Review of the literature on Hg toxicity
The reference numbering system was changed to roman numbers and I couldn't change them to arabic numbers, when copying a word document all formatting is lost.
Any way I just want to share with you six months of hard work, the following is a review of the scientific literature, part of a series of articles in collaboration with Dr Ilona Visser (President of the South African Chapter of the IAOMT) that was due to be published but due to the sensitivity of the topic even the Dental Journal that had initially committed to publishing it, decided against it.
Once absorbed, what are the health effects of Hg?
Orlando Rojas, DDS (Sept, 2008)
Mercury (Hg) is an ubiquitous environmental toxin that causes a wide range of adverse health effects in humans[i],2. Three forms of Hg exist: elemental, inorganic and organic. Each of these forms has its own profile of toxicity. Exposure to Hg typically occurs by inhalation or ingestion[ii]
Exposure to methyl-mercury (MeHg) through fish consumption is widely recognized, the public is less aware of the sources of mercuric chloride (HgCl(2)). Oral and laboratory thermometers, barometers, small batteries, thermostats, gas pressure regulators, light switches, dental amalgam (Hg-Am) fillings, cosmetic products, medications, cultural/religious practices, and gold mining all represent potential sources of exposure to inorganic forms of Hg. The route of exposure, the extent of absorption, the pharmacokinetics, and the effects all vary with the specific form of Hg and the magnitude and duration of exposure[iii],[iv],[v],[vi].
Microleakage of Hg from Hg-Am has been conclusively confirmed over the past 30 years[vii]. This microleakage, has generally been considered to occur via either erosion or evaporation directly from the surface of fillings, followed by ingestion. The importance of the direct migration of Hg through the tooth into the pulp and blood stream has also been widely demonstrated[viii],[ix],[x],[xi],[xii],[xiii],[xiv],[xv]. There are pointers to show that Hg vapour is more neurotoxic than methyl-Hg in fish1.
There is a large body of scientific literature that shows that Hg-Am spreads around the body, and that Hg-Am typically provides the greatest portion the Hg to be found in the human body1. Several autopsy studies showed a correlation between the Hg concentration in various tissues and organs of the human cadavers and the number of fillings or surfaces of Hg-Am present[xvi],[xvii],[xviii],[xix],[xx],[xxi],[xxii].
Many studies are reporting findings of Hg toxicity well below the most recent exposure levels reported in the literature[xxiii],[xxiv],[xxv].
Hg’s toxic effects have been associated with human death and several ailments that include cardiovascular diseases, anaemia, kidney and liver damage, developmental abnormalities, neurobehavioral disorders, autoimmune diseases, and cancers in experimental animals, oxidative stress, autism, skin and mucosa alterations or non-specific symptoms and complaints.1,11,[xxvi].
Basic metabolic process of Hg toxicity
At the cellular level, Hg has been shown to interact with sulphydryl groups of proteins21,[xxvii] and enzymes, to damage DNA, and to modulate cell cycle progression and/or apoptosis (Disintegration of cells into membrane-bound particles)21.
One hypothesis to explain Hg toxicity is that it induces the production of free oxygen radicals and modifies the redox potential of the cell[xxviii].
This is confirmed in a study[xxix] that found that mice exposed to 0.8 µg of HgCl2 in two oral doses per week, for two weeks, had increased lipidoxidation in the kidneys, testicles and epididymides, and an elevated concentration of glutathione (GSH) and superoxide dismutase in the testicles. Administering a dose 10 times as large resulted in a significant reduction in GSH concentration in the epididymides, and also in the activity of glutathione disulphide reductase (GR) and glutathione reductase (GPx) in the kidneys and epididymides.
Olivieri[xxx] confirmed the redox potential hypothesis when he found, that mercuric chloride (HgCl2) in a concentration of 50 µg/l reduces the cellular content of GSH by 30 per cent in neuroblastoma cells, thereby decreasing their reductive capacity. Another observation was an increased release of ß-amyloid (Aß) peptide and elevated phosphorylation of tau protein.
Hg, cadmium, and other heavy metals have a high affinity for sulfhydryl (-SH) groups, inactivating numerous enzymatic reactions, amino acids, and sulfur-containing antioxidants, with subsequent decreased oxidant defense and increased oxidative stress. Hg induces mitochondrial dysfunction with reduction in Adenosine triphosphate (ATP), depletion of GSH, and increased lipid peroxidation[xxxi].
The effects of particulate dental Hg-Am on cultured macrophages were studied to assess their plasma membrane permeability. Alterations in cellular morphology were slight during the first day. Prominent cellular damage was seen in cultures treated with particulate Ag2Hg3 (gamma 1-phase) for 1 week. A slight increase in lactate dehydrogenase (LDH) activity in the medium was seen one hour after the alloy treatment. Intraperitoneal phagocytosis did not cause any morphological changes in macrophages, but the percent of phagocytosis was diminished5.
After exposure to HgCl2 (0-20 µgr/ml) or MeHgCl (0-2 µgr /ml) for 24 hr, another study[xxxii] found:
- cell death was apparent,
- monocytes exhibited significant loss of viability,
- cells revealed early nuclear alterations characterized by:
- hyperchromaticity, nuclear fragmentation and condensation of nucleoplasm,
- destruction of cytoplasmic organelles,
- loss of membrane integrity,
- alterations in membrane structure.
- decrease in total phosphatide synthesis by treated cells.
Monocyte phospholipid synthesis appeared to be more sensitive to the presence of Hg than lymphocytes and a rapid and sustained elevation in the intracellular levels of Ca++ was elicited. These morphological and biochemical changes are consistent with the notion that Hg initiates cytotoxic changes associated with programmed cell death.
Rosenspire et al.[xxxiii] found that 0.13 µM HgCl2 boosted phosphorylation of tyrosine in proteins from B-cell lymphoma cells from mouse. They also reported[xxxiv] that 0.6 µM HgCl2 inhibits T cell-receptor-mediated activation of RAS in Jurkat cells, which are a human T cell line. Königsberg et al.[xxxv] studied the effect of 0.5µM on mitochondrion function in a foetal liver-cell line. They found ultrastructural modification of the mitochondria. The respiratory functions of the cell remained intact, but they found that the modification had involved uncoupling from signal links in the cell.
Effect on DNA
Dental fillings provide a major iatrogenic exposure to xenobiotic compounds (substances that are foreign to the body). Experimental data suggest that both Hg-Am and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance (Balance reduction-oxidation). The main mechanism underlying the genotoxicity (toxic to DNA) of dental restorative materials may be ascribed to the ability of both Hg-Am and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions. The association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time[xxxvi].
Wolfreys and Oliviera[xxxvii] (1997) found that the increase in sensitivity to IgE stimulation in the peritoneal mast cells of mercury-sensitive rats is due to intracellular increase of free oxygen radicals produced by mercury. Mice exposed to mercury vapour, at 0.5 mg/m3 for two hours, showed an elevated mercury concentration in motor neurons in the spine and signs of oxidative damage to DNA (Pamphlett et al. 1998).
Another study focused on genotoxic effects below a cytotoxic dose level of mercuric dichloride (HgCl2) in human samples of salivary glands and lymphocytes to elucidate a possible role in tumor initiation. In both cell types a significant increase in DNA migration could be shown starting from HgCl(2)concentrations of 5 µM in comparison to the negative control. The viability of the cell systems was not affected except at the highest concentration (50 microM) tested. It was concluded that there is genotoxicity without cytotoxicity or cell death[xxxviii].
Reichl et. al. [xxxix]investigated the cytotoxicity on human gingival fibroblasts (HGFs) of the following dental material components:
- hydroxyethylmethacrylate (HEMA),
- triethyleneglycoldimethacrylate (TEGDMA),
- urethanedimethacrylate (UDMA),
- bisglycidylmethacrylate (Bis-GMA),
- the autoimmunogen Hg(2+) component present in Hg-Am (as HgCl(2))
- and methyl HgCl(2) (MeHgCl)
MeHgCl was the most toxic substance, Hg(2+) was about fourfold less toxic than MeHgCl but Hg(2+) was about fourfold more toxic than BisGMA. A decreased toxicity was observed for Hg(2+) at 48 h, compared to the 24 h Hg(2+) exposure (P<0.05).
Reichl et.al. 12 also studied the cytotoxicities of the resin-based dental (co)monomers hydroxyethylmethacrylate (HEMA), triethyleneglycoldimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and bisglycidylmethacrylate (BisGMA) compared with methyl HgCl(2) (MeHgCl) and the Hg-Am component mercuric chloride (HgCl2). The results of this study indicate that resin composite components have a lower toxicity than Hg from Hg-Am in human gingival fibroblasts (HGF).
Effects on the immune system.
The composition of dental materials available indicates that there is the potential for adverse biological effects, in reactions mediated via the amplifying mechanisms of the immune system; small amounts may lead to clinical manifestations of allergic contact dermatitis and urticaria[xl].
Macrophages play a central role in the pathogenesis of inflammation. In this study the macrophages were inoculated with alloy particles prepared from silver and tin (Ag3Sn), the gamma-phase of Hg-Am. Ten minutes after inoculation with alloy particles, about 58% of the macrophages had ingested particles. When the cultures had been inoculated for ten days, a marked reduction in phagocytosis was observed probably due to cytolysis of those macrophages which initially had phagocytized the alloy particles. The study[xli] suggests that macrophage function is compromised by alloy particles and it seems to worsen in time.
Hg exposure is known to induce autoimmune reactions in susceptible animals,[xlii],[xliii],[xliv],[xlv] different investigations show the same for Hg-Am[xlvi],[xlvii],[xlviii],[xlix],[l],[li].
Hultman et. al.46 implanted gelatin coated particles of either finished Hg-Am or unmixed silver alloy in the peritoneal cavity of mice known to be genetically susceptible to Hg–induced autoimmune reactions. Over the course of the experiment, both groups displayed their characteristic reactions of hyperimmuno-globulinemia, serum autoantibodies targeting nucleolar proteins, and systemic immune complex deposits. The authors ascribed the reactions in the alloy–only group to the silver component.
Patients with autoimmune and allergic diseases (systemic lupus, multiple sclerosis, autoimmune thyroiditis or atopic eczema) often show increased lymphocyte stimulation with low doses of inorganic Hg in vitro[lii]. Hg is immunotoxic [liii],[liv] and causes hypersensitivity[lv].
Hg-containing compounds are immunomodulatory[lvi],[lvii], moreover, the decrease in T-cell function following exposure to Hg indicates that this metal is immunotoxic at very low exposure levels.
In a different study[lviii] he clearly showed that susceptibility to the immunotoxic effects of HgCl2 is, in part, dependent upon intracellular GSH levels and further that Hg inhibits GSH generation by lymphocytes and monocytes45,[lix],[lx],[lxi],[lxii].
Demethylation (The enzymatic removal of methyl groups) has been described in the organs, especially in phagocytic cells, but mainly in the flora of the intestinal tract. While most of the Hg(2+) formed in the intestine is excreted, a fraction is reabsorbed which together with the local demethylation increases the organ Hg(2+) concentration. MeHg is a well-known immunosuppressive agent, while Hg(2+) is associated with immunostimulation and autoimmunity especially in genetically susceptible rodents, creating a syndrome, i.e. Hg-induced autoimmunity (HgIA)[lxiii],[lxiv].
There are differences in either dose requirement or induction mechanisms for the different HgIA parameters. The selective accumulation of Hg(2+) in lymph nodes following MeHg treatment should be taken into account when the effect of MeHg on the immune system is evaluated.
After a study on the effect of dose, treatment time, gender and non-H-2 genes on immune parameters and toxicokinetics in murine HgIA; it was concluded that conditions exist for a rapid modification of fibrillarin, followed by a T cell-dependent immune response, which is consistent with the presence of anti-fibrillarin antibodies (AFA) in serum after 2 weeks. AFA developed in a dose-dependent pattern. Serum IgE showed a dose-dependent increase with a maximum after 1-2.5 weeks followed by a distinct decline towards the baseline level. It was also concluded that genetic factors outside the H-2 region, and not related to toxicokinetics, modify the autoimmune response[lxv],[lxvi].
HgCl(2) induces in mice an autoimmune syndrome (HgIA) with T cell-dependent polyclonal B cell activation and hypergammaglobulinemia, dose- and H-2-dependent production of autoantibodies targeting the 34 kDa nucleolar protein fibrillarin (AFA), and systemic immune-complex deposits. MeHg and ethylHg (EtHg in the form of thimerosal) induce AFA. In conclusion, while metabolically formed Hg(2+) might be the main AFA-inducing factor also after treatment with EtHg, the quality of the Hg-induced AFA response is modified by the species of Hg as well as the dose[lxvii].
Individual differences in susceptibility may be caused by genetical as well as external variables. In mice as well as rats, autoimmune response can be induced by Hg or gold. The autoimmune response depends qualitatively on the H-2 haplotype (set of any one of a series of two or more different genes that occupy the same position on one chromosome). If a strain of mice does not have the susceptible haplotype, no autoimmunity will be induced. A clear correlation exists between renal Hg deposition and the autoimmune response in susceptible strains of mice. There was a threshold for induction of the autoimmune response in genetically susceptible mice. When exposure was interrupted, the Hg deposition decreased as did the antinucleolar antibody titre. Thresholds exist within susceptible strains below which no autoimmune response is induced[lxviii].
Multiple Sclerosis (MS)
A study56 compared blood findings between MS subjects who had their Hg-Am removed to MS subjects with Hg-Am. MS subjects with Hg-Am were found to have significantly lower levels of red blood cells, haemoglobin (key chemical compound that combines with oxygen from the lungs and carries the oxygen from the lungs to cells throughout the body), and hematocrit (a measure of how much of the blood is made of red cells) compared to MS subjects with Hg-Am removal. Thyroxine (T4, a form of thyroid hormone) levels were also significantly lower in the MS Hg-Am group and they had significantly lower levels of total T Lymphocytes and T-8 (CD8) suppressor cells. The MS Hg-Am group had significantly higher blood urea nitrogen and lower serum IgG. Hair Hg was significantly higher in the MS subjects compared to the non-MS control group. A health questionnaire found that MS subjects with Hg-Am had significantly more (33.7%) exacerbations during the past 12 months compared to the MS volunteers with Hg-Am removal[lxix].
In another study Siblerud RL57 compared the mental health status of 47 MS patients with Hg-Am to that of 50 patients with their fillings removed. On the Beck Depression Inventory the MS subjects with Hg-Am suffered significantly more:
· symptoms of depression,
· obsessive-compulsive behaviour.
These data suggested that the poorer mental health status exhibited by multiple sclerosis subjects with dental Hg-Am fillings may be associated with Hg toxicity from the Hg-Am[lxx].
Objective biochemical changes like changes in cerebrospinal fluid protein have been documented following the removal of Hg fillings in MS patients[lxxi].
Oral Lichen Planus (OLP)
The US population demonstrates allergy levels of 5-8% to Hg by skin patch testing[lxxii].
By using antibody – antigen flocculation tests on blood serum, the number is over 90%. In another study [lxxiii] 180 subjects with Hg-Am fillings were patch tested, and found that 16.1% of those without allergic disease, and 22.5% of those with allergic disease, tested positive for Hg allergy. Of sixty subjects without Hg-Am fillings, none tested positive for Hg allergy. And at Baylor College of Dentistry, of 171 dental students patch tested, 32% were positive for Hg allergy. The percentage of positive tests correlated with the students’ own Hg-Am scores, and with the length of time they had been in dental school[lxxiv].
A case-control study[lxxv] determined the association of dental materials with OLP and, particularly, the effects of Hg-Am, Hg-Am corrosion status, gold and dissimilar metals in continuous contact. The findings suggested that although the presence of Hg-Am or gold themselves is not associated with increased risk of OLP, corrosion of Hg-Am and the presence of a 'galvanic effect' from dissimilar dental materials in continuous contact (bimetallism) are associated with an increased risk of OLP.
29 patients with OLP and Hg-Am fillings were patch tested for contact allergy to dental materials. 18 of these patients (62%) had a contact allergy to Hg. In a control material, the frequency of Hg allergy was 3.2%. In 3 of the patients the lesions healed completely after removal of the Hg-Am fillings. On the basis of these findings it is recommended that all Hg-Am fillings be removed after a positive patch test to Hg, as a step in the treatment of oral lichen planus[lxxvi].
Oral lichenoid lesions caused by hypersensitivity to Hg in Hg-Am fillings may mimic oral lichen planus on clinical and histologic examination. A positive patch test reaction to more than one mercurial allergen increases confidence in the diagnosis and justifies the removal and replacement of all Hg-Am fillings with those made of other materials. A complete remission may be expected about 3 months after the last Hg-Am filling is removed[lxxvii].
174 patients referred for lichenoid lesions and evaluation of a possible connection with Hg-Am restorations were invited to a clinical re-examination. 159 of the patients were re-examined with the purpose of evaluating the long-term effect upon performed substitution therapy. Partial or total removal of Hg-Am had been recommended according to a set of given criteria. The re-examination showed that 62 patients had performed partial and 69 patients total removal of Hg-Am fillings. 28 patients had not performed any substitution therapy. There was a difference between recommended and performed therapy. The results demonstrated that 92% of patients with lichenoid lesions only in contact with Hg-Am fillings healed or improved clinically following removal of Hg-Am. No statistical difference was found in healing between patients who only removed fillings in contact and those who had removed all Hg-Am restorations. More than 60% of buccal lichenoid lesions without contact with Hg-Am at time of referral disappeared following Hg-Am substitution. 3 out of 17 patch-tested patients demonstrated a hypersensitivity reaction to Hg. All lichenoid lesions in these patients healed following total substitution. Partial or total removal of Hg-Am fillings was also performed on 10 patients with completely negative patch-tests. 6 out of these patients demonstrated complete healing of their lichenoid reactions at re-examination[lxxviii].
In 97% of all patients with oral lichenoid reactions (OLR) associated with dental Hg-Am a removal of the fillings leads to a decline of the lesions, as a minimum[lxxix].
The potential harmful effects of Hg on cardiovascular diseases (CVD) have raised a cause for concern in the last 10 years, mostly due to the proposed role of Hg in oxidative stress propagation. Some epidemiological studies have indeed found an association between increased levels of Hg in the body and risk of CVD[lxxx],[lxxxi].
Results from recently published studies have shown an independent association between the Hg concentration in the human body and the risk of coronary heart disease68,[lxxxii],[lxxxiii],[lxxxiv],[lxxxv].
· may promote atherosclerosis
· it may increase the risk of acute coronary events in several ways
· stimulates the production of free radicals
· binds to sulfhydryl groups of enzymes
· forms an insoluble complex with selenium
· induces lipid peroxydation
· increases oxidized low-density lipoprotein concentration in blood.
Comparisons between subjects with and without Hg-Am68 showed Hg-Am-bearing subjects had significantly:
· higher blood pressure,
· lower heart rate,
· lower haemoglobin,
· lower haematocrit,
· lower haemoglobin, haematocrit, and red blood cells when correlated to increased levels of urine Hg.
· greater incidence of chest pains, tachycardia, anaemia, fatigue, tiring easily, and being tired in the morning.
The data suggest that inorganic Hg poisoning from dental Hg-Am does affect the cardiovascular system68.
Exposure to Hg at nanomolar level also affects cardiac function, Wiggers et. al. [lxxxvii] investigated those effects on vascular reactivity, the results suggest that at nanomolar concentrations HgCl2 increase the vascular reactivity to Pressor responses (Causing, or giving rise to pressure or to an increase of pressure) to phenylephrine (PHE). This response is endothelium mediated and involves the reduction of Nitric Oxide (NO) bioavailability and the action of reactive oxygen species. The local angiotensin converting enzyme (ACE) participates in Hg actions and depends on the angiotensin II generation.
The effects of continuous exposure to 5 and 20 nM of HgCl(2) on the cardiac contractility were investigated[lxxxviii]. In perfused hearts beating spontaneously, isovolumic systolic pressure reduced progressively and the diastolic pressure increased. Results suggested that cardiac effects may be observed after continuous exposure to very small concentrations of Hg, probably as a result of the cell capacity to concentrate Hg.
Significant deposits of Hg, previously non-existent, were found in the lungs, kidneys, endocrine organs, liver, and heart with abnormal low-voltage electro cardiograms (ECGs) in all the limb leads and V1 (but almost normal ECGs in the precordial leads V2-V6) the day after removing Hg-Am[lxxxix] hence the importance of following a strict protocol for Hg-Am removal.
The overall vascular effects of Hg include:
· oxidative stress,
· vascular smooth muscle dysfunction,
· endothelial dysfunction,
· immune dysfunction,
· mitochondrial dysfunction.
The clinical consequences of Hg toxicity include:
· coronary heart disease (CHD),
· myocardial infarction (MI),
· increased carotid IMT and obstruction,
· cerebrovascular accident (CVA),
· generalized atherosclerosis,
· renal dysfunction with proteinuria.
· Hg diminishes the protective effect of fish and omega-3 fatty acids.
· Hg, cadmium, and other heavy metals inactivate Catechol O-Methyl Transferase (COMT) which increases serum and urinary epinephrine, norepinephrine, and dopamine. This effect will increase blood pressure and may be a clinical clue to heavy metal toxicity.
Heavy metal toxicity, especially Hg and cadmium, should be evaluated with a urine challenge test, in any patient with hypertension, CHD, or other vascular disease[xc].
A study by Fillion et.al. adds further support for Hg cardiovascular toxicity[xci].
Effect on kidneys
A comprehensive analysis of published data indicates that inorganic Hg is one of the most environmentally abundant toxic metals, is a potent and selective nephrotoxicant that preferentially accumulates in the kidneys, and is known to produce cellular injury in the kidneys. Binding sites are present in the proximal tubules, and it is in the epithelial cells of these tubules that toxicants such as inorganic Hg are reabsorbed, due to the high affinity binding of divalent mercuric cations to thiols of sulfhydryl groups of proteins. This can affect the enzymatic activity and the structure of various proteins. Hg may alter protein and membrane structure and function in the epithelial cells and this alteration may result in long term residual effects[xcii],[xciii].
From the nephrotoxicity point of view, dental Hg-Am is an unsuitable filling material, as it may give rise to Hg toxicity. Renal damage is possible and may be assessed by urinary excretions of albumin, NAG, and gamma-GT[xciv].
In a sheep experiment, the organ that accumulated the greatest amount of Hg was the kidneys, 7438 nanograms per gram of tissue (ng/g). The urine concentration was only 4.7 ng/g , demonstrating the inadequacy of plain urine samples as an indicator of Hg storage in internal organs[xcv]. The appropriate test will be discussed later in this series.
The same experiment was repeated using a monkey[xcvi], which would eat much the same food and chew in much the same way as humans. The results were virtually identical to those found with the sheep. Within twenty eight days, the radioactive Hg had spread around the monkey’s body, yielding tissue concentrations that were highly similar to the sheep’s. The monkey experiment was confirmed by Danscher et. al. [xcvii]
A study by Boyd et. al.[xcviii] demonstrated how much stress is placed upon the kidneys by the presence of Hg-Am, and suggested how patients with kidney malfunction may be endangered by Hg-Am fillings.
Studies[xcix] were undertaken to investigate the principal actions underlying Hg-induced oxidative stress in the kidney. Mitochondria from kidneys of rats treated with HgCl2 (1.5 mg/kg i.p.) demonstrated a 2-fold increase in hydrogen peroxide (H2O2) formation for up to 6 hr following Hg(2+). Together with increased H2O2 formation, mitochondrial GSH content was depleted by more than 50% following Hg(2+) treatment. These events, coupled with Hg(2+)-mediated GSH depletion and pyridine nucleotide oxidation, create an oxidant stress condition characterized by increased susceptibility of mitochondrial membranes to iron-dependent lipid peroxidation. Since increased H2O2 formation, GSH depletion and lipid peroxidation were also observed in vivo following Hg(2+) treatment, these events may underlie oxidative tissue damage caused by Hg compounds. Moreover, Hg(2+)-induced alterations in mitochondrial Ca2+ homeostasis may exacerbate Hg(2+)-induced oxidative stress in kidney cells.
Cytotoxicity experiments indicated that inorganic Hg is highly toxic. The percentages of cells undergoing early apoptosis were increasingly high at treatments of 0, 1, 2, 3, 4, 5, and 6 microg/mL of Hg. This indicates a dose-response relationship with regard to Hg-induced cytotoxicity and the externalization of phosphatidyl-serine in human renal proximal tubule HK-2 cells[c],[ci],[cii]. Both glomerular and tubular damage may occur at exposure levels lower than those giving rise to central nervous system effects[ciii].
It was demonstrated that HgCl2 potently stimulates renal generation of hydrogen peroxide in vitro and in vivo and such generation of peroxide contributes to renal dysfunction in vitro and in vivo. It was also demonstrated that in response to HgCl2, redox sensitive genes are expressed including heme oxygenase and members of the bcl family[civ].
Expression of specific stress proteins in rat kidney exhibits regional heterogeneity in response to Hg(II) exposure, and a positive correlation exists between accumulation of some stress proteins and acute renal cell injury[cv].
Susceptibility to renal injury induced by Hg(2+) increases significantly as a result of compensatory renal growth (following reductions of renal mass). This phenomenon is related in part to increased basolateral uptake of Hg(2+) by proximal tubular cells. The specific transporters involved in the process have been also identified[cvi].
Effect on liver
Hg-induced hepatotoxicity is associated with the modulation of specific gene expressions in liver cells that can lead to several disease states involving immune system dysfunctions. Dormant genes in the liver are capable of reactivation. Hierarchical cluster analysis identified 2,211 target genes that were affected.
Further analyses of affected genes identified genes located on all human chromosomes with higher than normal effects on genes found on chromosomes 1-14, 17-20 (sex-determining region Y). These genes are categorized as control and regulatory genes for metabolic pathways involving the cell cycle (cyclin-dependent kinases), apoptosis, cytokine expression, Na+/K+ ATPase, stress responses, G-protein signal transduction, transcription factors, DNA repair as well as metal-regulatory transcription factor 1
Significant alterations in these specific genes provide new directions for deeper mechanistic investigations that would lead to a better understanding of the molecular basis of Hg-induced toxicity and human diseases that may result from disturbances in the immune system21.
Apoptosis arises from the active initiation and propagation of a series of highly orchestrated specific biochemical events leading to the demise of the cell. It is a normal physiological process, which occurs during embryonic development as well as in the maintenance of tissue homeostasis. Diverse groups of molecules are involved in the apoptosis pathway and it functions as a mechanism to eliminate unwanted or irreparably damaged cells. However, inappropriate induction of apoptosis by environmental agents has broad ranging pathologic implications and has been associated with several diseases including cancer. The toxicity of several heavy metals such as Hg has been attributed to their high affinity to sulfhydryl groups of proteins and enzymes, and their ability to disrupt cell cycle progression and/or apoptosis in various tissues[cvii].
The following are but a few of several studies that explain the different mechanisms by which heavy metals with specific reference to Hg cause liver toxicity and damage to the immune system:
- Korashy HM and El-Kadi AO: Glutathione transferase (GST) is a phase II detoxifying enzyme that plays a protective mechanism against oxidizing substances and toxic contaminants. Among these contaminants, heavy metals and polycyclic and halogenated aromatic hydrocarbons (PHAHs) have been shown to exert their toxic effects through the modulation of detoxifying enzymes, including the GSTs[cviii],[cix].
· Exposure to metals (Hg(2+), Pb(2+), and Cu(2+)) and polycyclic aromatic hydrocarbons (PAHs) mixtures would differentially modulate PAHs-mediated carcinogenicity in human hepatoma HepG2 cells[cx].
· Hg2+ and Pb2+ and Cu2+ significantly increased the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. Heavy metals differentially modulate the constitutive and the inducible expression of aryl hydrocarbon receptor (AHR) regulated genes[cxi] and differentially modulate the GSH activity through oxidative stress- and AhR-mediated mechanisms[cxii].
· Cells were exposed to Hg for 10 and 48 hours respectively at doses of 0, 1, 2, and 3 microg/mL on a study by Sutton and Tchounwou. The study data indicated a dose response relationship between Hg exposure and the degree of early and late-stage apoptosis in HepG2 cells. There is a gradual increase in apoptotic cells with increasing doses of Hg as well as a gradual increase in Caspase positive cells with increasing doses of Hg[cxiii].
· Thiols are known to influence the metabolism of GST. This paper looked at the effects of thiols as well as the interaction between thiols and Hg ions in cultures of both HeLa and hepatoma cells. The presence of dithiols, either DTT or dihydrolipoate (the metabolite of LA), increased the effects of Hg ions on GSH concentrations in hepatoma cells, whereas monothiols such as NAC or GSH did not. Thus, metabolic effects of Hg ions were observed in hepatoma cells as well as in HeLa cells at a lower concentration than the supposed toxicity threshold for Hg in blood[cxiv],[cxv].
- Mechanisms of methylHg (MeHg) and inorganic Hg (Hg) uptake were examined in HepG2 cells, a human hepatoma-derived cell line. MeHg uptake was faster when it was present as the l-cysteine complex, as compared to the GSH, CysGly, gamma-GluCys, d-cysteine, N-acetylcysteine, l-penicillamine, or albumin complexes. Uptake of MeHg-l-cysteine was independent of Na(+), stereoselective, and was inhibited by the amino acid transport system l substrates l-leucine, l-valine, and l-phenylalanine (5 mM). These findings demonstrate that Hg uptake by HepG2 cells is dependent on the chemical structure of the Hg compound, the thiol ligand, and the activity of gamma-glutamyl transpeptidase. gamma-Glutamyl transpeptidase appears to play a key role in the disposition of MeHg-SG by facilitating the formation of MeHg-l-cysteine, which is readily transported into the cells on an amino acid-type carrier[cxvi].
Part of the neurotoxic effects of Hg(2+) and MeHg was attributed to their interaction with voltage-activated calcium channels. Tarabová et.al. investigated effects of Hg(2+) and MeHg on neuronal Ca(v)3.1 (T-type) calcium channel stably expressed in the human embryonic kidney (HEK) 293 cell line; they concluded that, interaction with the Ca(v)3.1 calcium channel may significantly contribute to neuronal symptoms of Hg poisoning during both acute poisoning and long-term environmental exposure[cxvii].
Hg(2+) and MeHg are neurotoxic upon chronic and acute exposure, these toxic manifestations interfere with divalent cation regulation in neuronal systems[cxviii].
MeHg disrupts intrasynaptosomal polyvalent cation homeostasis by at least two mechanisms. The first involves release of endogenous non-Ca2+ polyvalent cations, while the second is due to increased Ca2+ permeability of the plasma membrane[cxix],[cxx],[cxxi].
A severe loss of intracellular calcium (Ca2+(i)) homeostasis, apparently contributes to neuronal death of cerebellar granule cells in culture. This is due to the activation of M3 muscarinic receptors with subsequent generation of IP3 that evidently contributes to elevated [Ca2+]i and subsequent cytotoxicity of cerebellar granule cells by MeHg106.
Sweeney et. al. [cxxii]estimated the amount of Hg(2+) released into both air and saliva from fresh and aged, abraded Hg-Am discs and then investigated neurotoxic effects of inorganic Hg upon sensory neuronal cultures. Little change was noted in the neuronal cultures treated with 1 µM mercuric chloride. However, the cultures exposed to high level (10 µM) mercuric chloride showed cells that became rounded and clumped together indicating pathological change. Hg-Am placement appears to present minimal Hg exposure risk. The neurotoxic effect of Hg appears to be related to concentration, as only in the cultures treated with 10 microM mercuric chloride showed striking qualitative and quantitative cellular changes. Unfortunately Sweeney did not follow up those cultures for a period of time to assess chronic exposure to Hg-Am as it happens with dental patients.
On Alzheimer’s disease
The development of Alzheimer's disease (AD) or multiple sclerosis has also been linked to low-dose Hg exposure1,[cxxiii],[cxxiv]. Epidemiological and demographical studies, the frequency of amalgam application in industrialized countries, clinical studies, experimental studies and the dental state of Alzheimer patients in comparison to controls suggest a decisive role for inorganic mercury in the etiology of neurodegeneration like AD.
People showing a genetically determined subgroup of transportation protein for fats (apolipoprotein E4) have an increased AD risk. Apoliprotein E (APO E2,E3,E4) is found in high concentrations in the central nervous system. Individuals with apo-E2/2 almost never get AD, while those with apo-E4/4 tend to have early onset of the disease. Apo-E3 is intermediate. The increased AD risk through APO E4 might be caused by its reduced ability to bind heavy metals.
Age of onset of AD in the population is associated with APO4. What’s the difference among the genotypes? At amino acid position 112 and 158, apo-E2 has two of the sulfhydryl containing cysteine molecules. Apo-E3 has arginine at position 158, and apo-E4 has arginine at both places. In other words, apo-E2 has the most capacity to bind and remove divalent toxic metal atoms such as Hg as it moves from the brain into the cerebrospinal fluid, and out into the blood. Apo-E3 has less, and apo-E4 has none108,[cxxv].
A recent study[cxxvi] aimed to elucidate the relationships between on the one hand MeHg and inorganic Hg (I-Hg) in human brain and other tissues, including blood, and on the other Hg exposure via dental amalgam in a fish-eating population. There was a significant correlation between MeHg in blood and occipital cortex. Also, total-Hg in toenails correlated with MeHg in both blood and occipital lobe. I-Hg in both blood and occipital cortex, as well as total-Hg in pituitary and thyroid were strongly associated with the number of dental amalgam surfaces at the time of death. They concluded that in a fish-eating population, intake of MeHg via the diet has a marked impact on the MeHg concentration in the brain, while exposure to dental amalgam restorations increases the I-Hg concentrations in the brain113,[cxxvii]. Discrimination between mercury species is necessary to evaluate the impact on Hg in the brain of various sources of exposure, in particular, dental amalgam exposure.
The AD brain:
· Is deficient in tubulin synthesis, firmly linked to Hg[cxxviii],[cxxix]
· the binding site on β-tubulin is uniquely blocked by Hg, at extremely low concentrations in the 10-7 M range.
· Cadmium has a smaller effect, by orders of magnitude, aluminum and lead have no effect at all.
· zinc had a slight effect, but greatly increased the inhibitory action of the low concentrations of Hg108,[cxxx],[cxxxi],[cxxxii].
· aluminum, copper, cadmium, manganese, iron, and chrome are not able to elicit all of these deteriorations in low levels, yet they aggravate the toxic effects of Hg108.
· Exibits a variety of trace mineral changes as compared to controls from patients with other psychiatric diseases or normal brains.
· consistent elevated concentrations of Hg110,[cxxxiii],[cxxxiv],[cxxxv],[cxxxvi],[cxxxvii],[cxxxviii],[cxxxix],[cxl].
Olivieri, et. al.25 and Mutter et. al.108 reported that adding a very low concentration of Hg, 36 x 10-9 M, to neuroblastoma cells in tissue culture caused them to exhibit all the biochemical lesions of AD (inhibited tubulin synthesis, drop in intracellular GSH, excretion of phosphorylated tau protein, and finally, excretion of β-amyloid).
Most contemporary researchers think that amyloid is the cause of AD, here we have vanishingly small quantities of Hg causing amyloid in turn. The authors of this study suggest that Hg is the ultimate cause of these events.
Research shows[cxli] that this phenomenon can be induced in living animals. HgCl(2) has been shown to get into rat brains and inhibit the binding of GTP to β-tubulin, and the same for elemental Hg vapor[cxlii],[cxliii]. Rats breathing 300 μg Hg0 per cubic meter of air, a concentration that has been found in the mouths of people with lots of Hg-Am, for just four hours a day for fourteen days, had 75% inhibition of the photolabeling of β-tubulin.
On Parkinson’s disease
Several metals have toxic actions on nerve cells and neurobehavorial functioning. Lead, mercury, manganese and copper, have been implicated in amvotrophic lateral sclerosis (ALS) and Parkinson's disease[cxliv],[cxlv]. Evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant GSH and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. Redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease[cxlvi].
The sparse epidemiologic data point toward the possibility of a risk of lung, kidney, and central nervous system tumors. Me-HgCl(2) causes kidney tumors in male mice. HgCl(2) has shown some carcinogenic activity in male rats136
Epidemiologic data are available for chloralkali workers, dentists and dental nurses, and nuclear weapons workers, three groups occupationally exposed to low levels of Hg and its compounds[cxlvii].
Deposits of Hg in the lungs, kidneys, endocrine organs, liver, G.I. system and then the blood circulation may contribute to intractable infections or pre-cancer. Hg has been found to exist in cancer and pre-cancer cell nuclei[cxlviii].
Because mercuric components reach the circulating blood it eventually may contribute to tumorigenesis in a variety of target cells. The data indicated genotoxic effects of mercuric dichloride in human salivary glands and lymphocytes at concentrations not leading to cytotoxic effects or cell death. A contributory role in oral salivary gland tumor initiation warrants further investigation27.
Ayensu WK and Tchounwou PB. demonstrated that Hg exposure induces cytotoxicity and apoptosis, modulates cell cycle, and transcriptionally activates specific stress genes in human liver carcinoma cells21.
Galvanic corrosion is an electrochemical reaction between dissimilar metals that has the potential to cause unpleasant and even painful biological effects intra-orally[cxlix].
Galvanic effects as well as metal particles may induce a series of heterogeneous symptomatology, (local or systemic pathological phenomena in sensitive individuals)[cl],126. The occurrence of pathologically acting galvanic effects is influenced not only by the composition and combination of different dental alloys, but to a significant degree also by the quality of used materials and processing. Abrasion and corrosion not only damage dental alloys but also burden the organism by release of metallic particles[cli].
In dentistry there is allergic intolerance for the given metals and galvanic currents appearance in the oral cavity. Although they often have similar symptoms their differential diagnostics is very important for determination of correct tactics of treatment and further patient's course[clii].
In recent years there has been an increase in the use of dental casting alloys in prosthodontic treatment. Many patients have metals or alloys, as well as Hg-Am fillings, in their mouth, and will have them for many years[cliii]. Increasing the risk of developing symptoms related to oral galvanism, such as the following125:
· facial pain,
· pain from the teeth (periodontitis, pulpitis and pulpal necrosis),
· pain from temporomandibular joints (TMJ),
· Pain from masticatory muscles,
· TMJ clicking and locking,
· smarting (pain associated with a burn or a sore) in the oral mucosa and tongue
· unpleasant taste, a metallic taste or a battery taste.
· joint symptoms,
· pain in the back, neck and shoulders
· general muscular pain
· difficulty in concentrating,
· depression and insomnia.
· functional disturbances of the masticatory system.
The basic mechanism of dental Hg-Am corrosion has been thoroughly studied[cliv] during the last 50 years by various experimental techniques, most often carried out in vitro with electrochemical methods. It is recognized that a gradual dealloying of the more electroactive components, Zn, Sn, and to a lesser extent Cu, contributes to change the surface composition.
Galvanic current-time transients are typically characterized by an immediate and rapid rise to a peak current, followed by an exponential decay to a much lower value at 15s. A wide range of galvanic currents result from electrical contact of restorations in vivo. These currents are influenced by restoration age and total surface area of the galvanic couple[clv].
Galvanic interaction between cast gold and Hg-Am is reduced with time and surface treatments, but is increased considerably when the Hg-Am contains zinc[clvi],[clvii] .
The ions released from conventional and high-copper Hg-Am in contact with titanium were quantitatively analyzed when the surface area ratio of titanium/Hg-Am was set up as 1/10, 1/1, or 10/1. The corrosion potentials of the Hg-Am and titanium were measured under the same conditions. The potential of the conventional Hg-Am was always lower than that of titanium; the potential of the high-copper Hg-Am was reversed during the early stage of immersion and remained lower. When the surface area ratio of titanium grew at 10/1, tin and copper ions released from the conventional and high-copper Hg-Am, respectively, increased significantly compared with those of each Hg-Am that was not in contact with titanium. The galvanic corrosion in such a large surface area of titanium possibly led to the heavy corrosion of the Hg-Am131.
Bimetallic cells consisting of Remanium CS and Remanium GM 380 alloys (Dentaurum medical products) have a very low EMF (a few mV) and is not a potential source of galvanic currents in the oral cavity. However, galvanic cells prepared from Amalcap plus and Remanium CS or Remanium GM 380 showed a much greater EMF: 104 and 109mV, respectively. This clearly indicates that in these latter cases it is possible to expect some metal ions in the saliva solution as a result of the work of galvanic currents. It was found, by adsorptive stripping voltammetry analysis, that nickel or cobalt, depending on the alloy used, appeared in the saliva solution and increased in concentration over time[clviii].
Due to the various and multi-faceted symptomathology of galvanism, they tend to be a source of significant problems not only for the patient but also for the attending dentist. Very discreet and uncharacteristically objective diagnosis during a regular examination frequently causes this state to be ascribed to a completely different illness[clix].
A case control study showed that corrosion of Hg-Am and the presence of a 'galvanic effect' from dissimilar dental materials in continuous contact (bimetallism) are associated with an increased risk of OLP[clx].
Despite the extensive amount of published literature upon burning symptoms in patients with clinically healthy appearance of the oral mucosa, as well as burning mouth syndrome (BMS) itself, they both remain still challenging topics. It is important to consider in those patients candidal infection, salivary flow rate, presence of oral galvanism and parafunctional habits as well as complete blood count, serum ferritin, serum glucose levels, serum antibodies to Helicobacter pylori together with detailed medical history with special regard to medication intake[clxi].
In a study by the Harvard School of Public Health[clxii], was concluded that, women who delivered before 35 weeks' gestation were more likely to have hair mercury levels at or above the 90th percentile (> or = 0.55 microg/g), even after adjusting for maternal characteristics and fish consumption.
Scientists in Spain[clxiii] assessed in a prospective birth cohort study, the prenatal exposure to total mercury (T-Hg), both inorganic and organic, in newborns by analyzing the T-Hg concentration in cord blood, and evaluated the role of maternal fish consumption in this exposure. They concluded that newborns from a Mediterranean area presented elevated levels of T-Hg in cord blood.
Studies of the toxicokinetics of mercury in humans, including pregnant and lactating women, confirm the picture previously obtained from animal experiments, and have provided quantitative information. The mother’s amalgam fillings are reflected in the quantities of inorganic mercury in the placenta[clxiv], in umbilical-cord blood, in breast milk[clxv] and in amniotic fluid[clxvi].
There is little information about Hg concentration in human amniotic fluid (AF) of pregnant women and its potential toxic effect on the fetuses. A study by Luglie et.al[clxvii]. on Seventy-two pregnant women assessed:
· the relationship between the presence of detectable Hg concentration in human AF, number and surface areas of amalgam fillings of pregnant women;
· obstetric history and perinatal complications.
One dentist recorded the dental status, presence, number and surface areas of amalgam fillings. To estimate the dependence of the explanatory variables (such as number and surface areas of amalgam fillings, fish consumption, presence of liver or neurological diseases and smoking habits) on mercury concentration several linear regression models were built up.
A dependence of mercury concentration on number of amalgam fillings (p=0.03), surface area of the amalgam fillings (p=0.04) and fish consumption (p=0.04) was observed. They concluded that the number and surface areas of amalgam fillings influenced positively Hg concentration in AF but not at a significant level. Moreover Hg levels detected in AF were low and no adverse outcomes were observed through pregnancies and in the newborns.
This prospective study unfortunately did not follow up the offspring’s development, since we know that the neuro toxic effects of heavy metals are only apparent after one year of age.
Effects on dental personnel
Dental personnel are occupationally exposed to Hg vapour in their working environment and this exposure constitutes a potential risk to people in the dental surgery, mainly from the inhalation of Hg vapour and fine particles of Hg-Am. In this study, the urinary Hg excretion levels of 20 dentists and nine control subjects, matched for age, were determined by cold-vapour atomic absorption spectrometer. The levels of Hg in the non-challenged urine samples of the dentists were about three times higher than the control subjects. It is not clear if the Hg-Am used by these dentists was encapsulated or not. Some 90% of dentists wore both gloves and masks. The results showed that dentists had significant exposure to Hg vapour compared to control subjects and therefore might be subject to possible adverse effects due to Hg toxicity[clxviii].
Samples, mainly from occipital cortex and pituitary gland, but also from rental cortex, olfactory bulbs, thyroid gland and liver were collected from autopsies of 8 dental staff cases and 27 controls. These samples were analysed for total mercury content. The results revealed high mercury concentrations on the dental staff cases compared to controls[clxix].
Another study[clxx], 180 dentists were compared with an equal number of controls of university employees. Mean urinary mercury secretion was four times as large among the dentists as among the controls and five times as large as that in the dental-care personnel above before chelation. Statistically significantly more often than the controls, the dentists showed memory impairment and deterioration in psychomotor function. These changes were not, however, correlated with the mercury secretion in their urine.
Personal exposure to Hg vapor and the release of Hg from or during removal of Hg-Am dental fillings increases its blood and plasma concentration. Continuous professional exposure to Hg followed by its absorption might have toxicological consequences affecting cardiac function[clxxi].
In one study, dentists with high baseline urinary Hg levels showed neuropsychological and motor control deficits[clxxii]. In another, dentists and staff with high Hg levels, proven by DMPS challenge, had altered porphyrin (hemoglobin) metabolism, as well as neurobehavioral changes, including impairment of attention, motor and perceptual skills, and increased irritability[clxxiii],[clxxiv].
The urinary Hg levels of 4272 dentists were measured at random at dental conventions by Naleway et. al.[clxxv], between 1975 to 1983. They found that dentists on average did not have urinary Hg concentrations outside “acceptable limits” and came to the conclusion that there was no problem with their occupational exposure due to Hg-Am. However, the urinary concentrations correlated significantly (p<.001) with the number of Hg-Ams each dentist placed per week, and the range was tremendous. The general population has a range of 0 – 5 μg Hg per liter of urine, while 10.9% of the dentists in this study had over 30 μg per liter, including 1.3% with over 100 μg per liter! If the proportionality of Hg in urine to total body burden, as shown by the sheep and the monkey studies, holds true for humans, the dentists who use the most Hg-Am are storing prodigious quantities of Hg in their bodies.
Information on the reproductive effects of chemical exposures in dental work is sparse or inconsistent. The study population included 222 cases of miscarriage and 498 controls (births). A slightly increased risk was found for exposure to Hg Hg-Am, some acrylate compounds, solvents and disinfectants. These findings indicate that the possibility of a weak association between exposure to these agents and an increased risk of miscarriage cannot be excluded[clxxvi].
In a survey of 7,000 female dental assistants, a subgroup of 418 women who placed over 30 Hg-Ams per week, and had poor Hg hygiene habits, had a fertility rate of 63% that of control women not exposed to Hg[clxxvii]. Many other studies point to a negative effect of Hg vapor exposure on reproductive outcomes[clxxviii],[clxxix],[clxxx],[clxxxi].
In a group of 465 patients diagnosed as having chronic Hg toxicity (CMT), 32.3% had severe fatigue, 88.8% had memory loss, and 27.5% had depression. A significant correlation was found between CMT and the Apo-lipoprotein E4 genotype (p=0.001). An investigation into an additional 864 consecutively seen general practice patients, resulted in 30.3% having evidence consistent with CMT, and once again a significant correlation was found with the APO-E4 genotype (p=0.001). Removal of Hg-Am Hg fillings when combined with appropriate treatment resulted in a significant symptom reduction (p<0.001) to levels reported by healthy subjects[clxxxii].
Exposure to volatile free Hg in dental clinics should be controlled to eliminate occupational risk6,145. To reduce the amount of Hg released into air, however, Hg-Am should be polished in a moist atmosphere with high volume aspiration108.
The bioavailability and accumulation of Hg from external environmental exposure to mixed, cured, milled, sieved and proportioned dental Hg-Am typically found within the dental wastewater discharge stream was proven to be bio-available to fish[clxxxiii].
Available data suggests that dental Hg-Am is an unsuitable material for medical, occupational and ecological reasons1,39.
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[xcv] Hahn, LJ; Kloiber, R; Leininger, RW; Vimy, MJ; Lorscheider, FL. Dental "silver " tooth fillings: a source of Hg exposure revealed by whole body scan and tissue analysis. FASEB J, 3:2641-6, 1989.
[xcvi] Hahn, LJ; et al. Whole-Body Imaging of the Distribution of Hg Released from Dental Fillings into Monkey Tissues. FASEB J. 4:3256-609 1990.
[xcvii] Danscher, G; et al. Traces of Hg in Organs from Primates with Hg-Am Fillings Experim Molec Pathol,:291-9, 1990.