Mercury toxicity and antioxidants: part I: role of glutathione and alpha-lipoic acid in the treatment of mercury toxicity - Me

Mercury exposure is the second-most common cause of toxic metal poisoning. Public health concern over mercury exposure, due to contamination of fish with methylmercury and the elemental mercury content of dental amalgams, has long been a topic of political and medical debate. Although the toxicology of mercury is complex, there is evidence for antioxidant protection in the prevention of neurological and renal damage caused by mercury toxicity. Alpha-lipoic acid, a coenzyme of pyruvate and alpha-ketoglutarate dehydrogenase, has been used in Germany as an antioxidant and approved treatment for diabetic polyneuropathy for 40 years. Research has attempted to identify the role of antioxidants, glutathione and alpha-lipoic acid specifically, in both mitigation of heavy metal toxicity and direct chelation of heavy metals. This review of the literature will assess the role of glutathione and alpha-lipoic acid in the treatment of mercury toxicity. Mercury: Sources of Exposure According to the Agency for Toxic Substances and Disease Registry (ATSDR) of the U.S. Department of Health and Human Services, mercury is listed as the third-most frequently found (lead and arsenic are first and second), and the most toxic substance in the United States. (1) This figure originates from the U.S. Government's Priority List of Hazardous Substances. This list includes, in order of priority, substances that have been found at hazardous waste sites on the National Priorities List (Superfund sites) that "pose the most significant potential threat to human health due to their known or suspected toxicity and the frequency of exposure." Of 1,467 hazardous waste sites listed on the National Priorities List in 1998, toxic levels of mercury were identified in 714. Mercury toxicity is also considered the second-most common cause of acute heavy metal poisoning, with 3,596 cases reported in 1997 by the American Association of Poison Control Centers. (2) Annual worldwide emissions of mercury into the atmosphere have been estimated at 2,200 metric tons. (3) One-third of these emissions are estimated to originate from natural sources (volcanic eruptions and decay of mercury-containing sediment) and two-thirds from manmade sources. Twenty-five percent of total worldwide emissions come from fossil fuel combustion. In the United States, 26 percent (64.7 tons/year) of atmospheric mercury emissions come from medical waste incineration, such as cremation. (4) There are currently 1,782 advisories (one per body of water) issued by the U.S. Environmental Protection Agency (EPA) in 41 states in the United States restricting the consumption of any locally caught fish or shellfish due to their mercury content. Sixteen states have issued statewide or statewide-coastal advisories recommending restricting the consumption of fish caught in the state or along the coastline due to methylmercury contamination. (4) The Environmental Working Group, in a presentation to the Food Advisory Committee of the U.S. Food and Drug Administration (FDA), recently presented data warning of the consequences for fetuses of women who follow the current FDA's fish consumption advisory and eat 12 ounces of "safe" fish per week. The Environmental Working Group estimates that more than 25 percent of children in utero in the United States would be exposed to levels of mercury above the EPA sale reference dose (0.1 [micro]g methylmercury/kg body weight/day) for at least 30 days during gestation and would have an increased risk for neurological damage. (5) The ATSDR considers anyone who lives in close proximity to a former mercury mining site, recycling facility, municipal or medical incinerator, or coal-fired electric generating plant to be at risk for mercury toxicity. Anyone who routinely consumes contaminated fish, subsistence hunters who consume meat or organ tissues of marine mammals or feral wildlife, individuals with a "large number" of dental amalgams, pregnant or nursing women (and their developing fetuses and breast-fed babies), those who use consumer products containing mercury (skinlightening creams or antiseptic facial products, mercury containing diuretics or laxatives, and teething powders), or those living or working in buildings painted with mercury-containing latex paint are also considered at significant risk. Mercury-containing latex paint was removed from paint manufacturing in 1991 but may still be available in the reserve inventories of contractors and warehouses. (4) Mercury is found in the environment in three basic states: elemental mercury or mercury vapor, inorganic mercury, and organic mercury (ethyl-, methyl-, alkyl-, or phenylmercury). Each form has an individual toxicological profile and metabolic fate. The most frequent source of mercury exposure is open to debate. On an individual exposure basis, the estimated intake and retention of elemental mercury vapor (from dental amalgams and atmospheric pollution) in non-occupationally exposed individuals has a much broader range (3.9-21.0 [micro]g/day) than either inorganic (4.3 [micro]g/day) or methylmercury (1-6 [micro]g/day) exposure. (6) Elemental Mercury Elemental mercury, found in thermometers, thermostats, dental amalgams, and mercury added to latex paint, eventually enters a vaporized state. Eighty percent of inhaled elementary mercury vapor is absorbed and can cross the blood-brain barrier or reach the placenta. (2) Mercury vapor in the gastrointestinal tract is converted to mercuric sulfide and excreted in the feces. (6) Mercury vapor in the kidneys, however, the main repository for elemental mercury, is carried to all parts of the central nervous system as a lipid-soluble gas. Mercury vapor can also be oxidized to inorganic mercury by catalase and can attach to the thiol groups in most proteins--enzymes, glutathione, or almost any structural protein. (7) Elemental mercury can also be methylated by microorganisms in soil and water and potentially the human gastrointestinal track, (8) where it can then be transformed into organic methylmercury, the form found in fish, fungicides, and pesticides. Elemental mercury and its metabolites have the toxic effect of denaturing biological proteins, inhibiting enzymes, and interrupting membrane transport and the uptake and release of neurotransmitters. (7) Chronic exposure most commonly manifests as a triad of increased excitability and irritability, tremors, and gingivitis. (2) Less commonly, chronic exposure causes central and peripheral nervous system damage, manifesting as a characteristic fine tremor of the extremities and facial muscles, emotional lability, and irritability. Rarely, significant exposure can cause acrodynia or "pink disease," involving a pink rash on the extremities, pruritis, paresthesias, and pain. (9) Inorganic Mercury Inorganic mercury (mercury salts) is found in cosmetic products, laxatives, teething powders, diuretics, and antiseptics. (2) Inorganic mercury can be formed from the metabolism of elemental mercury vapor or methylmercury. (7) Although inorganic mercury does not normally reach the placenta or cross the blood brain barrier, it has been found in the neonatal brain due to the absence of a fully formed blood-brain barrier. (6) Inorganic mercury is complexed with glutathione in the liver and secreted in the bile as a cysteine-mercury or glutathionemercury complex. Chronic exposure to inorganic mercury salts primarily affects the renal cortex (10) and may manifest as renal failure (dysuria, proteinuria, hematuria, oliguria, and uremia) or gastrointestinal problems (colitis, gingivitis, stomatitis, and excessive salivation). Irritability and occasionally acrodynia can occur. (2) Organic Mercury Considered the most toxic and most frequent form of mercury exposure, organic mercury is found in fish, poultry that has been led fishmeal, pesticides, fungicides, insecticides, and thimerosal-containing vaccines. Thimerosal, which is 49.6-percent ethylmercury (a form of organic mercury), has been used as a preservative in vaccinations since the 1930s. It is currently mixed with DTaP, HIB, and hepatitis B vaccines or is used in the manufacturing process for vaccines, with resultant trace amounts being present in the final product. Based on existing Centers for Disease Control (CDC) recommendations for vaccinations, a typical six-month-old child, if receiving all thimerosal-containing vaccines, could potentially be injected with as much as 187.5-200 [micro]g of methylmercury; the equivalent of more than 1.0 [micro]g per day. This amount exceeds the reference limits for exposure to mercury set by the EPA of 0.1 [micro]g/kg/day. (11) In the United States, at the FDA's request, all vaccines are currently being produced as thimerosal-free or thimerosal-reduced (> 95-percent reduction) vaccines. Thimerosal-preserved vaccines are still available and used in clinical practice. Methylmercury is almost completely absorbed (95-100 percent) in the human gastrointestinal tract, (2,7) 90 percent of which is eventually eliminated through the feces. Methylmercury is present in the body as a water-soluble complex, mainly with the sulfur atom of thiol ligands, (7) and crosses the blood-brain barrier complexed with L-cysteine in a molecule resembling methionine. Methylmercury is absorbed into the placenta and stored in the fetal brain in concentrations that exceed maternal blood levels. (12) After being released from cells in a complex with reduced glutathione, methylmercury is degraded in the bile duct to an L-cysteine complex. Only 10 percent of methylmercury is eliminated through the kidneys. The rest either undergoes enterohepatic recycling or demethylation by microflora in the intestine and immune system and eventual elimination through the feces. Most methyl mercury in animal exposure studies is degraded to, and eliminated as, inorganic mercury at the rate of one percent per day. (7) At least one study has demonstrated the capacity of two common forms of gastrointestinal yeast to convert inorganic mercury to methylmercury. (8) Demethylation by intestinal microflora is a crucial step in the elimination of methylmercury from the body, but research has not yet identified the mechanisms or the microbes responsible for this detoxification system. (7) Enterohepatic reabsorption is also a significant event in the metabolism of methylmercury; more than 70 percent is reabsorbed from the gut and returned to the liver. (7,13) Inorganic mercury has been found as the major form of mercury in brain tissue in humans fatally exposed to methylmercury. (14) The conversion of methylmercury to inorganic mercury is thought to take place in phagocytic cells in the liver or in the astroglial cells of the brain. (7) The majority of toxicity due to methylmercury exposure involves the central nervous system. Methylmercury can cause demyelination, autonomic dysfunction, sensory nerve conduction delay, abnormal neuronal migration, and abnormal central nervous system cell division. Chronic toxicity symptoms include paresthesia, peripheral neuropathy, cerebellar ataxia, akathisia, spasticity, memory loss, dementia, constricted vision, dysarthria, impaired hearing, smell and taste, tremors, and depression. (2,7) Methylmercury exposure also appears to increase risk for cardiovascular disease. In a longterm prospective study, both intake of nonfatty freshwater fish and hair mercury content demonstrated a statistically significant correlation with increased risk for acute myocardial infarction. (15) Men with the highest hair mercury had a 2.9-fold increased risk for cardiovascular death. An examination of the same cohort found a significant correlation between hair mercury and increased risk for progression of carotid atherosclerosis. (16) Prenatal exposure to methylmercury has been correlated with significant blood pressure elevations in seven-year-old children as a result of maternal fish intake. (17) Ethylmercury (fungicides, thimerosal in vaccines, and gamma-globulin) also causes renal and central nervous system toxicity and is deposited in the liver, kidneys, skin, brain, spleen, and plasma. (7) Ethylmercury, like methylmercury, is metabolized to the inorganic form and accounts for 50 percent of the mercury eliminated in urine. Ethylmercury may actually be converted to inorganic mercury in the tissues in greater amounts and more rapidly than methylmercury. (7) As with methylmercury, the feces are the main natural route of elimination. Table 1 summarizes the forms of mercury and their pharmacokinetics. Mechanisms of Mercury Toxicity Mercury can cause biochemical damage to tissues and genes through diverse mechanisms, such as interrupting intracellular calcium homeostasis, disrupting membrane potential, altering protein synthesis, and interrupting excitatory amino acid pathways in the central nervous system. (19) Mitochondrial damage, lipid peroxidation, microtubule destruction), (20) and the neurotoxic accumulation of serotonin, aspartate, and glutamate are all mechanisms of methylmercury neurotoxicity. (19) Over time, both methylmercury and elemental mercury vapor in the brain are transformed to inorganic mercury, and become firmly bound to sulfhydryl-containing macromolecules. (21) Both methylmercury and inorganic mercury bind to various molecular weight thiol-containing proteins (glutathione, cysteine, albumin, etc.). The binding and dissociation of these mercurythiol complexes are believed to control the movement of mercury and its toxic effects in the body. (7) Mitochondrial damage from oxidative stress may be the earliest sign of neurotoxicity with methylmercury. A study in neural tissue indicates the electron transport chain appears to be the site where free radicals are generated, leading to oxidative damage induced by methylmercury. (19) Mercury-Thiol Binding Because the stability constants (energy necessary to form and break bonds) for mercury and thiol complexes (glutathione, albumin, cysteine, etc.) are so high, mercury will bind to any free thiol available and the thiol in the highest concentration will be the most frequently-bound. (22) The reaction rate is almost instantaneous. (7) Although the mercury-sulfhydryl bond is stable, it is labile in the presence of other free sulfhydryl groups; therefore, methylmercury will be redistributed to other competing sulfhydryl-containing ligands. (23) This is the basis for chelation of heavy metals with sulfhydryl compounds like DMPS and DMSA--providing free sulfhydryl groups in high concentrations to encourage the metal to move from one sulfhydrylcontaining ligand to another. The endogenous thiol-containing molecules--glutathione, cysteine, homocysteine, metallothionein, and albumin--all contain reduced sulfur atoms that bind to mercuric ions and determine the biological fate of mercury compounds in the body. (24) The complex of methylmercury and cysteine may act as a "molecular mimic" for the amino acid methionine and gain entry into the central nervous system via the same mechanism methionine uses to cross the blood-brain barrier. (25) Endogenous thiols transport mercury compounds and act to protect them from binding to other proteins, preventing functional damage in that tissue. In general, the higher the cysteine or thiol concentration in a cell medium, the lower the concentration of intracellular divalent mercury. In other words, higher concentrations of thiols appear to protect against accumulation of mercury, both in vivo and in vitro. (22) Glutathione in Heavy Metal Binding Glutathione is the most common low-molecular weight sulfhydryl-containing compound in mammalian cells, present in millimolar amounts in most cells. (26) As a result of the binding of mercury to glutathione and the subsequent elimination of intracellular glutathione, levels of reduced glutathione are lowered in several specific types of cells on exposure to all forms of mercury. Glial cells, (27) human erythrocytes, (28) and mammalian renal tissue (24) have all been found to have significantly lowered levels of reduced glutathione, a major source of oxidant protection. Mercury, as well as cadmium, generates highly toxic hydroxyl radicals from the breakdown of hydrogen peroxide, which further deplete glutathione stores. (27) There is evidence that glutathione depletion can lead to neurological damage; low levels of glutathione have been found in Parkinson's disease and cerebral ischemia-reperfusion injury. (29) Glutathione, as both a carrier of mercury and an antioxidant, has three specific roles in protecting the body from mercury toxicity. First, glutathione, specifically binding with methylmercury, forms a complex that prevents mercury from binding to cellular proteins and causing damage to both enzymes and tissue. (30) Glutathione-mercury complexes also reduce intracellular damage by preventing mercury froth entering tissue cells and becoming an intracellular toxin. Second, glutathione-mercury complexes have been found in the liver, kidney, and brain, and appear to be the primary form in which mercury is transported and eliminated from the body. (24) The transport mechanism is unclear, but complexes of glutathione and mercury are the predominant form of mercury in both the bile and the urine. (31) Glutathione and cysteine, acting as carriers of mercury, actually appear to control the rate of mercury efflux into bile; the rate of mercury secretion in bile appears to be independent of actual bile flow. When bile flow rate is increased or decreased, the content of mercury in the bile changes inversely so net mercury efflux from the liver remains unchanged. (32) However, increasing bile levels of both glutathione and cysteine increases the biliary secretion of methylmercury in rats. (13) Other studies have confirmed this data in animal models. (33-35) Conversely, glutathione depletion inhibits biliary secretion of methylmercury in animal models and blocking glutathione production appears to shut down biliary release of mercury. (35) Cells of the blood-brain barrier (brain capillary endothelial cells) release mercury in a glutathione complex. Inhibiting glutathione production in these cells inhibits their ability to release mercury. (23) Mercury accumulates in the central nervous system primarily in astrocytes, the cells that provide the first line of defense for the central nervous system against toxic compounds. (36) Astrocytes are the first cells in brain tissue to encounter metals crossing the blood brain barrier. They also contain high levels of metallothionein and glutathione, both carriers for heavy metals. It is hypothesized that astrocytes are the main depot of mercury in the brain. (37) In studies with astrocytes, the addition of glutathione, glutathione stimulators, or glutathione precursors significantly enhances the release of mercury from these cells in a complex with glutathione. Fujiyama et al (38) also suggest that conjugation with glutathione is the major pathway for mercury efflux from astrocytes. Glutathione also increases mercury elimination from renal tissue. Studies in mammalian renal cells reveal glutathione is 50 percent as effective as the chelating agent DMSA (2,3-dimercaptosuccinic acid) in preventing inorganic mercury accumulation in renal cells. (39) Third, glutathione increases the antioxidant capacity of the cell, providing a defense against hydrogen peroxide, singlet oxygen, hydroxyl radicals, and lipid peroxides produced by mercury. (30) The addition of glutathione to cell cultures exposed to methylmercury also prevented the reduction of cellular levels of glutathione peroxidase, a crucial antioxidant enzyme necessary for protection against the damaging effects of lipid peroxidation. (30) As an antioxidant, glutathione appears to protect against renal damage resulting from inorganic mercury toxicity. The co-incubation of rat renal cells with glutathione and inorganic mercury was significantly more protective of renal cell injury when compared to inorganic mercury exposure alone. (40) Antioxidant levels--specifically glutathione, vitamin E, and ascorbic acid--are depleted in renal tissue exposed to mercuric chloride (inorganic mercury), and the addition of glutathione increased levels of both vitamin E and ascorbic acid in renal cells exposed to mercuric chloride. (24) Mammalian cell lines resistant to mercury toxicity have been cloned. (41) They do not readily accumulate mercury and are resistant to the toxic effects of methylmercury or inorganic mercury. An outstanding characteristic of this cell line is that glutathione levels are five times greater in these cells than the parent cells from which they originated. The authors of this study conclude that the mechanisms of resistance were primarily due to glutathione's ability to facilitate mercury efflux from cells and the protective binding of mercury by glutathione to prevent cellular damage. The Role of alpha-Lipoic Acid In 1966, German physicians began using alpha-lipoic acid (ALA) therapeutically in patients with diabetic polyneuropathy and liver cirrhosis because of their observation that these patients had lower levels of circulating lipoic acid. (42) The application was subsequently extended to heavy metal intoxication and toxic mushroom poisoning. According to Jones and Cherian, (43) an ideal heavy metal chelator should be able to enter the cell easily, chelate the heavy metal from its complex with metallothionein or other proteins, and increase the excretion of the metal without its redistribution to other organs or tissues. Although no human clinical trial has investigated the use of ALA as a chelating agent in mercury toxicity, there is evidence ALA satisfies at least two of the above criteria; i.e., absorption into the intracellular environment and complexing metals previously bound to other sulfhydryl proteins. ALA produced endogenously is bound to proteins, but can also be found unbound in the circulation, after exogenous lipoic acid supplementation. (41) In this form it is chemically able to trap circulating heavy metals, thus preventing cellular damage caused by metal toxicity. (41) Lipoic acid is lipophilic and is able to penetrate cell membranes and reach high intracellular concentrations within 30 seconds of its administration. (44) The fact that free ALA crosses the blood-brain barrier is significant because the brain readily accumulates lead and mercury, where these metals are stored intracellularly in glial tissue. (36,45) Oral doses of 10 mg/kg ALA in rats have reached peak levels in the cerebral cortex, spinal cord, and peripheral nerves within 30 minutes of administration, and studies of chronic daily dosing conclude ALA reaches all areas of the CNS and peripheral nervous system. (46) ALA has been shown to decrease lipid peroxidation in brain and sciatic nerve tissue (47) and when given orally to rats, decreased lipid peroxidation in brain tissue by 50 percent. (46) In diabetic neuropathy, free lipoic acid may prevent glucose-related oxidative damage by entering nerve tissue where it acts as both an antioxidant and heavy metal-binding agent. (28) ALA has been administered to humans in doses up to 1,200 mg intravenously without toxicity, and in oral daily doses of as much as 600 mg three times daily. The only side effects reported are infrequent nausea and vomiting. No side effects have been reported in oral administration of up to 1,800 mg daily. (41,48) Doses of 500-1,000 mg have been well tolerated in placebocontrolled studies. (49) Extrapolation of pharmacokinetic and toxicity data demonstrate safe human dosages would not be exceeded with oral doses of several grams per day. (41) ALA has been shown to increase both intraand extracellular levels of glutathione in T-cell cultures, human erythrocytes, glial cells, and peripheral blood lymphocytes. (50) In rats, oral dosing of 150 mg/kg/day for eight weeks significantly increased glutathione levels in the blood and liver. (51) ALA has been shown to increase intracellular glutathione by 30-70 percent in murine neuroblastoma and melanoma cell lines, and in the lung, liver, and kidney cells of mice that had received intraperitoneal injections of 4, 8, or 16 mg/ kg ALA for 11 days. (52,53) Levels of intracellular glutathione have been shown to increase by 16 percent in T-cell cultures at concentrations of 10-100 [micro]M (concentrations achievable with oral and intravenous supplementation of ALA). (50) A single oral dose of 600 mg ALA was able to produce a serum concentration of 13.8 [+ or -] 7.2 [micro]M and levels of 100-200 [micro]M have been reported after 600 mg intravenous administration. (54) Increases in glutathione levels seen with ALA administration are not only from the reduction of oxidized glutathione (one of the functions of ALA) but also from the synthesis of glutathione. (46) ALA is reduced to dihydrolipoic acid (DHLA), itself a potent antioxidant. DHLA is able to regenerate oxidized ascorbate, glutathione, coenzyme Q, and vitamin E, (28) and is responsible for the ability of ALA to increase intracellular glutathione levels (Figure 1). (55)