause. The cause is exposure to environmental toxicants like mercury that attack enzymes with the most reactive thiol groups.
The data on the effects of mercury on the nucleotide binding properties and the abnormal partitioning of two very important brain nucleotide binding proteins proven to be aberrant in AD brain first suggested that mercury must be considered as an exacerbating factor to the condition classified as AD. This has been strongly supported by the recent finds that nanomolar levels of mercury causes neuroblastoma cells to secrete b-amyloid protein and increase phosphorylation of the microtubulin associated protein Tau, both major biochemical observations related to AD. Also, neurons in culture exposed to Hg2+ at the 10-7 to 10-10 M levels have conclusively been visually shown to rapidly produce abnormal tubulin aggregation, resulting in particulate partitioning as observed in AD brain. Also, this stripping of tubulin from the neurofibrils results in the formation of NFTs that are indistinguishable from those observed in AD brain. and used as a diagnostic marker of the disease(18). These facts alone warrant serious consideration of mercury as a certain exacerbating factor for AD, if not causal.
Consideration of mercury as a causal or exacerbating factor for AD is especially relevant when mercury is present in combination with other heavy metals such as zinc (Zn) cadmium (Cd) and lead (Pb). Synergistic toxicity is not an exception but is observed as a general rule (19). This obviates the argument that mercury must be significantly elevated in AD brains to be considered causal or contributing to the disease state. Further, the reaction of oral mercury from amalgams with toxic thiols produced by periodontal disease bacteria very likely enhances the toxicity of the mercury being released. Humans are likely the only mammals with amalgam fillings and periodontal disease. Bluntly, the determination of safe body levels of mercury by using animal data where the animals have not been exposed to other heavy metals is not scientifically justifiable. Mercury is much more toxic to individuals with other heavy metal exposures. It is my opinion that one of the major unanswered questions concerning the toxic effects of mercury is "does the combination of mercury with different heavy metals lead to different clinical observations of toxicity?"
Finally, mercury biochemically mimics numerous observations seen in AD brain tissues including inducing the formation of widely accepted diagnostic hallmarks of the disease. Further, the synergistically toxicity of mercury with other heavy metals, microbial produced oral toxins and certain metal chelators is obvious. It is also a scientific fact that amalgams contribute greatly to overall mercury body burden and are capable of producing cytotoxic solutions with properties like mercury solutions. Therefore, it seems very reasonable to consider a hypothesis that mercury would be the major contributor to early onset AD.
1. Khatoon, S., Campbell, S.R., Haley, B.E. and Slevin, J.T. Aberrant GTP b-Tubulin Interaction in Alzheimer's Disease. Annals of Neurology 26, 210-215 (1989).
2. David, S., Shoemaker, M., and Haley, B. Abnormal Properties of Creatine kinase in Alzheimer's Disease Brain: Correlation of Reduced Enzyme Activity and Active Site Photolabeling with Aberrant Cytosol-Membrane Partitioning. Molecular Brain Research 54, 276-287 (1998).
3. Duhr, E.F., Pendergrass, J. C., Slevin, J.T., and Haley, B. HgEDTA Complex Inhibits GTP Interactions With The E-Site of Brain b-Tubulin Toxicology and Applied Pharmacology 122, 273-288 (1993).
4. Pendergrass, J.C. and Haley, B.E. Mercury-EDTA Complex Specifically Blocks Brain b-Tubulin-GTP Interactions: Similarity to Observations in Alzheimer"s Disease. pp98-105 in Status Quo and Perspective of Amalgam and Other Dental Materials (International Symposium Proceedings ed. by L. T. Friberg and G. N. Schrauzer) Georg Thieme Verlag, Stuttgart-New York (1995).
5. Pendergrass, J. C., Haley, B.E., Vimy, M. J., Winfield, S.A. and Lorscheider, F.L. Mercury Vapor Inhalation Inhibits Binding of GTP to Tubulin in Rat Brain: Similarity to a Molecular Lesion in Alzheimer's Disease Brain. Neurotoxicology 18(2), 315-324 (1997).
6. Pendergrass, J.C. and Haley, B.E. Inhibition of Brain Tubulin-Guanosine 5'-Triphosphate Interactions by Mercury: Similarity to Observations in Alzheimer's Diseased Brain. In Metal Ions in Biological Systems V34, pp 461-478. Mercury and Its Effects on Environment and Biology, Chapter 16. Edited by H. Sigel and A. Sigel. Marcel Dekker, Inc. 270 Madison Ave., N.Y., N.Y. 10016 (1996).
7. Pendergrass, J.C., David, S. and Haley, B. Aberrant GTP-Tubulin Interactions and Aberrant –Tubulin Partitioning in Alzheimer's Disease Brain are Induced In Vitro by Micromolar Mercury, Zinc and other Sulfhydryl Reactive Heavy Metals. (in preparation 1998).
8. Wataha, J. C., Nakajima, H., Hanks, C. T., and Okabe, T. Correlation of Cytotoxicity with Element Release from Mercury and Gallium-based Dental Alloys in vitro. Dental Materials 10(5) 298-303, Sept. (1994)
9. Chew, C. L., Soh, G., Lee, A. S. and Yeoh, T. S. Long-term Dissolution of Mercury from a Non-Mercury-Releasing Amalgam. Clinical Preventive Dentistry 13(3): 5-7, May-June (1991).
10. Thompson, C. M., Markesbery, W.R., Ehmann, W.D., Mao, Y-X, and Vance, D.E. Regional Brain Trace-Element Studies in Alzheimer's Disease. Neurotoxicology 9, 1-8 (1988).
11. Deibel, M. A., Ehmann, W.D., and Markesbery, W. R. Copper, Iron and Zinc Imbalances in Severely Degenerated Brain Regions in Alzheimer's Disease: Possible Relation to Oxidative Stress. J. Neurol. Sci. 143, 137-142 (1996).
12. Gunnersen, D.J. and Haley, B. Detection of Glutamine Synthetase in the Cerebrospinal Fluid of Alzheimer's Diseased Patients: A Potential Diagnostic Biochemical Maker. Proc. Natl. Acad. Sci. USA, 88, 11949-11953 (1992).
13. Pendergrass, J. C., Cornett, C.R., David, S. and Haley, B. Mercury and Zinc Levels in Frontal Pole and Hippocampus of Alzheimer's Disease Brain: Relationship to Abberant GTP-b-Tubulin Interactions. Submitted to Neurotoxicology (1998).
14. Jayaram, B. and Haley, B. Identification of Peptides Within the Base Binding Domains of the GTP and ATP Specific Binding Sites of Tubulin. J. Biol. Chem. 269 (5) 3233-3242 (1994).
15. Olcott, M. and Haley, B. Identification of Two Peptides From the ATP-Binding Domain of Creatine Kinase. Biochemistry, 33, 11935-11941 (1994).
16. Tumani, H., Shen, G-Q., Peter, J. and Bruck, W. Glutamine Synthetase in Cerebrospinal Fluid, Serum and Brain: A Diagnostic Marker for Alzheimer Disease? Arch. Neurol. 56, 12411246, 1999.
17. Olivieri, G., Brack, Ch., Muller-Spahn, F., Stahelin, H.B., Herrmann, M., Renard, P; Brockhaus, M. and Hock, C. Mercury Induces Cell Cytotoxicity and Oxidative Stress and Increases b-amyloid Secretion and Tau Phosphorylation in SHSY5Y Neuroblastoma Cells. J. Neurochemistry 74, 231-231, 2000.
18. Leong, CCW, Syed, N.I., and Lorscheider, F.L. Retrograde Degeneration of Neurite Membrane Structural Integrity and Formation of Neruofibillary Tangles at Nerve Growth Cones Following In Vitro Exposure to Mercury. NeuroReports 12 (4):733-737, 2001.
19. Schubert, J., Riley, E.J. and Tyler, S.A., Combined Effects in Toxicology—A Rapid Systemic Testing Procedure: Cadmium, Mercury and Lead. J. of Toxicology and Environmental Health v4;763-776, 1978.
20. Kingman, A., Albertini, T. and Brown, L.J. Mercury Concentrations in Urine and Whole Blood Associated with Amalgam Exposure in a US Military Population. J. of Dental Research v77(3): 461-471, 1998.
21. B. E. Haley, "Development and Utilization of 8-Azidopurine Nucleotide Photoaffinity Probes," Federation Proceedings, 42, 2831-2836 (1983).
22. K. Hensley, P. Cole, R. Subramaniam, M. Aksenov, M. Aksenova, P. M. Bummer, B. E. Haley, J. M. Carney and D. A. Butterfield, , "Oxidatively-Induced Structural Alteration of Glutamine Synthetase Assessed by Analysis of Spin Labeled Incorporation Kinetics: Relevance to Alzheimer's Disease," J. Neurochem., 68, 2451-2457 (1997).
23. Begerow, J., Zander, D., Freier, I. And Dunemann, L. Long-term Mercury Excretion in Urine after Removal of Amalgam Fillings. Int. Arch. Occup. Environ. Health v66 (3), 209-212, 1994.
24. (a)Roses, A.D. Scientific American. Science and Medicine. 16-25, 1995. (b)Roses, A.D. Apolipoprotein-E and Alzheimer's Disease. The Tip of the Susceptibility Iceberg. Annals of the N.Y. Academy of Science 855, 738-743, 1998.
25. Saxe, S.R., Wekstein, M.W., Kryscio, R.J., Henry, R.G., Conrett, C.R., Snowdon, D.A., Grant, F.T., Schmitt, F.A., Donegan, S.J., Wekstein, D.R., Ehmann, W.D. and Markesbery, W.R. Alzheimers' Disease, Dental Amalgam and Mercury. JADA 130, 191-199, 1999.
26. Lorscheider, F.L., Vimy, M.J. and Summers, A.O. Mercury Exposure from Silver Tooth Fillings: Emerging Evidence Questions a Traditional Dental Paradigm. FASEB J. 9, 504-508, 1995.
27. Hock, C. Drasch, G. Golombowski, S., Muller-Spahn, F., Willershausen-Zonnchen, B., Schwarz, P., Hock, U. Growdon, J.H. and Nitsch, R.M. Increased Blood Mercury Levels in Patients with Alzheimer's disease. J. of Neural Transmission v105L 1) 59-68, 1998.
28. Devanand, D.P., Michaels-Marston, K.S., Liu, X., Pelton, G.H., Padilla, M., Marder, K., Bell, K., kStern, Y., and Mayeux, R. Olfactory Deficits in Patients with Mild Cognitive Impairment Predict Alzheimer's Disease at Follow-up. Am. J. Psychiatry 157(9): 1399-1405, 2000.
29. Kovacs, T., Cairns, N.J., Lantos, P.L. Olfactory Centres in Alzheimer's disease: Olfactory Bulb is Involved in Early Braak's Stages. Neuroreport 12(2): 285-288, 2001.
30. Gray, A.J., Staples, V., Murren, K., Dahariwal, A. and Bentham, P. Olfactory Identification is Impaired in Clinic-Based Patients with Vascular Dementia and Senile Dementia of Alzheimer's type. Int. J. Geriatr. Psychiatry 16(5):513-517, 2001.
31. World Health Organization (WHO) report on Environmental Health Criteria 118, Inorganic Mercury, WHO, Genevia, 1991.
32. Brouwer, D.A., Clinical Chemistry of Common Apoprotein-E Isoforms. J. Chromatography, Biomed. Applications, v678(1) 23-41, 1996.