Glutathione as an antioxidant in inorganic mercury induced nephrotoxicityAT Jan1, A Ali2, QMR Haq3
1 Department of Bioscience, Jamia Millia Islamia, New Delhi; Department of Biotechnology, Jamia Millia Islamia, New Delhi, India
2 Department of Biotechnology, Jamia Millia Islamia, New Delhi, India
3 Department of Bioscience, Jamia Millia Islamia, New Delhi, India
Heavy metal toxicity represents an uncommon but clinically significant medical condition, which if unrecognized or inappropriately treated results in significant morbidity and mortality. Among heavy metals, mercury is recognized as a potent and widely distributed toxicant having the ability to accumulate at various levels of food chain besides possessing ability to cross placental and blood-brain barrier. Symptom picture of mercury (Hg 2+ ) toxicity is characterized mainly by a series of renal disorders. Mechanism of inorganic mercury toxicity includes production of reactive oxygen species (ROS) capable of damaging lipids in membrane, proteins or enzymes in tissues, and DNA to induce oxidative stress as balance between generation, and elimination of ROS is essential for maintaining the functional integrity of a cell. Mitigation of endogenous mercury depends as a part on the presence of antioxidants such as glutathione - most abundant intracellular non-protein thiol that plays a central role in the maintenance of cellular redox status by quenching free radicals generated during oxidative stress. Ability of a cell to survive the threat posed by endogenous mercury represents a biological adaptation fundamental to survival. This review describes the current understanding and the mechanisms involved by different forms of mercury in eliciting their toxicity in kidney along with the knowledge of major intracellular reductant that plays important role in the mitigation of mercury toxicity for the maintenance of homeostasis within the body of living organisms.
Keywords: Glutathione, intracellular reductants, mercury toxicity, reactive oxygen species
Human evolution has led to immense scientific and technological progress. However, global development has raised new challenges especially in the field of environmental protection and conservation as demand for economic, agricultural, and industrial development has bypassed safety demand of natural environment.  Living organisms are continuously exposed to non-nutritional foreign chemical species that interact deleteriously with an organism, causing toxic and sometimes carcinogenic effects. Mercury, sixth most abundant toxic element present in the earth's crust as elemental form and as a variety of binary minerals such as cinnabar, is released into the environment as a result of both natural as well as anthropogenic activities.  In nature, mercury exists in three forms: metallic or elemental form (Hg 0 ), inorganic (Hg 2+ or Hg + ), and organic form (R-Hg + or R-Hg-X, where "R" is alkyl or phenyl moiety). Most of the mercury found in nature occurs as metallic and inorganic mercury compounds. They become part of air from mercury and mercury-based salt spills, e-wastes (CFL) mining deposits of ores containing mercury, emissions of coal-fired plants, incineration of municipal and medical wastes, and from untreated industrial effluents.  Elemental or metallic mercury has got its application in the extraction of gold from its ores, as an electrode in the production of chlorine and caustic soda from saline, and in equipments like thermometers, barometers, etc., while as inorganic salts of mercury are used as fungicides besides being used as an antiseptic or disinfectant agent.
Mercury pollution is an emerging problem in the present day world as mercury concentration is rising continuously as a result of increased industrial, medicinal, and domestic use. Tremendous increase in the use of mercury over the past few decades has resulted in an increased influx of mercury into the environment. Mercury levels prescribed by the bureau of Indian standards and WHO after a survey in 2003 are 0.001 mg/l for drinking water and 0.01 mg/l for industrial effluents.  Mercury levels in many parts of the world including India have reached at the verge of exceeding threshold level in soil as well as in water bodies and are still increasing because of agricultural runoff and sludge produced by industries and population centers.  It enters human body through a variety of sources, the most common being contaminated food and drinks, as a part of vaccines, or through direct contact with the skin. After entering the body, it gets accumulated in the target organs like kidneys, liver, and brain, whereby degrade health without being noticed or diagnosed. Enormous rise in mercury levels has become a growing concern for the developing world due to its high toxicity, translocation, and ability to get accumulated at various steps of food chain.  Mercury toxicity occurs as a part due to capability of mercury in its ionic form to bind with sulfhydryl, thioether, and imidazole groups of macromolecules whereby resulting in their inactivation. , Compared to organic, transport of inorganic mercury across the biological membrane occurs with the involvement of certain protein carriers present as integral part of membrane. Inorganic mercury exerts its toxic consequences mainly on getting methylated to methylmercury compounds (non-enzymatically by bacteria through the transfer of methyl group from methylcobalamin to mercuric ion), having ability to get accumulated in soft target organs main being brain and kidneys. ,, Mercury exerts its nephrotoxic effects through alterations in intracellular thiol status in turn associated with the generation of free radicals, disruption of protein synthesis leading to inactivation of various enzymes, structural protein alteration that affect transport functions by disturbing cell membrane permeability and by causing functional abnormalities of T-lymphocytes.
Various journals were consulted at national medical library. Besides that, Pubmed was also used for locating, selecting, and extracting data. Papers in last 20 years were mostly consulted. Above 150 papers were searched, but only 43 were relevant.
Search words: Heavy metal toxicity, mercury toxicity, heavy metal induced oxidative stress, glutathione as an antioxidant, antidotes for mercury toxicity.
Risk of humans being exposed to mercury is significant. Humans are exposed to mercury not only at occupational and environmental settings but also through dental amalgams, and medicinal and dietary sources ( http://www.atsdr.cdc.gov ).  Occupational mercury exposures generally occur when workers inhale metallic mercury vapors. Upon significant inhalation exposure to metallic mercury vapors, people (primarily children) suffer from a disease known as "Kawasaki" disease, characterized by fever, photophobia, pharyngitis, oral lesions, skin rashes, and peripheral extremity changes. ,, Other symptoms may include kidney dysfunction (Fanconi syndrome) or neuropsychiatric symptoms (Bradley Coyne syndrome) such as emotional lability, memory impairment, or insomnia. Nature and severity of toxicity resulting from mercury exposure are functions of the magnitude and duration of exposure, route of exposure, and form of the mercury or mercury compound to which exposure occurs.  It is primarily the differences in the delivery to target sites that result in the spectrum of effects. Since the ultimate toxic species for all mercury compounds is thought to be mercuric ion, the kinetics of the parent compound is the primary determinant of the severity of mercury toxicity.
Mercuric ions have a greater affinity to bond with reduced sulfur atoms especially those present on the endogenous thiol-containing molecules such as glutathione (GSH), cysteine, metallothionein, homocysteine, N-acetylcysteine, S-adenosyl-methionine, and albumin [Figure 1]. The affinity constant for mercury binding to thiolate anions is of the order of 10 15 -10 20 . High-affinity binding of the divalent mercuric ion to thiol or sulfhydryl groups of proteins is believed to be a major mechanism for the biological activity of mercury as sulfhydryl groups constitute an integral part in the structure or function of proteins present in both extracellular and intracellular membranes and organelles. , Besides that, it has also been found that binding of mercury may also occur to other sites (e.g., amine, carboxyl groups) generally less favored than sulfhydryl groups. However, affinity constant for mercury bonding to oxygen (e.g., carbonyl) or nitrogen (e.g., amino) containing ligands is approximately 10 times lower than that for the thiol-containing compounds. Therefore, biological effects of inorganic or organic mercury are related to interactions mostly with sulfhydryl-containing residues. Within the kidney, pars recta of proximal tubule of nephron is most vulnerable to the toxic effects of mercury. Toxicological activity of mercurous and mercuric ions in the kidney is largely driven by molecular interactions that occur at nucleophillic sites in cells. Molecular interactions of mercury with the sulfhydryl groups of thiol-containing molecules have been implicated in mechanisms involved in the proximal tubular uptake, accumulation, transport, and toxicity of mercuric ions.
Molecular mimicry with endogenous substrates plays a role in the transport of mercury to different cells and tissues.  Hg 2+ -GSH complex, that is, dicysteinyl-Hg, resembles with endogenous compound cystine while as that of CH 3 -Hg with cysteine, CH 3 -Hg-Cys, resembles with methionine. , Both these mercury complexes compete with carrier proteins of cystine and methionine for their transport in to the cell and tissues. It is supposed to be the possible explanation for specific transport and tissue-specific accumulation of these two forms of mercury over proximal tubular epithelia into the kidneys and into endothelial cells of blood-brain barrier. A large number of studies have suggested that mercury (Hg 2+ ) interaction with thiol-containing compounds following entry into the proximal tubular epithelial cell results in the alteration of membrane permeability to calcium ions and inhibition of mitochondrial function. Mercury-induced alterations in mitochondrial calcium homeostasis has been found to exacerbate Hg 2+ -induced oxidative stress in kidney cells. ,, Oxidative damage to kidneys results in numerous biochemical changes such as excretion of excess porphyrins in the urine (porphyrinuria) as mercury-thiol complexes possess redox activity, promoting oxidation of porphyrinogen.  Acute renal failure resulting from mercury exposure has been proposed to result from decreased renal reabsorption of sodium and chloride in proximal tubules and increased concentrations of these ions at the macula densa. This increase in ions at the macula densa in turn results in the local release of renin, vasoconstriction of the afferent arteriole, and filtration failure.
Apart from the currently accepted models of mercuric conjugates of GSH and cysteine being primarily involved in the luminal and basolateral uptake of inorganic mercury along the proximal tubule (after exposure to mercuric chloride), it is clear that other thiols, especially homologues of cysteine, such as homocysteine and N-acetylcysteine, can significantly influence the manner in which inorganic mercury is being handled in the kidney. It has been found that inorganic mercury on administration with cysteine into kidneys does not affect the luminal or basolateral uptake of mercury. However, when inorganic mercury was administered with homocysteine, much lower uptake occurs at the luminal surface than at basolateral membrane. Compared to inorganic mercury administered with homocysteine, when administered with N-acetylcysteine, greater difference in the levels of luminal versus basolateral was observed. In short, if inorganic mercury is administered with negatively charged molecule, all renal uptake of mercury occurs at the basolateral membrane as it prevents or impedes intake of mercury conjugates at luminal surface, thereby promoting urinary excretion of mercury. The urine and feces are the main excretory pathways of metallic and inorganic mercury in humans, with a body burden half-life of approximately 1-2 months.  Elimination of metallic and inorganic mercury in animals occurs predominantly in the fecal matter following inhalation of metallic mercury vapor, but as mercury concentrations increase in the kidneys, urinary excretion increases. Excretion of metallic mercury via, exhalation accounts for only 10-20% of the total excreted metallic mercury.  Elimination rate of inorganic mercury from the body is same as the rate of elimination from the kidneys, where most of the body burden is localized.
Under normal physiological conditions, balance between generation and elimination of ROS maintains the functional integrity of a cell as altered redox homeostasis leads to oxidative stress. Oxidative stress, an imbalance between free radical generation and the antioxidant defense system, represents a common threat and danger for all aerobic organisms. Reactive oxygen species (ROS) like H 2 O 2 and superoxide anions generated continuously get converted into highly reactive hydroxyl radical via Fenton and Haber-Weiss type reactions in the presence of redox active transition metals. ,
O 2 + e - → O 2· -
2O 2· - + H + →H 2 O 2 + O 2
H 2 O 2 → 2·OH
Fe 3 + + O 2· - → Fe 2+ + O 2
Fe 2+ + H 2 O 2 → Fe 3 + + ·OH + OH - (Fenton reaction)
O 2· - + H 2 O 2 → O 2 + ·OH + OH - (Haber-Weiss reaction)
NO + O 2· - → ONOO - + H + → ·OH + ·NO 2
R-H + ·OH → R· + H 2 O
R· + O 2 → ROO·
ROO· + R-H ; ROOH + R·
ROOH + Fe 2+ → RO· + - OH + Fe 3+
Reduced GSH, a low-molecular-weight tripeptide (γ-glutamylcysteinyl-glycine) is the most abundant source of nonprotein thiols in mammalian cells.  It is primarily synthesized in liver from its three amino acid precursors - l-glutamate, l-cysteine, and glycine - via a complete pathway starting from methionine through homocysteine and cysteine to GSH. Glutamate cysteine ligase (formerly called γ-glutamylcysteine synthase) catalyzes the rate-limiting step of GSH synthesis. GSH synthesis from cysteine and glutamate via glutamylcysteine occurs at the expense of two ATP molecules. Normal GSH content of a cell that is imperative to maintain balance between depletion and synthesis ranges from 1 to 10 mM.  GSH form metal complexes via non-enzymatic reactions. Once mercuric conjugates of GSH are formed in hepatocytes, they enter into systemic circulation whereby they are delivered to kidneys. Bonding characteristic between mercuric ions and thiol-containing molecules in plasma appears labile within the body of living organisms which results in rapid decrease in the plasma burden of mercury with an increase in the concurrent uptake of mercuric ions by kidneys through various mechanisms [Figure 2]. GSH acts both as a carrier as well as an antioxidant in kidney as it performs a complex role in the regulation of renal cellular disposition and cytotoxicity of Hg 2+ .  It not only protects renal cells from Hg 2+ -induced cellular injury by preventing its binding to other essential cellular thiols, but also enhances the renal cellular accumulation of mercury as mercury uptake in renal epithelial cells occur primary as conjugates with extracellular GSH rather than as free Hg 2+ ions. Astrocytes and neurons do not express the enzyme γ-cystathionase and therefore are unable to synthesize cysteine. As a result, astrocytes and neurons are dependent on plasma cysteine derived primarily from the liver to synthesize GSH. 
Metal bound to sulfhydryl group of GSH is stabilized if it gets coordinated to other binding sites present within the tripeptide. Generally, metals form stable structures when they form 1:2 metal sulphydryl complexes. Compared to organic mercurials such as methylmercury having tendency to form 1:1 complex with the thiol-containing molecules, mercuric ions have a high propensity toward the linear coordination of two such molecules in a situation of abundant thiol-containing molecules, that is, each mercuric ion will bind to two molecules of GSH through sulfur atom on the cysteinyl residue of GSH molecules.  GSH acts as an important line of defense against oxidative stress. It performs a series of important physiological and metabolic functions in mammalian cells particularly being detoxification of free radicals, metals, and other electrophilic compounds.  Mitochondria lacking enzymes necessary for GSH synthesis imports it from cytosol. It increases antioxidant capacity of mitochondria, thereby protects it by providing defense against H 2 O 2 , singlet oxygen, hydroxyl radicals, and lipid peroxides generated by mercury.
Intracellular levels of oxidized glutathione (GSSG) increase from the metabolism of H 2 O 2 by GSH peroxidase and decrease from export of GSSG from cell and from GSH reductase and NADPH-mediated reconversion of GSSG to GSH. Under severe oxidant stress, increase in GSSH promotes the oxidation of cellular protein cysteinyl thiols, ultimately resulting in impaired protein function.  GSSG is associated with an increase in the activity of glucose-6-phosphatase, acid phosphatases, and fructose-1,6-bisphosphatase enzymes and inhibition of pyruvate kinase, adenylate cyclase, ribonucleotide reductase, phosphofructokinase, and fatty acid synthase activity. , Hg 2+ -mediated GSH depletion creates an oxidative stress condition characterized by increased susceptibility of the mitochondrial membrane to iron-dependent lipid peroxidation. Epithelial cell damage is believed to occur as the result of enhanced free radical formation and lipid peroxidation. , Depletion of mitochondrial GSH and increases in mitochondrial hydrogen peroxide at the inner-mitochondrial membrane contribute to acceleration of the turnover of potassium and magnesium ions.  Thiol-disulfide equilibrium within a cell regulates metabolic pathways by activating or inactivating key enzymes. As thiol transfer reactions are bidirectional, equilibrium is determined by redox state of the cell. Many enzymes in antioxidant defense systems protect imbalance between pro-oxidant and antioxidant. Antioxidant enzymes such as GSH reductase (GR), GSH peroxidase (GPx), superoxide dismutase, etc. containing sulfhydryl groups at their active sites besides carrying essential metal ions such as zinc, selenium, etc. as cofactors, are more prone to attack by mercury that ultimately leads to suspension of their activities. ,,
GSH being synthesized in the cell cytosol, its degradation occurs outside the cell with the involvement of two membrane-bound enzymes: γ-glutamyltransferase and cysteinylglycinase. γ-Glutamyltransferase catalyzes γ-glutamylcysteine bond in GSH. It is the only enzyme that removes γ-glutamyl moiety from GSH under physiological conditions. Cysteinylglycinase causes the removal of glycine moiety. Breakdown products of GSH (glutamate, cysteine, and glycine) enter the cell through defined transporter for use in GSH synthesis. A pivotal role for extracellular GSH and membrane-bound γ-glutamyltransferase has also been identified in the renal disposition, toxicity, and excretion of inorganic mercury (Hg 2+ ) in rats.  Evidences supporting the role of γ-glutamylcysteine in renal tubular uptake of mercury comes from in vivo experiments performed with acivicin. Exposure to acivicin causes inhibition of γ-glutamylcysteine, thereby showing profound effect on renal disposition of mercury followed by subsequent increase in urinary excretion of mercury. ,
Mercury - a metal with a widespread use in industries and agriculture - has been recognized as one of the most toxic element, principally in relation to its series of effects on humans following acute or prolonged occupational exposure or from a number of environmental accidents. It has created large havocs in the past, particular being the minimata disaster of Japan and of Iraq. Exposure to mercury is an inescapable consequence of human life. Despite toxic potential of mercury being widely known, its existence in the environment and in several man-made applications makes human exposure inevitable. Concerns regarding potential risk to human population from environmental sources are growing at a steady rate. Mercury toxicity is partly due to its ability to produce a variety of deleterious health effects, ranging from single to multiple target effects inside the body of living organisms. Exposure to mercury has profound effect on renal cellular function and consequently on renal handling of mercury.
Current contentious issue on health risks of mercury underlies a major public health dilemma. Most fascinating of the mercury mysteries is bonding to biological molecules such as proteins, thereby modulating their reactivity as well as their biological effects. Once incorporated in an organism, its physiological and toxicological effects are regulated by two general mechanisms: binding to specific ligands (chelating agents) such as cysteine, homocysteine, GSH, etc., and excretion. Drugs tentatively used as specific chelators against mercury include British Anti-lewisite (BAL), meso-2,3-dimercapto succinic acid (DMSA), sodium salt of 2,3-dimercapto-1-propane sulfonic acid (DMPS), d-penicillamine, etc. However, for being an effective therapeutic chelating agent, it needs to be water-soluble as lipophillic chelators often have a redistribution effect to different organs of the body. GSH, being one of the most versatile and pervasive mercury-binding tripeptide of γ-glutamylcysteinyl-glycine, plays an important role in mercury transport, storage, and distribution. Manipulation of intracellular thiols alters accumulation of mercury and as such modulates the effect of mercury at the target site. GSH acts as an important line of defense against oxidative stress as it increases antioxidant capacity of mitochondria, thereby protects it by providing defense against H 2 O 2 , singlet oxygen, hydroxyl radicals, and lipid peroxides generated by mercury. In biological system, binding of mercury with specific ligands such as GSH provides concentration-dependent protection from mercury-induced cytotoxicity as conjugation with GSH limits and regulates its reactivity besides facilitating its transport and elimination from the cell. Despite numerous studies attempted to elucidate the mechanisms implicated in mercury toxicity, further studies are still needed in order to improve pharmacological treatment. Chelating agents such as GSH are the only drugs nowadays available to limit metal toxicity. However, their use is often limited by their lack of selectivity as they cause removal of essential metal ions, thereby making it urgent to identify novel natural substituents that allow the removal of toxic mercury from the body without affecting physiological ionic homeostasis.
Authors would like to acknowledge CSIR, India, for financial assistance in terms of SRF to one of the fellow (Arif Tasleem Jan).
[Figure 1], [Figure 2]