Biomedical Implications of Heavy Metals Induced Imbalances in Redox Systems

Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 640754, 26 pages http://dx.doi.org/10.1155/2014/640754

Review Article Biomedical Implications of Heavy Metals Induced Imbalances in Redox Systems

Bechan Sharma,1 Shweta Singh,2 and Nikhat J. Siddiqi3

1 Department of Biochemistry, University of Allahabad, Allahabad 211002, India 2 Department of Genetics, SGPGIMS, Lucknow 226014, India 3 Department of Biochemistry, King Saud University, Riyadh 11451, Saudi Arabia

Correspondence should be addressed to Bechan Sharma; [email protected]

Received 28 February 2014; Revised 28 May 2014; Accepted 10 July 2014; Published 12 August 2014

Academic Editor: Hartmut Jaeschke

Copyright © 2014 Bechan Sharma et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Several workers have extensively worked out the metal induced toxicity and have reported the toxic and carcinogenic effects of metals in human and animals. It is well known that these metals play a crucial role in facilitating normal biological functions of cells as well. One of the major mechanisms associated with heavy metal toxicity has been attributed to generation of reactive oxygen and nitrogen species, which develops imbalance between the prooxidant elements and the antioxidants (reducing elements) in the body. In this process, a shift to the former is termed as oxidative stress. The oxidative stress mediated toxicity of heavy metals involves damage primarily to liver (hepatotoxicity), central nervous system (neurotoxicity), DNA (genotoxicity), and kidney (nephrotoxicity) in animals and humans. Heavy metals are reported to impact signaling cascade and associated factors leading to apoptosis. The present review illustrates an account of the current knowledge about the effects of heavy metals (mainly arsenic, lead, mercury, and cadmium) induced oxidative stress as well as the possible remedies of metal(s) toxicity through natural/synthetic antioxidants, which may render their effects by reducing the concentration of toxic metal(s). This paper primarily concerns the clinicopathological and biomedical implications of heavy metals induced oxidative stress and their toxicity management in mammals.

1. Introduction Heavy metals are natural components of the earth’s crust. These elements are the oldest toxins known to humans, Many different definitions for heavy metals have been pro- having been used for thousands of years. Potential sources posed, some based on density, some on atomic number or of heavy metals exposure include natural sources (e.g., atomic weight, and some on chemical properties or toxicity. groundwater, metal ores), industrial processes, commercial The group of heavy metals can include elements lighter products, folk remedies, and contaminated food and herbal than carbon and can exclude some of the heaviest metals. products. Virtually all heavy metals are toxic in sufficient One of such definitions entails that heavy metals are those quantities. Because of their concentrations in the environ- inorganic elements which have five times the specific gravity ment lead, mercury, and arsenic are of particular interest. of water. Among the heavy metals having serious health Entering our bodies by way of food, drinking water, and implications are arsenic, lead, cadmium, and mercury [1]. air, metals produce toxicity by forming complexes with Theaccumulationoftheseelementscancauseseveredamage cellular compounds containing sulfur, oxygen, or nitrogen. to mucus tissues and intestinal tract and skeletal, central The complexes inactivate enzyme systems or modify critical nervous, and reproductive systems. In addition to the toxic protein structures leading to cellular dysfunction and death. and carcinogenic effects of some metals in humans and The most commonly affected organ systems include animals, they also play pivotal role in mediating a balanced central nervous, gastrointestinal (GI), cardiovascular, biological functioning of cells. hematopoietic, renal, and peripheral nervous systems. The 2 BioMed Research International

Table 1: Brief description of some reactive oxygen species (ROS). Reactive oxygen species (ROS) Oxidant Description ROS contain nonradicals ...and radicals One-electron reduction state of O2,formed H O H in many autoxidation reactions and by the Molecules O O − electron transport chain. Rather unreactive Hydroxyl ∙O2 , H ∙ but can release Fe2+ from iron-sulfur Hydrogen radical (OH ) O O H superoxide proteins and ferritin. Undergoes peroxide (H2O2) anion Hydroperoxyl dismutation to form H O spontaneously or − − ∙ 2 2 radical (HO 2 ) by enzymatic catalysis and is a precursor for Ions: O Cl O O metal-catalyzed ∙OH formation. Hypochlorite Superoxide − ∙− Two-electron reduction state, formed by ion (OCl ) anion (O 2 ) ∙ − H2O2, hydrogen dismutation of O2 or by direct reduction peroxide of O2. Lipid soluble and thus able to diffuse across membranes. Figure 1: Reactive oxygen species (ROS) existing in radicals and Three-electron reduction state, formed by nonradicals forms. ∙OH, hydroxyl Fenton reaction and decomposition of radical peroxynitrite. Extremely reactive and will attack most cellular components. natureandseverityoftoxicityvarywiththeheavymetal ROOH, organic Formed by radical reactions with cellular involved, its exposure level, chemical and valence states hydroperoxide components such as lipids and nucleobases. (inorganic versus organic), mode of exposure (acute versus Oxygen centred organic radicals. Lipid RO∙,alkoxy, chronic), and the age of the individual. Children with their forms participate in reactions. Produced in and ROO∙, developing nervous systems are particularly vulnerable to the presence of oxygen by radical addition peroxy radicals heavy metal intoxication (especially lead). to double bonds or hydrogen abstraction. The heavy metals induced toxicity has been extensively Formed from H2O2 by myeloperoxidase. studied and reported by various workers. They have demon- HOCl, Lipid soluble and highly reactive. Will strated that the exposure of an animal to excess concen- hypochlorous readily oxidize protein constituents, trations of these elements may augment production of free acid including thiol groups, amino groups, and radicals which may cause oxidative stress [2–5]. In fact methionine. − generation of oxidative stress has been considered as one Formed in a rapid reaction between ∙O2 of the major mechanisms behind heavy metal toxicity. The and NO∙. Lipid soluble and similar in − heavy metals have potential towards production of the highly ONOO , reactivity to hypochlorous acid. Protonation reactive chemical entities such as free radicals having the peroxynitrite forms peroxynitrous acid, which can ability to cause lipid peroxidation, DNA damage, oxidation undergo homolytic cleavage to form of sulfhydryl groups of proteins, depletion of protein, and hydroxyl radical and nitrogen dioxide. several other effects [3]. The heavy metals have been found to generate reactive oxygen species (ROS), which in turn results in their toxic effects in the form of hepatotoxicity and pentavalent arsenate (As (V)), and elemental. Arsenite is ten neurotoxicity as well as nephrotoxicity in both animals and times more toxic than arsenate; elemental arsenic is non- humans [2, 4].Someofthereactiveoxygenspeciesareshown toxic. Arsenic also exists in three chemical forms: organic, in Figure 1. Their few characteristic properties and impact on inorganic, and arsine gas, with organic arsenic having little cellular systems are summarized in Table 1. toxicity whereas inorganic arsenic and arsine gas are toxic. Thisreviewsummarizesanupdatedaccountofavailable It is widely distributed in nature and utilizes its three most information on heavy metals induced imbalance in the redox common oxidation numbers (+5, +3, and –3) to form chemi- systems in the body and their clinical and pathophysiological cal complexes in body. A summary of environmental sources implications as well as use of certain natural and synthetic ofarsenicaswellastheirpotentialhealtheffectsismen- products to mitigate their toxicity. It illustrates toxic effects of tioned in the National Research Council report on arsenic only four key heavy metals such as arsenic, lead, cadmium, in drinking water. Most major US drinking water supplies and mercury, which are ubiquitous in air and water pollutants contain levels lower than 5 𝜇g/L. It has been estimated that that continue to threaten the quality of public health around about 350,000 thousand people might drink water containing the world. These elements are associated with many adverse more than 50 𝜇g/L [7]. Exposure to arsenic primarily occurs health effects generated through oxidative damage and dis- by ingestion, but inhalation and absorption through the skin ease processes [5]. are possible. Arsenic occurs naturally in seafood as nontoxic organic compounds, such as arsenobetaine, which can cause 2. Arsenic elevated urine arsenic levels. The lethal dose of inorganic arsenic has been estimated to be 0.6 mg/kg. Arsenic is particularly difficult to characterize as a single About 80–90% of a single dose of arsenite As (III) element because its chemistry is really complex. Arsenic or arsenate As (V) has been shown to be absorbed from occurs in three oxidation states: trivalent arsenite (As (III)), the gastrointestinal (GI) tract of humans and experimental BioMed Research International 3 animals. Arsenic compounds of low solubility such as arsenic As5+ Anion transporter Diffusion As3+ selenide, lead arsenide, and gallium arsenide are absorbed less efficiently than dissolved arsenic. Systemic skin toxicity As5+ Plasma membrane has been reported in persons having extensive acute dermal As3+ contact with solutions of inorganic arsenic [8]. The airborne arsenic is largely trivalent arsenic oxide. Its deposition in Activation of signal transduction pathway (JNK, MAP4K, MAP3K) airways and absorption from lungs are dependent on particle MEKK 2, 3, 4 size and chemical form [9].Ithasbeenreportedasanagent, MEKK 3, 4 which may indirectly activate other carcinogens responsible Transcriptional regulation to generate tumors into key organs of the body such as lungs, skin, liver, bladder, and kidney [10–14]. After absorption, Biochemical/physiological effects inorganic arsenic rapidly binds to hemoglobin in erythrocytes. Blood arsenic is redistributed quickly (within 24 h) to the liver, kidneys, heart, and lungs and to a lesser degree to Figure 2: Effect of activation by arsenate (As(V)) and arsenite the nervous system, GI tract, and spleen. Arsenic undergoes (As(III)) on the signal transduction pathways. As(V) and As(III) hepatic biomethylation to form monomethylarsonic and activate different proteins to regulate c-Jun N-terminal kinase dimethylarsinic acids that have relatively lower toxicity [15]. (JNK), which functions in the stress-activated protein kinase pathway (SAPK). A SAPK pathway is a sequential protein kinase cascade where mitogen-activated protein (MAP) kinase kinase kinase kinase 2.1. Arsenic Mediated Modulation of Transcription Factors. (MAP4 K) phosphorylates and activates a MAP kinase kinase Arsenichasbeenfoundtomodulatethefunctionsof kinsase (MAP3 K), which repeats the cycle by phosphorylating and transcription factors (as nuclear factor kappa-light chain activating the next kinase in the cascade. The small GTP binding enhancer of activated B-cells (NF-𝜅B), activator protein 1 proteins (G-proteins: Ras, Rac, Cdc-42, and Rho) are localized (AP-1), and tumor suppression protein (p53)). NF-𝜅B, AP- upstream of the sequential protein kinase cascade. The anion 1, and p53 bind certain consensus sequences of DNA and transport protein regulates entry of arsenate into the cell, while regulate the cellular responses. As reviewed by Valko et al. arsenite, which is an uncharged arsenic species, enters the cell by diffusion. The small G-proteins that are regulated by As(V) and [3], the heavy metals exhibit differential impacts on the three As(III) do not appear to play a significant role in As(V) and As(III) major mitogen activated pathway (MAP) kinases (such as signaling to JNK. The p-21-activated kinase (PAK) plays a role in extracellular signal regulated protein kinases ERKs; c-Jun As(III)-dependent JNK activity. MEKK3 and MEKK4 are involved N-terminal kinase JNK; and stress activated protein kinase in both As(V) and As(III) activation of JNK, while MEKK2 may be knownasSAPKorMAPKorp38).Forexample,arsenichas involved in the activation of JNK by As(III) (Porter et al.) [6]. been shown to induce ERKs and JNKs in JB6 cells; the former may promote carcinogenesis while the latter may promote apoptosis [16, 17]. Further, it was demonstrated that the effects redox-active flavoproteins all contribute a small portion to of arsenic mediated through MAPK pathways and PKC may the overall production of oxidants under normal conditions. cause induction of AP-1, a transcriptional factor involved in Other enzymes capable of producing superoxide are xanthine tumour promotion [18–20]. The negative impact of arsenic oxidase, NADPH oxidases, and cytochromes P450. Hydrogen on the TNF𝛼-induced NF-𝜅B activation via inhibition of peroxide (H2O2) is produced by a wide variety of enzymes the IkB, an inhibitory protein of NF-𝜅B kinase complex, including several oxidases. ROS play important roles in cell has been demonstrated [3, 21, 22]. Very recently, Sun et al. signalling, a process termed as redox signaling. The best [23] have shown that carcinogenic metalloid arsenic induces studied cellular antioxidants are the enzymes superoxide dis- expression of mdig (mineral dust-induced gene) oncogene mutase (SOD), catalase, and glutathione peroxidase (GPx). through JNK and STAT3 (signal transducer and activator of Few well studied antioxidants include the peroxiredoxins and transcription 3) activation. The regulation of transcription by therecentlydiscoveredsulfiredoxin.Otherenzymesthathave arsenic and its biological effects are summarised in Figure 2. antioxidant properties (though this is not their primary role) include paraoxonase, glutathione-S transferases (GST), and 2.2. Oxidative Stress Induced by Arsenic. The oxidative stress aldehyde dehydrogenases (ADH). iscausedduetoanimbalancebetweentheproductionof Arsenic is known to produce ROS and hence to generate free radical species and antioxidant defense system of an oxidative stress [24]. One of such studies conducted in human organism that is the ability of any biological system to readily populations exposed to varying concentrations of arsenic detoxify the reactive oxygen intermediates or easily repair the indicated that this metal caused disruption of cell signaling − resulting damage. pathways via ROS generation. The arsenic induced2∙ O was One source of reactive oxygen under normal condi- found to be playing crucial role in this process as a chief tionsinhumansistheleakageofactivatedoxygenfrom participating species. It was later on transformed to the more mitochondria during oxidative phosphorylation. However, reactive oxygenated species such as H2O2 and ∙OH.Theinter- E. coli mutants that lack an active electron transport chain action of these species with biological macromolecules may produced as much hydrogen peroxide as wild-type cells, furtherleadtoDNAdamageanditshypo-orhypermethy- indicating that other enzymes contribute to the bulk of lation in addition to lipid peroxidation (LPO) and alterations oxidants in these organisms. One possibility is that multiple in regulatory mechanisms of cell proliferation and death. This 4 BioMed Research International study indicated that arsenic induced oxidative stress results such as ROS. In this process, GSH is converted to its oxidized in alterations in the levels of some antioxidant enzymes such form, glutathione disulfide (GSSG). GSH is found almost as superoxide dismutase (SOD), catalase (CAT), glutathione exclusivelyinitsreducedform,sincetheenzymethatreverts peroxidase (GPx), and heme oxygenase-1 (HO-1), as well as it from its oxidized form, glutathione reductase (GRx), is the nonenzymatic factors such as sulfhydryl group containing constitutively active and inducible upon oxidative stress. peptides and proteins in human systems, which could be Therefore, the decreased ratio of reduced glutathione (GSH) associated with toxicity of arsenic [24]. to oxidized glutathione (GSSG) within cells is often used The oxidative stress has been recognized as a major scientifically as a measure of cellular toxicity or an indicative component in the chain of pathogenic events that cause of OS generation in the cells or tissues [3, 41]. late complications in diabetes mellitus [25]. Several other The arsenic exposure is reported to cause depletion of workers have opined that it may also be considered as a major GSH in several biological systems [42–45]. Some important contributor to vascular and neurological complications in chemical events such as high affinity of As (III) to GSH, capa- patients with diabetes mellitus [26–31]. The involvement of bility of GSH to reduce As (V) into As (III), and conversion oxidative stress in the cytotoxicity and genotoxicity of arsenic of GSH to ROS generation by arsenic collectively contribute [32] has been reported. It could be due to generation of to oxidation of GSH into GSSG thereby reducing its net NO causing DNA damage and activating poly (ADP-ribose) concentration in the cell. The network of biochemical reac- polymerase (PARP) [32], a major cause of islet cell damage in tions having influence on arsenic cellular reduction systems, diabetes [33]. ROS production, lipid peroxidation (LPO), and levels of GSH The lipid peroxidation (LPO) has been found tobe (through involvement of vitamins C and E), including impact significantly increased in arsenic treated animals as com- on the activities of antioxidant enzymes (such as glutathione pared to control thus corroborating well with the earlier reductase (GPx), superoxide dismutase (SOD), and GRx), are speculations that arsenite induces oxygen free radical or shown by other workers [3, 42]. Using the TRL 1215 cell line promotes formation of lipid peroxides [34]. High levels of (a rat epithelial liver cell line originally derived from the liver such lipid peroxidation products have been indicated in of 10-day-old Fisher F344 rats), Santra et al. [45]haveshown the development of diabetes [35–37]. This finding reflects that the methylated form of arsenic (dimethylarsinic acid, the possibility that the chronic oral arsenic exposure may DMA) is a complete carcinogen in rodents. They found that cause significant damage to the pancreas, particularly to arsenite was very cytotoxic in these cells (LC50 =35𝜇Mafter the endocrine cellular components of pancreatic islets. It 48 h of exposure). With arsenite exposure, most dead cells therefore may be responsible for transforming the normal showed histological and biochemical evidence of necrosis. physiological features of the oral glucose tolerance test to a Arsenite cytotoxicity increased markedly when cellular GSH diabetic form. In 2002, Tibbetts has shown that exposure to was depleted [45]. It was found that the trivalent, methylated, arsenic causes oxidative stress that damages cellular health and relatively less ionizable arsenic metabolites might be and triggers onset of many diseases [38]. unusually capable of interacting with cellular targets such as In addition, monomethyl arsenous compounds produced proteins and even DNA [46]. Their findings indicated arsenic when arsenic covalently binds to the reactive thiols of induced OS as a possible mode of action for carcinogenesis. endothelial NO synthetase (NOS) result in inactivation Arsenic can affect the central nervous system (CNS) through of enzyme activity [39]. A correlation among the arsenic several pathways and hence induce significant alterations in induced ROS generation, interaction with NOS, and influ- the behavioural pattern of experimental animals. Arsenic ence on cell signaling cascade through mitogen activated pro- enters the CNS, accumulates, and undergoes biomethylation tein kinases (MAPKs) and other factors leading to apoptosis in the brain. The neurotoxicity of arsenicals observed could and cytotoxicity have been demonstrated by Flora et al. [5]. be attributed to the adverse effects of the parent compound Glutathione (GSH) acts as a natural antioxidant and or to the effects of the methylated metabolites on antioxidant potential reducing agent and is reported to protect the body enzymes and neurotransmitter metabolism [3]. systems as a cellular protagonist from damaging effects of oxidative stress (OS) [40, 41]. It is an intracellular tripeptide known as nonprotein thiol, consisting of three amino acids 3. Clinical and Pathophysiological such as glutamic acid, cysteine, and glycine and is chemically Aspects of Arsenic Toxicity recognized as 𝛾-l-glutamyl-l-cysteinyl-glycine. It contains an unusual peptide linkage between the amine group of 3.1. Toxicity. A small amount of inorganic arsenic is excreted cysteine and the carboxyl group of the glutamate side chain. unchangedasithasbeenobservedthatabout50%ofingested The thiol groups are kept in a reduced state at a concentration arsenic can be eliminated in the urine in 3 to 5 days with of approximately ∼5 mM in animal liver cells. In healthy cells residual amounts remaining in the keratin-rich tissues, such and tissue, more than 90% of the total glutathione pool is as nails, hair, and skin. Depending on its oxidative states, in the reduced form (GSH) and less than 10% exists in the arsenic poisons cells by one of two key mechanisms: (1) by disulfide form (GSSG). In effect, GSH is capable of reducing bindingthesulfhydrylgroupsoncriticalenzymesand(2) any disulfide bond formed within cytoplasmic proteins to through depletion of lipoate by trivalent arsenite. Lipoate is cysteines by serving as an electron donor. In fact, in the involved in the synthesis of key intermediates in the Krebs reduced state, the thiol group of cysteine is able to donate a cycle. Hence the depletion of lipoate results in inhibition + − reducing equivalent (H +e ) to other unstable molecules, of the Krebs cycle and oxidative phosphorylation leading BioMed Research International 5 to ATP depletion. Pentavalent arsenate, on the other hand, nails may persist for prolonged periods, but external contami- can replace the stable phosphate ester bond in ATP with the nation may render interpretation difficult and unreliable [47]. arsenate ester bond, thereby rapidly hydrolyzing (arsenolysis) uncoupling oxidative phosphorylation and depleting ATP 3.4. Management of Toxicity of Arsenic. Treatment of arsenic stores. The combination of inhibiting cellular respiration poisoning begins with the removal from the exposure source. and uncoupling oxidative phosphorylation results in cellular Supportive measures and chelation therapy are the main- energy depletion, resulting in cell death in high-energy stays of management. Volume resuscitation is of paramount dependent tissues [47]. importance in the severely poisoned patient, and chelation with dimercaprol or succimer (2,3-dimercaptosuccinic acid, 3.2. Clinical Features. Acute arsenic poisoning may produce DMSA) should be considered in patients who have symptoms disturbance in gastrointestinal (GI) tract involving nausea, or increased body burden of arsenic. Hemodialysis may be vomiting, abdominal pain, and bloody rice water diarrhea. considered for patients who have renal failure. However, Hypovolemic shock may follow in severe cases as a result of using A375 cells, the role of arginine, a component of aqueous endothelial damage and third spacing of fluid. Hematologic garlic extract, has been shown in remediation of sodium abnormalities (bone marrow depression, pancytopenia, ane- arsenite induced toxicity [48]. In addition, coadministration mia, and basophilic stippling) usually appear within 4 days of of folic acid and vitamin B12 has been shown to prevent large ingestions. QT interval (a measure of the time between cardiac tissues from arsenic induced oxidative damage in the start of the Q wave and the end of the T wave in the heart’s vivo.[49] electrical cycle) prolongation and ventricular arrhythmias such as torsade de pointes (a French term that literally means “twisting of the points”) can occur several days after 4. Lead initial improvement in GI symptoms. Arsenic poisoning may Lead (Pb; Latin: plumbum, atomic number 82) is another cause distal symmetric peripheral neuropathy with burning ubiquitous toxic metal detectable practically in all phases of and numbness in the hands and feet and a syndrome of the inert environment and biological systems [50–52]. Lead is rapidly developing ascending weakness similar to Guillain- frequently used in the production of batteries, metal products Barre(Guillain-Barr´ e´ syndrome is a disorder in which the (solder and pipes), ammunition, and devices to shield X-rays body’s immune system attacks part of the peripheral nervous leading to its exposure to the people working in these indus- system). Along with the peripheral nervous system, CNS may tries. Use of lead in gasoline, paints and ceramic products, also be affected, with the development of encephalopathy. caulking, and pipe solder has been dramatically reduced in The chronic arsenic exposure results into dermatologic recent years because of health concerns. Ingestion of contam- changes (hyperpigmentation and keratosis on the palms and inated food and drinking water is the most common source soles); nails may exhibit transverse white bands known as of lead exposure in humans. Exposure can also occur via Mees lines (the result of interruption of the nail matrix) and inadvertent ingestion of contaminated soil/dust or lead-based are not specific to arsenic. Cardiovascular effects include an paint. The most susceptible population to lead poisoning is increased incidence of hypertension and peripheral vascular children, particularly soldiers, infants in neonatal periods, disease. Sporadic outbreaks of peripheral vascular gangrene and the fetus [53–56]. Several reviews and multiauthored knownasblackfootdiseasehaveoccurredinTaiwandueto books on toxicology on lead are available [57–61]. It gets high levels of arsenic in the drinking water. Chronic arsenic into water from the corrosion of plumbing materials. Lead exposure has been associated with various malignancies poisoning (also known as saturnism, plumbism, Devon colic, including skin, lung, liver, bladder, and kidney. Inorganic or painter’s colic) is a medical condition caused by increased arsenic crosses the placenta and may be teratogenic in levels of lead in the blood [62]. animals. The Centers for Disease Control and Prevention (CDC) and others state that a blood lead level (BLL) of 10 𝜇g/dL 3.3. Diagnosis of Arsenic Toxicity. Some arsenic compounds oraboveisacauseforconcern[63, 64]. However, lead are radiopaque and visible on abdominal radiograph. An can impair development of animals even at BLLs below electrocardiogram is indicated to assess the QT interval, 10 𝜇g/dL [65, 66].AccordingtotheWorldHealthOrganiza- which may be prolonged. Nerve conduction testing typically tion (WHO) [67] and United Nations Food and Agriculture shows evidence of distal symmetric sensorimotor axonopa- Organization (UNFAO) Joint Expert Committee on Food thy. The most important and reliable diagnostic test isa Additives (JECFA) [68], the provisional tolerable weekly −1 −1 quantitative 24 h urinary arsenic excretion. Normal values are intake (PTWI) for Pb should not exceed 25 mg kg d .After lessthan50mg/L.Theseafoodingestion,however,maycause exceeding this safe concentration limit, lead can induce a significantly elevated urinary arsenic values, and speciation broad range of physiological, biochemical, and behavioral of the urinary arsenic can be performed to differentiate dysfunctions in humans and laboratory animals including inorganic from organic forms. Although blood arsenic may peripheral and CNS, haemopoietic system, cardiovascular be elevated initially in the acute poisoning, levels rapidly system, kidney, liver, and reproductory systems of males decline in the next 24 to 48 h despite continued symptoms and females [3, 69]. However, some workers have reported and increased urinary arsenic excretion. Whole blood arsenic that lead at even low concentration may cause behavioural level is usually less than 1 mg/dL. Residual arsenic in hair and abnormality, impairment in hearing, learning, and cognitive 6 BioMed Research International functions in humans and experimental animals [69, 70]. It of red blood cell (RBC) membrane proteins and lipids and to has been shown to cause permanently reduced cognitive inhibit hemoglobin synthesis [3, 81]. capacity (intelligence) in children, with apparently no lower Inthehemebiosynthesispathway,therearefour threshold to the dose-response relationship (unlike other majorkeyenzymesthatarethetargetsoftoxiceffectsby heavy metals such as mercury) [71]. This neurotoxicity was heavy metal. These enzymes are ALA dehydratase (ALAD, later found to be associated with lead-induced production of also known as porphobilinogen synthase, cytosolic in ROS which caused increase or decrease in the levels of lipid nature), uroporphyrinogen decarboxylase (UROD, cytosolic peroxidation or antioxidant defense mechanisms in the brain in nature), protoporphyrinogen oxidase (PPO, mitochondrial of experimental animals, and the effects were concentration in nature), and ferrochelatase (FeC, mitochondrial enzyme). dependent [72–74]. Among these, ALAD, which catalyses the conversion of Since lead has no known physiologically relevant role in two molecules of 𝛿-aminolevulinic acid (ALA, which is the body, its toxicity comes from its ability to mimic other synthesized into mitochondria and released into cytoplasm) biologically important metals, most notably calcium, iron, into one molecule of porphobilinogen (PBG) at the second and zinc which act as cofactors in many enzymatic reactions. step of heme biosynthesis pathway, represents one of the Lead is able to bind to and interact with many of the same most sensitive sites of inhibition of heme biosynthesis by lead 2+ enzymes as these metals but, due to its differing chemistry, (Pb ), though several other heavy metals including arsenic 2+ 2+ 2+ does not properly function as a cofactor, thus interfering (III), cadmium (Cd ), mercury (Hg ), silver (Ag ), and 2+ with the enzyme’s ability to catalyze its normal reaction(s). copper (Cu ) also influence its activity82 [ , 83]. These metals Moreover, lead is now known to induce production of ROS bind to the thiol groups of allosteric sites and, according to and RNS and hence this heavy metal exhibits ability to their structure, provoke allosteric transitions to the active generate oxidative stress in the body of those occupationally or inactive form of the enzyme. The lead poisoning causes exposed to lead and experimental animal models treated with an increase in ALA in the circulation in the absence of an varying concentrations of lead [72–75]. increase in porphobilinogen. The accumulation of ALA has beenshowntobeinvolvedinlead-inducedoxidativedamage 4.1. Mechanisms of Lead-Induced Oxidative Stress. The mech- by causing formation of ROS. The ALAD is a cytosolic anisms of lead-induced oxidative stress primarily include enzyme containing thiol (SH) groups and requires Zn for its damage to cell membrane and DNA as well as the enzy- optimal activity. Since Pb exhibits binding affinity to thiol matic(catalase,SOD,GPx,andglucose-6-phosphatedehy- groups, hence it has ability to inhibit activities/functions drogenase (G6PD)) and pool of nonenzymatic antioxidant of all those enzymes/proteins including ALAD possessing molecules such as thiols including GSH of animals and sulfhydryl groups accessible to this metal ion. Like ALAD, FeC which is responsible for catalyzing the joining of proto- human systems [3, 5]. 2+ porphyrin IX and Fe finally to form heme is also vulnerable to inhibition by lead and mercury [84, 85]. Disruption of 4.2. Lead-Induced Membrane Damage. It is known that the FeC activity leads to an accumulation of Zn-protoporphyrin polyunsaturated fatty acids constituting the cellular mem- in erythrocytes, which serves as the major biomarker and a brane are highly prone to react with ROS and get peroxidized diagnostic tool in detecting childhood lead poisoning. in completely different chemical entities which perturbs varied key functions of the cell membrane such as membrane permeability and receptors, activities of membrane bound enzymes/transmembrane proteins [76, 77], ion channels 4.4. Influence of Lead on Divalent Metals Working as Cofactors and Their Binding Sites. The mechanisms of toxicity of lead and transport of ions, and exo- and endocytosis as well 2+ as signal transduction. Some in vitro and in vivo animal chiefly (1) involve substitution of Ca by lead, thereby studies have indicated that lead-induced oxidative damage affecting all those physiological/biochemical processes of significantly contributes to enhancement of erythrocytes the cell which requires calcium and (2) cause neurotoxicity. membrane fragility during lead intoxication [78, 79]. Lead can cross the cell membrane in various ways, which arenotyetentirelyunderstood.Leadtransportthroughthe erythrocyte membrane is mediated by the anion exchanger 4.3. Lead-Induced Perturbations in Hematological Indices. in one direction and by the Ca-ATPase pump in the other. Lead has multiple hematological effects. In lead-induced In other tissues, lead permeates the cell membrane through anemia, the red blood cells are microcytic and hypochromic voltage-dependent or other types of calcium channels. After as,inirondeficiency,thereareanincreasednumberof reaching the cytoplasm, lead continues its destructive mim- reticulocytes with basophilic stippling. The anemia results icking action by occupying the calcium binding sites on from two basic defects: (1) shortened erythrocyte life span numerous calcium-dependent proteins. Lead has high affin- and (2) impairment of heme synthesis. Shortened life span of ity for calcium-binding sites in the proteins; picomolar the red blood cells is due to the increased mechanical fragility concentration of lead can replace calcium in micromolar of the cell membrane. The biochemical basis for this effect is concentrations. Lead binds to calmodulin, a protein which not known but the effect may be accompanied by inhibition in the synaptic terminal acts as a sensor of free calcium in the activity of sodium and potassium dependent ATPases concentration and as a mediator of neurotransmitter release. [79, 80]. Lead is shown to induce changes in the composition The chemical basis for lead mimicking calcium is not clear. BioMed Research International 7

Calcium and lead radically differ in their electronic struc- the astrocytes that provide signals for the maintenance of tures and ionic radii. Calcium lacks coordination chemistry integrity of BBB [91]. Lead uptake through BBB into the brain whereas lead has a broader coordination complex formation proceeds at an appreciable rate, consistent with its action as a property. Calcium prefers oxygen ligands, whereas lead may potent central neurotoxin [92]. − − − also complex with other ligands such as OH ,Cl ,NO3 ,and The BBB is a system of tightly joined endothelial cells that 2− CO3 , especially the sulfhydryl group. Furthermore, it alters form a transport barrier for certain substances between the the functioning of the enzyme protein kinase C, a virtually cerebral capillaries and the brain tissue. Under these condi- ubiquitous protein which is of crucial importance in numer- tions, solutes may gain access to brain interstitium via only ous physiological functions. Kinase C is normally activated one of two pathways: (i) lipid-mediated transport (restricted by modulators outside the cell (hormones, neurotransmitters, to small molecules with a molecular weight <700 Da and etc.) through an enzyme chain and in a calcium-dependent generally proportional to the lipid solubility of the molecule) manner. Besides many other functions, the activated kinase or (ii) catalyzed transport (carrier-mediated or receptor- directly affects the expression of the immediate early response mediated transport) processes. The transport mechanism is nottotallyunderstood,butitmostlikelyinvolvespassive genes (IERG). + uptake of PbOH ions. In the brain, lead accumulates in astroglia cells, which function as a lead sink, protecting the 4.5. Neurotoxicity of Lead. Lead as a systemic toxicant is more vulnerable neurons. Astroglia cells (or astrocytes or a neurotoxin that has been linked to visual deterioration, neuroglia) are the nonneuronal cells of the nervous system. central and peripheral nervous system disorders, [86]renal They not only provide physical support, but also respond to dysfunction [87],andhypertensivecardiovasculardisease injury, regulate the ionic and chemical composition of the [88]. Khalil-Manesh et al. [88] have presented the interplay of extracellular milieu, participate in the BBB and blood-retina reactive oxygen species and nitric oxide in the pathogenesis barriers (BRB), form the myelin insulation of nervous path- of experimental lead-induced hypertension pointing out ways, guide neuronal migration during development, and towards the role of oxidative stress (OS) as a major mediator exchange metabolites with neurons. Astrocytes are the largest in this disease. Lead affects virtually every organ system in and most numerous neuroglial cells in the brain and spinal general and the central nervous system (CNS) of developing cord. They have high-affinity transmitter uptake systems and brain in particular. The children therefore are more prone to voltage-dependent and transmitter-gated ion channels and the risk of lead toxicity than adults. can release transmitter, but their role in signalling is not well understood. On the other hand, astrocytes may be vulnerable 2+ 4.6. Lead Exposure and Blood Brain Barrier. Lead has ability to the toxic effects of Pb .Bothinastrogliacellsandin to cross the blood-brain barrier (BBB) and to substitute for neurons lead uptake is mediated by calcium channels. calcium ions. It interferes with the regulatory action of calcium on brain cells functions and disrupts many intracellular biological activities. Lead-induced damage in the prefrontal 4.7. Lead Adversely Influences Neurotransmission Systems. cerebral cortex, hippocampus, and cerebellum regions can The effects of lead on the brain, including mental retardation lead to a variety of neurologic disorders [89]. Experimen- and cognitive deficit, are mediated by its interference with tal studies have shown that Pb-induced neurotoxic effects three major neurotransmission systems: (1) the dopaminer- could potentially be mediated through alterations in the gic, (2) cholinergic, and (3) glutamatergic systems. The effects interactions of glucocorticoids with the mesocorticolimbic of lead on the first two systems are well established, but their dopamine system of the brain [90]. Many of the neurotoxic mechanisms have not yet been described exhaustively. Lead effectsofleadappearrelatedtotheabilityofleadtomimicor is known to directly interfere with the action of glutamate, in some cases inhibit the action of calcium as a regulator of the brain’s essential excitatory neurotransmitter at more cell function as discussed above. At a neuronal level, exposure than half of the synapses in the brain, and is critical for to lead alters the release of neurotransmitter from presynaptic learning. The glutamate receptor, thought to be associated nerve endings. Spontaneous release of neurotransmitter is with neuronal development and synaptic plasticity, is the enhanced probably due to activation of protein kinases in N-methyl-D-aspartate (NMDA) type receptor (NMDAR). It the nerve endings. On the other hand its evoked release is serves as a direct target for lead and is blocked selectively inhibited possibly due to the blockade of voltage-dependent by lead. This disrupts long-term potentiation, which compro- calcium channels. This disruption of neuronal activity may mises the permanent retention of newly learned information. lead to alterations in the developmental processes of synapse Glutamate binds to membrane receptors of different types. formation resulting into a less efficient brain with cognitive Micromolar concentration of lead can block the ion flux deficits. through the membrane channel associated with NMDAR, Brain homeostatic mechanisms are disrupted by exposure which is also associated with neural network creation and to higher levels of lead. The final pathway appears to be a memory and learning functions. In fact, the adverse impact breakdown in BBB. Lead-induced mimic or mobilization of of lead on NMDAR has been shown to alter calcium sig- calcium and activation of protein kinases may alter the func- nalling pathways which are involved in learning and memory. tion of endothelial cells in immature brain and disrupt BBB. The NMDAR-mediated calcium signaling can activate pro- In addition to a direct toxic effect upon the endothelial cells, tein kinase A (PKA), the mitogen-activated protein kinase lead may alter indirectly the microvasculature by damaging (MAPK), and the calcium-/calmodulin-dependent protein 8 BioMed Research International kinase II (CAMKII) pathways [5, 93]. These pathways finally systems via reduction of glutathione disulfide (GSSG) to activate c-AMP response element binding protein (CREB), GSH by glutathione reductase (GR). Of greater quantitative a transcription factor which regulates the transcription of importance is the production of NADPH for tissues actively genes in nucleus essential for learning and memory [5, 94]. engaged in biosynthesis of fatty acids and/or isoprenoids, 2+ Lead also targets the activity of Ca -dependent protein such as the liver, mammary glands, adipose tissue, and the adrenal glands. G6PD is activated by its substrate glucose-6- kinaseC(PKC)bycompetingwithcalciumbindingsitesand + causes neurotoxicity. PKC is a critical enzyme involved in phosphate. The usual ratio of NADPH/NADP in the cytosol many cell signaling pathways. In neuronal cells of developing of tissues engaged in biosynthesis is about 100/1. Increased utilization of NADPH for fatty acid biosynthesis may increase rat brain, Pb has been shown to adversely influence the + activity of an enzyme, nitric oxide (NO) synthase (nNOS), the level of NADP andstimulatetheactivityofG6PDto responsible for catalysis of NO synthesis, which could be produce more NADPH. NADPH is utilized by mitochondria considered as another mechanism of neurotoxicity caused by as well as RBCs, which is deficient in containing mitochon- lead. The activation of NMDAR is associated with triggering dria. NADPH maintains the level of glutathione in RBCs synthesis of NO utilizing nNOS as a biocatalyst. Since lead that helps protect them against oxidative damage. Thus, the interferes in functioning of NMDAR via competing with inhibition of G6PD by lead may exert a serious bearing onto calcium, it is quite likely that lead would be also inhibiting the RBCs. However, inhibition of phosphoribosyltransferases nNOS activity and hence the biosynthesis of NO as was by lead has also been suggested to be a potential mechanism 2+ observed in hippocampus of Pb - exposed rats [5]. of lead toxicity in RBCs [77]. The studies on another cytolosic Recently, the investigation on effects of prenatal and NADPH utilising enzyme from rat’s kidneys, thioredoxin postnatalleadexposureonmonoamineoxidase(MAO) reductase-1, which is a selenoprotein involved in catalysis of activity in the different stages of rat brain development has reduction of oxidised thioredoxin utilising NADPH as an indicatedincreasedMAOactivityinmostofthebrainregions electron donor have shown it to be strongly inhibited due especially at early developmental stages (2 weeks of age) and to both the acute and chronic exposure of lead. The effect the toxicity was gradually decreased with the development of was mediated via oxidative stress as this metal ion altered rats. It has been postulated that the increased MAO activity significantly the levels of known markers of lead-induced by lead intoxication may contribute to the neurobehavioral oxidative damage, for example, the activities of antioxidant changes such as cognitive and attention deficit as well as enzymes, delta aminolevulinate dehydratase, glutathione S- hyperactivity [5, 90]. Similar results showing lead-induced transferase, nonprotein thiol groups, lipid peroxidation, and neurotoxicity were obtained with the mitochondrial MAO antioxidant enzymes in the kidney [96–100]. when investigated in different parts of the brain of lead Glutathionylation has been studied in the context of exposed rats of varying age groups. The activity of another key oxidative stress and, from this perspective, it is one of enzyme, acetylcholinesterase (AChE), responsible for smooth the many forms of oxidation of thiols of protein cys- function of nerve impulse transmission has been shown to teines. Mechanisms of glutathionylation include (1) ROS- be highly sensitive towards lead exposure in different parts dependent glutathionylation via sulfenic acid, (2) ROS- of developing rat brain as well as in the erythrocytes of dependent glutathionylation via SH/SS exchange reaction, (3) humans occupationally exposed to lead [5, 90]. Another ROS-dependent glutathionylation via radical reactions, and study observing the perinatal effects of lead exposure on (4) ROS-independent glutathionylation [97, 98]. However, a the monoaminergic, GABAergic, and glutamatergic systems groupofenzymeplayersareinvolvedinproteinthioloxidore- in the striatum, cerebral cortex (Cx), dorsal hippocampus duction, called protein disulfide oxidoreductases (PDOR) (d-Hipp), and basal-medial hypothalamus or rat brain has [98]asshowninTable 2 that are equipped with abilities to indicated regional alterations in monoamine contents with bring back the oxidized/inactivated thiol group containing increase in dopamine and serotonin or their metabolites. proteins back to normal and active state. These enzymes The glutamate levels decreased in all brain regions studied, often contain a CXXC redox active dithiol motif and catalyze thiol-disulfide exchange reactions with different substrate whilegamma-aminobutyricacid(GABA)contentdecreased specificities. In particular, Trx and Grx systems are generally onlyintheCx[90] showing depletion of neurotransmitters described as protein disulfide reductants and are assigned due to lead exposure, which may be associated with possi- an “antioxidant” function [99–101]. More recently, Srx, a ble neurobehavioral impairment. However, a separate study monothiol enzyme, was shown to reduce glutathionylated observing the effect of lead in pregnant rats concludes that 2+ proteins. The protein disulfide isomerases (PDIs) are thought maternal Pb treatment distorts the dopaminergic-nitrergic to play a major role in the formation of disulfide bonds, interaction in the nigrostriatal pathway, without involving for instance, during protein folding in the endoplasmic ROS production in this process [95]. reticulum [102]. Another target for lead attack could be a thiol group containing key enzyme of pentose phosphate pathway, that is, G6PD, which catalyses conversion of glucose 6-phosphate 4.8. DNA Damage and Altered Calcium Homeostasis. The into 6-phosphoglucono-𝛿-lactone by oxidation (a rate lim- results of some studies as compiled by Krejpcio and Wojciak iting step in pentose phosphate pathway) and generates [103]andFloreaandBusselberg̈ [65]haveshownthatlead NADPH. NADPH, as a reducing energy, is required to depletes glutathione and protein bound sulfhydryl groups maintain optimum levels of glutathione (GSH) in the cellular which induce excess formation of GSH from cysteine via BioMed Research International 9 GI: inorganic salts may be absorbedmay and be converted to organic mercurythe in gut by bacteria; inhalation: elemental Hg completely absorbed History of exposure; blood mercury Dissociation of salts precipitatesand proteins destroys mucosal membranes; necrosis of proximal tubular epithelium; inhibition of sulfhydryl (-SH) group containing enzymes Elemental Hg (vapor) crosses membranes well and rapidly moves from lung toOrganic CNS. salts (lipid soluble) aredistributed, intestinal evenly (intracellular)-fecal elimination. Inorganic salts concentrate in blood, plasma, and kidney (renal elimination) Environmental: electronics and plastic industry; seed fungicide treatment; dentistry g/dL 𝜇 80 > -acroglobulin 2 -antitrypsin 1 Inhalation 10 to 40%; GI 1.5 to 5% History of exposure, whole blood Cd level Initially bound to albumin and blood cells, subsequently to metallothionene in liver and kidney GI: pigments, polishes, antique toys; environmental: Electroplating, galvanization, plastics, batteries; inhalation: industrial, metal fumes, tobacco Inhalation: lung, local irritation, and inhibition of alpha associated with emphysema; oral: kidney: proximal tubular injury, (proteinuria) associated with beta g/dL 𝜇 80 Heavy metals > 25 ug/dL and adults > 40 ug/dL, urinary lead > 50 ug/dL), protoporphyrin levels in Skin: alkyl lead compounds, because of lipid solubility (methyl and tetraethyllead); inhalation: up to 90% depending upon particle size; GI: adultschildren 40% 5 to 10%, Initially carried in red cells and distributed to soft tissues (kidney and liver); bone, teeth, and hairphosphate mostly salt as a GI: paint, pottery, moonshine; inhalation: metal fumes skin: tetraethyl lead in gasoline Inhibition of heme biosynthesis;the heme essential is structural component of hemoglobin, myoglobin, and cytochromes. Binds to sulfhydrylgroups (-SH groups) of proteins History of exposure, whole blood level (children > RBCs 90%; organic: > Table 2: The physical, chemical, and clinical properties of heavy metals included in review. the 90%; inhalation: uptake is also bound as tri- and pentavalent > dependent upon particle size Arsenic (As) Lead (Pb) Cadmium (Cd) Mercury (Hg) GI inorganic: trivalent and pentavalent salts Accumulates in lung, heart, kidney, liver, muscle, and neural tissue; concentrates in skin, nails, and hair GI:wellwater,food.Environmental: by-product of smelting ore, assemiconductors, Ga herbicides, and in pesticides; inhalation: fumes and dust from smelting Membranes: protein damage of capillary endothelium increased vascular permeability leading to vasodilation and vascular collapse; inhibition of sulfhydryl group containing enzymes; inhibition of anaerobic and oxidative phosphorylation (substitutes for inorganic phosphate in synthesis of high-energy phosphates) History of exposure; blood and urinary levels (acute); hair or fingernail (chronic) Physical/chemical/clinical properties Absorption Distribution Half-lifeSources of exposure 7to10h 10to20yearsMechanism of toxicity Blood:30–60days;bone:20–30years 60to70days Diagnosis 10 BioMed Research International -dimethyl 𝛽 , 𝛽 (EPA reference) 1 − day 1 − gkg 𝜇 g/dL: 2,3-dimercaptopropanol (BAL), -dimethyl cysteine (penicillamine), 𝜇 𝛽 4 , cysteine (N-acetyl-penicillamine); methyl Hg-supportive treatment (nonabsorbable thiol resins can be given orallymethyl to Hg reduce level in gut) most effective is N-acetyl- 𝛽 Removalfromexposure;HgandHgsalts > 0.1–2 Acute: (a) inorganic salts degradationmucosa-GI pain, of vomiting, diuresis, anemia, hypovolemic shock, renal toxicity; (b) organic CNS involvement: vision, depression, irritability, blushing, intention tremors, insomnia, fatigue, diuresis. Chronic: CNS symptoms similar to acute organic poisoning with gingivitis, tachycardia, goiter, increased urinary Hg. 1 − day 1 − gkg EDTA, BAL but 𝜇 2 0.5–1 BAL-Cd complex is extremely toxic, so it is not used (EPA reference) Acute: oral: vomiting, diarrhea, abdominal cramps, inhalation: chest pains, nausea, dizziness, diarrhea, pulmonary edema, Chronic: oral: nephrotoxicity, inhalation: emphysema-like syndrome and nephrotoxicity Removal from exposure, chelation therapy with CaNa Heavy metals EDTA, BAL, 2 (EPA reference) 1 − Table 2: Continued. day 1 − gkg 𝜇 Acute: nausea, vomiting, thirst, diarrhea/constipation, abdominal pain, hemoglobinuria, and oliguria leading to hypovolemic shock Chronic: GI: lead colic (nausea, vomiting, abdominal pain) NMJ: lead palsy (fatigue, wrist drop) CNS: lead encephalopathy (headache, vertigo, irritation, insomnia, CNS edema) Removal from exposure, treatment with chelators like CaNa dimercaprol, D-penicillamine > )actsasa 3 (EPA reference) 5 1 − gkg 𝜇 Arsenic (As) Lead (Pb) Cadmium (Cd) Mercury (Hg) cirrhosis), melanosis, arrhythmias, peripheral neuropathy, peripheral vascular disease (blackfoot disease); carcinogenicity: epidemiologic evidence; liver angiosarcoma and skin and lung cancer Acute-damage to mucosa, sloughing, hypovolemic shock, fever, GI discomfort/pain, anorexia; chronic weakness, GI, hepatomegaly (jaundice Removal from exposure Acute: supportive therapy: fluid, electrolyte replacement, blood pressure support (dopamine); chronic: penicillamine w/o dialysis arsine gas (AsH 10–50 hemolytic agent with secondary to renal failure. Supportive therapy: transfusion; chelators have not been shown to be beneficial Physical/chemical/clinical properties Symptoms Treatments Minimal permissible dose for humans BioMed Research International 11

𝛾-glutamyl cycle (a metabolic cycle for transporting amino Leadhastwomaintoxiceffectsonthehematologic acidsintocells)inadditiontoROSproduction.However, system: reduction of erythrocyte lifespan and decreased GSH may not be efficiently supplied, if depletion continues hemoglobin biosynthesis. Lead causes inhibition of 󸀠 becauseofchronicexposureoflead. pyrimidine-5 -nucleotidase and inhibition of Na-K- ATPase leading to decreased energy use by the erythrocyte and a decrease in cell membrane stability. Pyrimidine- 󸀠 5 -nucleotidase is necessary for the removal of degraded 5. Clinical and Pathophysiological RNA, and its inhibition by lead causes erythrocytes to Aspects of Lead Toxicity form clumps, giving the cells the classic basophilic stippling appearance. Lead interferes with several enzymes in the 5.1. Pediatric Toxicity. The young children (up to 2 years heme synthesis pathway, including aminolevulinic acid of age) use hand-to-mouth activity to explore their envi- synthetase, d-aminolevulinic acid dehydratase (ALA-D), ronment. Children who play with objects containing lead ferrochelatase, and coproporphyrinogen decarboxylase. added paints or live in an environment that is contaminated ALA-D in particular can be inhibited by minimal lead withleadaremorelikelytosufferfromtheeffectsoflead exposure. poisoning such as central neurotoxicity. At lower levels (1– 50 mg/dL) lead may cause subtle cognitive and behavioral changes difficult to differentiate from normal developmental 5.2. Diagnosis. Blood lead levels are the best indicator of variance whereas at moderate levels (50–70 mg/dL) children lead exposure. Venous blood samples are necessary because may display a global decrease in activity, such as children capillary samples can give false positives because of skin who do not enjoy playing or who developmentally fall contamination. All children who fit into a high-risk category behind their peers. These symptoms have been classified (mainly based on socioeconomic factors) should be screened as preencephalopathic symptoms and are most prominent with a blood lead level at 1 year and 2 years of age. A complete between 1 year and 5 years of age. With severe lead toxicity blood count may show a hypochromic microcytic anemia and (>70 mg/dL) children may be encephalopathic with coma, stippling of the RBCs. seizures, altered mental status, and symptoms consistent with increased intracranial pressure. Encephalopathy from 5.3. Management. The most effective treatment for lead lead occurs most commonly between the ages of 15 and 30 toxicity is removal of the patient from the lead-containing months. Other symptoms of childhood lead toxicity include environment and cessation of exposure. Pediatric patients anemia; peripheral motor neuropathy; GI complaints, such as who present with symptoms of lead encephalopathy or with anorexia, vomiting, and abdominal pain; and growth delay. blood levels >70 mg/dL should be considered candidates Lead readily crosses the placenta and has been reported to for parenteral chelation therapy with either dimercaprol or cause fetal toxicity. The primary route of lead exposure in succimer and CaNa2EDTA. Chelation with oral succimer children is through the GI tract. Lead is probably taken up should be considered in children who are asymptomatic with at calcium absorption sites, which have increased activity blood lead levels between 45 and 69 mg/dL. at times of rapid growth. The central neurotoxicity caused by lead is caused by disruption of the intercellular junction that seals the capillary endothelium. The mechanism of this 5.4. Adult Toxicity. Most adult lead poisonings occur from disruption is interference with cellular calcium metabolism occupational respiratory exposures. Lead-induced hyper- andsecondmessengersignalingsystems.Withthelossof tension is the most common symptom attributed to lead its tight seal, the BBB is less effective and the capillaries exposure in adults. Patients can also develop anemia, gastric leak, resulting in an increase in intracranial fluid and a colic, muscle and joint pain, decreased fertility, renal failure, resultant increase in intracranial pressure. This effect of and peripheral motor neuropathy. Rarely, adults with blood lead is more prominent in young children because of their lead levels >100 mg/dL present with encephalopathy. Adults immature BBB before the exposure. Chronic exposure to more commonly suffer from subtle neurologic deficits, such lead affects numerous neurotransmitter systems, increasing as fatigue and emotional lability after lead exposure. The main the spontaneous release of dopamine, acetylcholine, and mechanism of lead-induced hypertension seems to be related GABA, blocking N-protein kinase C. These effects result to changes in vascular smooth muscle because of increased in an increase in random synaptic signals, termed synaptic activity of the Na-Ca exchange pump and interference of noise, and a decrease in ability of neuron to produce a Na-K ATPase activity. The progressive development of renal synaptic signal in response to a true stimulus. A human has failure may result after long-term environmental exposures themostneurologicsynapsesattheageof2,afterwhich or the chronic release of deposited bone lead. Lead disrupts the body, through apoptosis, rapidly starts to prune faulty mitochondrial phosphorylation and oxidation within the and unnecessary synapses. The determination of whether kidney, leading to a decrease in energy-dependent transport. a synapse is kept or destroyed is related to feedback from The end result of this disrupted transport is phosphaturia, neurotransmitters and neurotransmitter receptors. Because glycosuria, and aminoaciduria. With chronic lead exposure lead interferes with neurotransmitters and their receptors, the kidneys are found to have lead-protein complex inclusion it results in a disruption of synapse formation and synapse bodies which help in excretion of lead. As chronic lead destruction. exposure progresses fewer of these inclusion bodies are seen 12 BioMed Research International andtherenaltubulesbegintoshowsignsofinterstitial control neutrons in nuclear fusion, black and white television fibrosis. phosphors, and blue and green phosphors for color television Peripheral neuropathy from lead exposure is caused by picture tubes, and semiconductors and in molecular biology Schwann cell destruction followed by demyelination and to block voltage-dependent calcium channels from fluxing axonal atrophy. Upper extremity motor neurons are more calcium ions. susceptibletodamagefromleadthansensoryorlower Cadmium poisoning is an occupational hazard associated extremity neurons, resulting in the classic, albeit rare, and with industrial processes such as metal plating and the pro- presentation of bilateral wrist drop. Lead’s total body burden duction of nickel-cadmium batteries, pigments, plastics, and is stored mainly in bone, with 70% of a child’s and 95% of other synthetics. The main sources of exposure to cadmium an adult’s total body burden stored in bone. Within bone are specific professional atmospheres, diet, drinking water, there are two main storage areas: the cortical bone, which is andtobacco.Theprimaryrouteofexposureinindustrial a stagnant store, and the trabecular bone, which is a more settings is inhalation of cadmium-containing fumes which bioavailable store. Blood lead levels may increase during can result initially in metal fume fever but may progress times of increased bone metabolism during pregnancy, osteo- to chemical pneumonitis, pulmonary edema, and death. porosis,andfractures.Thehalf-lifeofleadinhumanboneis Cadmium possessing a long biological half-life (17–30 years) estimatedtobeupto30years. in humans accumulates primarily in liver and kidney [104]. This long half-life of Cd is mainly due to its low ratio of 5.5. Diagnosis. Those who display unexplained hypertension, excretion and its continued accumulation in the organism. encephalopathy, peripheral motor neuropathy, gastric colic, The local agricultural communities in Japan consuming and renal failure would be suffering from lead toxicity. Work- Cd contaminated rice developed itai-itai disease and renal ers with blood lead levels >60 mg/dL or three consecutive abnormalities, including proteinuria and glucosuria. It also levels >50 mg/dL should have a repeat level every month. causes osteoporosis, anemia, nonhypertrophic emphysema, Those with a level between 40 and 60 mg/dL should have irreversible renal tubular injury, eosinophilia, anosmia, and a repeat level every 2 months, and those with elevated chronic rhinitis. Cadmium is one of six substances banned by levels <40mg/dLshouldhaverepeatlevelsevery6months. theEuropeanUnion’sRestrictiononHazardousSubstances Currently the best screening test is a venous blood lead level. (RoHS) directive because of its carcinogenic potential in A complete blood count may show a hypochromic microcytic humans as it may cause cancers of lung, prostrate, pancreas, anemia and red blood cell stippling. Urine analysis and basic and kidney. The International Agency for Research on Cancer metabolicpanelmaybeusedtoscreenforrenaltoxicity.The of USA has classified Cd into the number 1 category of X-ray fluorescence technology may be a useful screening test carcinogens [105]. in the future to determine bone lead burden. The generation of ROS by Cd has been one of the known mechanisms by which this heavy metal induces mutagenesis [106]. Due to increase in the levels of ROS various 5.6. Management. The most effective therapy is limitation physiological perturbations develop, for example, increased of exposure at work place by using personal protective gear, permeability of BBB and alterations in synaptic transmission. improving industrial engineering, and adhering to safe work Further, at cerebral level, oxidative stress is associated with practices. Chelation usually is reserved for adults who are many degenerative diseases such as amyotrophic lateral scle- > symptomatic or who have a blood lead level 70 mg/dL. rosis and Alzheimer. ROS, continuously generated during the Mildlysymptomaticpatientswhohavelevelsbetween70and metabolism of oxygen, are transformed into highly reactive 100mg/dLmayrequireacourseoforalsuccimer,whereas hydroxyl radicals, attack the DNA, cause breaks into strands, > those patients who have encephalopathy or levels 100 mg/dL and even alter the bases. It induces mutations and causes the require intramuscular dimercaprol or oral succimer and subsequent development of tumours [107, 108]. In different 2+ intravenous CaNa2EDTA. A pregnant patient who has ele- cell systems, the ability of Cd to induce ROS generation has vatedleadlevelsshouldbetreatedusingthesamestandards been reported [109]. 2+ as a nonpregnant adult. Cd being a non-redox-active metal cannot initiate by itselftheFentonreactions[110]. However, it may generate 6. Cadmium nonradical hydrogen peroxide, which may become a source of free radical via the Fenton reaction. It therefore induces Another transition metal, cadmium (Cd), with atomic num- oxidative stress through indirect processes. Some of the ber 48 is soft, bluish-white, chemically similar to the two mechanisms through which Cd induces the formation of other metals in group 12, zinc and mercury. It shows similari- ROSincludethefollowing:(1)decreaseintheintracellu- ties with zinc and mercury in its preference of oxidation state lar GSH content, (2) Cd combines with thiol groups of +2 and low melting point, respectively. Cadmium is relatively enzymes involved in antioxidant mechanisms, such as SOD, the most abundant and toxic element. It was discovered in glutathione peroxidase (GPx), and catalase, and inhibits their 1817 by Friedrich Strohmeyer as an impurity in zinc carbon- activities [111], (3) Cd forms cadmium-selenium complexes in ate(calamine).Beinganimportantcomponentofmaking theactivecentreofGPxandinhibitstheenzymeactivity,and batteries, it is also used for cadmium pigments and coatings (4) Cd inhibits complex III of the mitochondrial electronic and plating and as stabilizers for plastics. Other applications transport chain and increases production of ROS [111]which include chemical stabilizers, metal coatings, alloys, barrier to may damage mitochondrial membrane and trigger onset of BioMed Research International 13 apoptosis. In addition, changes in mitochondrial oxidative down by the lysosomes and non-metallothionene bound metabolism may lead to energy deficit thereby affecting the calciumisreleasedinthetissue.Suchcadmiumstimulates essential cellular functions. Thus, Cd is capable of eliciting a renal metallothionene synthesis and thus there is constant variety of ROS (O2∙,H2O2,and∙OH), which could be the turnover of metallothionene. A majority of renal Cd is bound mainmechanismofcellulartoxicityinducedbythisheavy to metallothionene during long-term exposure situation. metal. Possibly Cd induced oxidative stress is involved in However, when the concentration of metallothionene bound (1) causing DNA damage/mutations [112], (2) oxidation of Cd exceeds a certain level at sensitive sites in the cell, toxicity proteins, and (3) lipid peroxidation (LPO), which may cause to renal tubules develops [120]. alterations in lipid composition of cellular membranes and functions. In this context, Lopez´ et al. [113]haveillustrated that Cd induces LPO in cortical neurons due to increased 6.1. Cadmium Induced Apoptosis. According to Kondoh et concentration of ROS. al., [121] Cd induces apoptosis both in vitro and in vivo. Cd can replace iron and copper from a number of However,themodeofactionremainsunclear.Theyhave cytoplasmic and membrane proteins like ferritin, thereby reported that Cd induced apoptosis was partly dependent causing rise in the concentration of iron and copper ions on mitochondria. They further reported the involvement of which may be associated with the production of oxidative caspase9,whichistheapexcaspaseinthemitochondria- stress via Fenton reaction [3, 110]. Another mechanism of Cd dependent apoptosis pathway, in Cd induced apoptosis toxicity may be carried out into the body by zinc binding in human promyelocytic leukemia HL-60 cells. However, proteins, that is, the proteins with zinc finger motifs into cadmium adaptation may inhibit cell apoptosis allowing thereby the overproduction of critical mutations [122]. For its structures. Zinc and cadmium are in the same group 2+ in the periodic table, contain the same common oxidation the management of the Cd toxicity, the natural antioxidants state (+2), and when ionized are almost the same in size. such as vitamins C and E [123, 124]aswellasotherantioxidant Due to these similarities, cadmium can replace zinc in many molecules like cysteine, melatonin, glutathione, and ascor- biological systems, particularly the systems that contain softer bate under certain conditions may induce more DNA damage ligands such as sulfur. Cadmium can bind up to ten times in vitro [125]. more strongly than zinc in certain biological systems and is notoriously difficult to remove. In addition, cadmium can 7. Clinical and Pathophysiological replace magnesium and calcium in certain biological systems, although these replacements are rare [114, 115]. Aspects of Cadmium Toxicity Taking into account the effect of Cd on the central 7.1. Toxicity. The EPA standard for maximum concentration nervous system (CNS) and endocrine system, it is currently of Cd in drinking water is 5 ppb (𝜇g/L). FDA has allowed classified as an endocrine/neuroendocrine disruptor116 [ , Cd in food coloring up to 15 ppb. Due to its long half-life 117]. Cadmium induced significant increase in the levels of (10–30 years in humans), Cd after accumulation poses serious malondialdehyde (MDA) and glutathione peroxide (GSH- threat to human health. Cd toxicity involves the organs such Px) has been reported by many workers in animal models as GI tract, lungs, kidney, and bone as well as pulmonary and [113]. The genotoxic potential of cadmium has also been stud- neurologic systems. Cd causes neurotoxicity and alters brain ied in vivo into cadmium workers, members of the general metabolism by alterations in the synthesis and/or metabolism population, and rodents. Although not always consistent, of biogenic amines and amino acid neurotransmitters in these results suggest that cadmium is a clastogenic agent, as the CNS. These effects have been evidenced by different judged by the induction of DNA damage, micronuclei, sister experimental protocols (in vivo and in vitro) in specific chromatid exchange (SCE), and chromosomal aberrations brain regions. Among the brain areas affected by cadmium [112, 113].Cadmiumisalsoknowntocauseitsdeleterious exposure, hypothalamus is the most affected. This region has effect by deactivating DNA repair activity112 [ ]. Studies have multiple roles: regulation of endocrine system and autonomic shown that the number of cells with DNA single strand breaks nervous system, modulation of body temperature, hunger- and the levels of cellular DNA damage were significantly satiety, emotional behavior, and so forth. higher in cadmium-exposed animals [112, 118, 119]. Cadmium may be present as a cadmium-albumin complex in blood, immediately after exposure3 [ ]. In this form 7.2. Clinical Symptoms. People chronically exposed to cad- thelivermaytakeupcadmium.Duringfirsthourofintake mium have headache, sleep disorders, and memory deficits. cadmium in the liver will not be bound to metallothionene. These diseases are related to alterations in neurotransmitters Cadmium metallothionene complex to a large extent remains (GABA, serotonin) by altering GABAergic and serotoniner- in the liver but a small portion of it is released in the gic systems. Other symptoms include increased salivation, blood and another small portion is constantly turned over, choking, throat dryness, cough, chest pain, restlessness, whereby non-metallothionene bound cadmium is released irritability, nausea, vomiting, kidney dysfunction (glucosuria, out, which all again induces new metallothionene and so proteinuria, and aminoaciduria), itai-itai disease, and renal on. Cadmium metallothionene complex that occurs in the and hepatic failures. Pulmonary involvement includes pneu- blood plasma is then quickly transported to the kidney, monitis, edema, and bronchopneumonia. Permanent lung where it is filtered through the glomeruli and reabsorbed damage and cardiovascular collapse may occur. Lung and by proximal tubule. The metallothionene moiety is broken prostate are the primary targets for the Cd induced cancer. 14 BioMed Research International

7.3. Management. Keeping the affected individuals away (I) counterparts because of their greater solubility in water fromthesourceofCdandchelationtherapythroughmela- and rapid absorption by the gastrointestinal tract [132]. tonin are the possible management practices suggested for cadmium toxicity. Chelation therapy is however contraindi- cated because it exposes the kidney to large quantities of 8.3. Organic Mercury Compounds. The organic compounds nephrotoxic cadmium. ofmercurytendtobemuchmoretoxicthantheelementitself and have been implicated in causing brain and liver damage. 8. Mercury The most dangerous mercury compound, dimethylmercury, is so toxic that even a small drop spilled on the skin, or even Mercury (Hg) occurs in nature in several physical and a latex glove, can cause death. Methylmercury is the major chemical forms, all of which can produce toxic effects in high source of organic mercury for all individuals [133]. It enters doses. It is a highly reactive and toxic transition element. the food chain through bioaccumulation in the environment, 0 Its zero oxidation state (Hg ) exists as vapor or as liquid reaching high concentrations among populations of some + metal,itscationicmercurousstateHg exists as inorganic species. Larger species of fish, such as tuna or swordfish, 2+ salts, and its mercuric state Hg may form either inorganic are usually of greater concern than smaller species. This salts or organomercury compounds. These three groups vary compound in excess can cause coronary heart disease deaths in effects. The different chemical forms of mercury include and suboptimal neural development in children. There is a elemental mercury vapor (Hg), inorganic mercurous (Hg II), longlatentperiod(upto5months)betweenexposureto mercuric (Hg III), and organic mercuric compounds [126]. methylmercury and the appearance of symptoms in adult These forms have toxic effects in a number of organs such poisoning cases. No explanation for this long latent period is as brain, kidney, and lungs [127]. Mercury poisoning can known. When the first symptom appears, typically paresthe- result in several diseases, including acrodynia (pink disease), sia (a tingling or numbness in the skin), it is followed rapidly Hunter-Russell syndrome, and Minamata disease. Though by more severe effects, sometimes ending in coma and death. the specific mechanism of action of damage by mercury is not The toxic damage appears to be determined by the peak value known, it has been shown that mercurous and mercuric ions of mercury, not the length of the exposure. It is cleared from impart their toxicological effects mainly through molecular the blood much more rapidly, with a half-life of 7 to 10 days, interactions by binding to the thiol groups present in different and metabolized much more quickly than methylmercury. molecules such as GSH, cysteine, and metallothionene (MT) It probably does not have methylmercury’s ability to cross [128]. BBB via a transporter but instead relies on simple diffusion to enter the brain. Other exposure sources of organic mercury includephenylmercuricacetateandphenylmercuricnitrate. 8.1. Elemental Mercury. Quicksilver (liquid metallic mer- These were used in indoor latex paints for their antimildew cury) is poorly absorbed by ingestion and skin contact. properties but were removed in 1990 because of cases of Itishazardousduetoitspotentialtoreleasemercury toxicity [134]. vapour. Animal data indicate that less than 0.01% of ingested mercury is absorbed through the intact gastrointestinal tract, though it may not be true for individuals suffering from 8.4. Disposition and Toxicokinetics of Mercury. The liquid ileus (a disruption of the normal propulsive gastrointestinal metallic mercury may be swallowed from a broken ther- motor activity from nonmechanical mechanisms). Cases of mometer, that is, only slowly absorbed by the gastrointestinal systemic toxicity from accidental swallowing are rare, and tract (0.01%) at a rate related to vaporization of the ele- attempted suicide via intravenous injection does not appear mental mercury, and is thought to be of no toxicological to result in systemic toxicity. Possibly the physical properties consequence. The vapor from metallic mercury is readily of liquid elemental mercury limit its absorption through absorbed in the lungs. Mercury’s dissolved form is absorbed intact skin and in light of its very low absorption rate from inthebloodstreamanddiffusestoalltissuesofthebody. the gastrointestinal tract, skin absorption would not be high The gastrointestinal absorption of compounds of mercury [129]. In humans, approximately 80% of inhaled mercury from food is 15% as has been found in a study with human vapor is absorbed via the respiratory tract where it enters the volunteers, whereas absorption of methylmercury is of the circulatory system and is distributed throughout the body. order of 90 to 95%. The distribution of this heavy metal Chronic exposure by inhalation, even at low concentrations also differs between red blood cells (RBCs) and plasma. For 𝜇 3 in the range 0.7–42 g/m ,hasbeenshownincasecontrol inorganic mercury the cell-to-plasma ratio ranges from <1to studies to cause effects such as tremors, impaired cognitive 2, but for methylmercury it is about 10. skills, and sleep disturbance in workers [130, 131]. Kidneys contain the greatest concentrations of mercury following exposure to inorganic salts of mercury and mercury 8.2. Inorganic Mercury Compounds. Mercury in the inor- vapor, whereas organic mercury has a greater affinity for the ganic form such as salts, for example, mercury (II) chloride, brain, the posterior cortex in particular. Mercury vapor also primarily affects gastrointestinal tract and kidneys. Since has greater predilection for the central nervous system than it cannot cross the BBB easily, mercury salts inflict little inorganic mercury salts. Excretion of mercury from the body neurological damage without continuous or heavy exposure. is by way of urine and faeces, which again differs for varying Mercury (II) salts are usually more toxic than their mercury forms of mercury, dose, and duration of exposure. BioMed Research International 15

8.5. Metabolic Transformation. The elemental or metabolic This large superfamily of proteins collectively referred to mercury is oxidized to divalent mercury after absorption as stress proteins include heat-shock proteins (hsps) and intothebodytissuesandisprobablymediatedbycatalases. glucose-regulated proteins (grps). This particular stress- InhaledmercuryvaporabsorbedintheRBCsistransformed protein response has evolved as a cellular strategy to protect, to divalent mercury, but a portion is transported as metallic repair, and chaperone other essential cellular proteins. The mercury to more distant tissues, particularly the brain, where differential expression of 4 HSPs has been observed in renal biotransformation may occur. cortex and medulla of mercury treated rats [140].

8.6. Therapy of Mercury Poisoning. For acute inorganic mercury poisoning chelation therapy can be done with 8.8. Mercury and Cardiovascular Death. Nearly ten years DMSA, 2,3-dimercapto-1-propanesulfonic acid (DMPS), D- ago,basedonclinicaltrialsundertakeninFinland[141], it penicillamine (DPCN), or dimercaprol (BAL) [135]. Only was concluded that a high intake of mercury from nonfatty DMSA is FDA-approved for use in children for treating freshwater fish and the consequent accumulation of mercury mercury poisoning. However, several studies found no clear in the body are associated with an excess risk of acute clinical benefit from DMSA treatment for poisoning due to myocardial infarction as well as death from coronary heart mercury vapor. No chelator for methylmercury or ethylmer- disease, cardiovascular disease. The increased risk may be cury is approved by the FDA; DMSA is the most frequently due to the promotion of lipid peroxidation by mercury. In used chelator for severe methylmercury poisoning, as it is agreement with the conclusions of this trial were two recent given orally, has fewer side effects, and has been found to epidemiological studies exploring possible participation of be superior to BAL, DPCN, and DMPS. Alpha-lipoic acid mercury in myocardial infarction [142]. This trial was related (ALA) has been shown to be protective against acute mercury to the fish intake, because fish is a major source of exposure poisoning in several mammalian species when it is given soon to mercury. It is assumed that the mercury content of fish 𝑛−3 after exposure. However, the correct dosage is required, as may counteract the beneficial effects of its fatty acids. inappropriate dosages increase toxicity. Although it has been The results led to the conclusion that the mercury levels were hypothesized that frequent low dosages of ALA may have directly associated with the risk of myocardial infarction, potentialasamercurychelator,studiesinratshavebeen andtheadiposetissueDHAlevelwasinverselyassociated contradictory. Glutathione and N-acetylcysteine (NAC) are with the risk. Thus high mercury content may diminish recommended by some physicians but have been shown to the cardioprotective effect of fish intake. A similar trial was increase mercury concentrations in the kidneys and the brain. conducted in order to investigate the association between the 𝑛−3 Experimental findings have demonstrated an interaction serum end product fatty acids docosahexaenoic acid between selenium and methylmercury, but epidemiological (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic studies have found little evidence that selenium helps protect acid and the risk of acute coronary events in middle-aged 𝑛 = 1871 against the adverse effects of methylmercury [136, 137]. men ( )inFinland[143]. The results displayed no Despite chelation therapy as advised in this case, one of the association between the proportion of eicosapentaenoic acid important molecules that helps scavenging and reducing the and the risk of acute coronary events. Both of the above toxic effects of mercury is metallothionene, which is a small, studies concluded that fish oil derived fatty acids reduce the low molecular weight protein and rich in sulfhydryl groups risk of acute coronary events. However, high mercury content [126]. in fish could attenuate this protective effect. The clinical manifestations of neurotoxic effects are (1) paresthesia, numbness and tightening sensation around the 8.9. Mercury Induced Apoptosis. It has been established that mouth, lips, and extremities, particularly the fingers and toes, apoptosis arises from the active initiation and propagation (2) ataxia, a clumsy, stumbling gait, and difficulty in swal- of a series of highly orchestrated specific biochemical events lowing and articulating words (3) neurasthenia, a generalized leading to the cell demise. Being a normal physiological pro- sensation of weakness, fatigue, and inability to concentrate, cess, it takes place in the maintenance of tissue homeostasis as (4) vision and hearing loss, (5) spasticity and tremor, and (6) well as during embryonic development. In apoptosis pathway coma and death. Neuropathologic observations have shown an array of diverse groups of molecules is involved. Being that cortex of the cerebrum and cerebellum are selectively a network of complex cascade of reactions, it functions to involved in focal necrosis of neurons, lysis, and phagocytosis eliminate irreparably damaged cells or the unwanted cells. It is and replacement by supporting glial cells. These changes important in this context that an inappropriate induction of are more common to renal cell necrosis due to various apoptosis by environmental agents may have broad ranging causes. Slight tubular cell injury, manifested by enzymuria pathologic implications which could be associated with and low molecular weight proteinuria, may occur in workers several diseases including cancer. The toxicity of mercury with low-level exposure to metallic mercury vapor [138]. occurs due to its high affinity to sulfhydryl groups of proteins The toxicity of mercury in different organs of rat and the and enzymes and its ability to disrupt cell cycle progression mitochondria [139]hasbeenreported. and/or apoptosis in various tissues. Recently a study to assess the potential of mercury to induce early and late- 8.7. Mercury and Stress Proteins. It is well established that stage apoptosis in human liver carcinoma (HepG2) cells using various stimuli, including toxic chemicals, enhance the syn- annexin-V and caspase 3 assays and flow cytometric analysis thesis of a class of proteins known as stress proteins [140]. to determine the extent of phosphatidylserine externalization 16 BioMed Research International and caspase 3 activation in HepG2 cells treated with subacute These patients may exhibit memory loss, drowsiness, with- doses of mercury (LD50 3.5 ± 0.6 𝜇g/mL for mercury in drawal, lethargy, depression, and irritability. Another com- HepG2 cells) for 10 to 48 h established a dose response mon finding in mercury poisoning is incoordination and a relationship between mercury exposure and the degree of fine motor intention tremor primarily involving the hands. early and late-stage apoptosis in HepG2 cells. The results Mercury inhibits the enzyme catechol-o-methyltransferase indicatedagradualincreaseinapoptoticcellsandcaspase by inactivation of the coenzyme, S-adenosyl methionine, positive cells with increasing doses of mercury [144]. producing elevated levels of catecholamines in the body. The increase in catecholamines results in hypertension, sweating, and tachycardia that may be clinically indistinguishable 9. Clinical and Pathophysiological from pheochromocytoma. A 24 h urine collection on these Aspects of Mercury Toxicity patients reveals elevated levels of urinary catecholamines, although typically to a lesser degree than that seen in true 9.1. Toxicity. There is rarely any toxicity of mercury due to its neuroendocrine disease. exposure to elemental mercury. The entry of mercury occurs by way of inhalation of the vapor, ingestion of the liquid, or 9.3. Diagnosis. The diagnosis of elemental mercury poison- cutaneous exposure. The metal is poorly absorbed in the gut ing involves incorporating clinical presentation (history and and is eliminated in the feces. Although, in patients who have physicalfindings),historyofexposure,andanelevatedbody normalGImucosa,toxicityrarelydevelopsafteringestion, burden of mercury. Blood mercury levels have limited useful- patients, who have abnormal GI mucosa, may absorb enough ness because of mercury’s short half-life in the blood. Whole mercury for toxicity to occur. Occasionally mercury becomes blood mercury concentrations typically are <10 mg/L. Urine trapped within the appendix, but without signs of mercury levels are obtained more commonly in chronically exposed toxicity or appendicitis it can be safely monitored and allowed patients, with 24 h collections more reliable than spot urine to pass on its own. If vomiting with subsequent aspiration of levels. Normal levels typically are <50 mg of mercury in a 24 h elemental mercury occurs, the risk for toxicity is increased period. Patients undergoing chelation therapy at the time of because mercury is well absorbed within the lungs. Toxicity collection have elevated levels of mercury in a 24 h sample from elemental mercury occurs from inhalation of the making interpretation difficult. vaporbecausemercuryiswellabsorbedintothepulmonary circulation allowing distribution to the brain, kidneys, gut, 9.4. Management. Removal of the patient from the source and lungs. Elemental mercury readily crosses BBB where of the toxic exposure is the most important intervention. it concentrates in neuronal lysosomal dense bodies. The As elemental mercury typically has minimal toxicity when manifestations of elemental mercury toxicity have great ingested, there is little role for GI decontamination. Although variability depending on the chronicity of the exposure. Acute chelation therapy is considered the mainstay of treatment toxicity may manifest within hours of a large exposure with several controversies remain regarding its use. Chelators GI upset, chills, weakness, cough, and dyspnea, with severe are charged molecules capable of binding the metal ion cases developing adult respiratory distress syndrome and forming a neutral complex excreted by the kidney. Several renal failure. agents are available such as succimer, dimercaprol, and D- penicillamine. Patients may require chelation for several 9.2. Clinical Symptoms. Chronic mercury toxicity may months depending on the total body burden of mercury. In develop over a period of weeks to months, depending on patients who have developed renal failure, hemodialysis may the level of exposure. Initial symptoms commonly include be required, with or without the inclusion of a chelation agent. GI upset, constipation, abdominal pain, and poor appetite and may mimic a viral illness. Other symptoms include dry 10. Treatment for Heavy Metals mouth, headache, and muscle pains. Chronic exposure results Poisoning by Chelation Therapy in two distinct clinical syndromes, acrodynia and erethism. Known as pink disease, Feer syndrome, Feer-Swift disease, Chelation therapy is the administration of chelating agents to erythroderma, and raw-beef hands and feet, acrodynia is a remove heavy metals from the body. Chelating agents were complex of symptoms occurring in chronic toxicity from introduced into medicine as a result of the use of poison elemental and inorganic mercury. It occurs more commonly gas in World War I. The term chelation comes from Greek in infants and children, but it has been reported in adults. word, “chelate” which means “claw.” It must be emphasized Symptoms include sweating, hypertension, tachycardia, pru- that treatment is only a secondary alternative to reduction or ritus, weakness, poor muscle tone, insomnia, anorexia, and prevention from exposure to toxic metals. an erythematous, desquamating rash to the palms and soles. Complexation is the formation of metal ion complex Other symptoms in mouth include reddened, swollen gums, in which the metal ion is associated with a charged or subsequent mucosal ulcerations, and possible tooth loss. By uncharged electron donor, referred to as a ligand. The ligand an unknown mechanism, mercury may result in proximal may be monodentate, bidentate, or multidentate; that is, it weakness primarily involving the pelvic and pectoral girdle. mayattachorcoordinateusingone,two,ormoredonor Patients who have mercury poisoning often develop char- atoms. Chelation occurs when bidentate ligands that include acteristic personality changes collectively termed erethism. the ligands forming ring structures bind to the metal ion BioMed Research International 17 and the two-ligand atom attached to the metal. Metal may toxicity to these metals. BAL has been used as an adjunct in − − − react with O ,S ,andN containing ligands present in the the treatment of the acute encephalopathy of lead toxicity. It form of OH, COOH, SH, NH2,NH,andN.Aresultant is a potentially toxic drug, and its use may be accompanied metal complex is formed by coordinate bonds in which by multiple side effects. Although treatment with BAL will both electrons are contributed by the ligand [145]. The first increase the excretion of cadmium, there is a concomitant experimental use of a chelator against metal poisoning was increase in renal cadmium concentration, so that its use in done by Kety and Letonoff [146]. They attempted to use citrate case of cadmium toxicity is to be avoided. It does, however, as an antidote towards acute lead intoxication in 1941. The remove inorganic mercury from the kidneys, but it is not concept of chelation therapy relies on treating heavy metal useful in the treatment of alkyl mercury or phenyl mercury intoxication by mobilization enabling it to pass through renal toxicity. BAL also enhances the toxicity of selenium and excretion. tellurium, so it is not to be used to remove these metals Chelating agents vary in their specificity for toxic metals. from the body. Other side effects of BAL include vomiting, Ideal chelating agents should be (1) water-soluble, (2) resistant headache, lachrymation, rhinorrhea and salivation, profuse to biotransformation, (3) able to reach sites of metal storage sweating, intense pain in the chest and abdomen, and anxiety without causing any side effect, and (4) capable of forming [147, 148]. nontoxic complexes with toxic metals and of being excreted fromthebodyand(5)theyshouldalsohavealowaffinity for essential metals particularly calcium and zinc. Several 12. DMPS chelating agents are available, having different affinities for 2,3-Dimercapto-1-propanesulfonic acid (abbreviated as different metals. Common chelating agents include alpha- DMPS) and its sodium salt (known as unithiol) are chelating lipoic acid (ALA), aminophenoxyethane-tetraacetic acid agents that form complexes with various heavy metals. They (BAPTA), deferasirox, deferiprone, deferoxamine, diethy- are related to dimercaprol, which is another chelating agent. lene triamine pentaacetic acid (DTPA), dimercaprol (BAL), DMPS is a water-soluble derivative of BAL developed in dimercaptopropane sulfonate (DMPS), dimercaptosuccinic response to BAL’s toxicity and unpleasant side effects. DPMS acid (DMSA), ethylenediaminetetraacetic acid (calcium dis- hasbeenshowntoreducebloodleadlevelsinchildren[149]. odium versenate) (CaNa2-EDTA), ethylene glycol tetraacetic It has an advantage over ethylenediaminetetraacetic acid acid (EGTA), and D-penicillamine. Some of the conventional (EDTA) which is that it is administered orally and does chelators, their applications, and side effects are described not appear to have toxic effects. It has been widely used here. A list of specific chelators used as drugs against toxicity in the former Soviet Union to treat many different metal induced by corresponding heavy metals is presented in intoxication cases. DMPS has been used experimentally Table 3. to estimate the real burden of lead and inorganic mercury [150, 151]. Its effectiveness in mobilizing metals from the kidney may be because it is transported into kidney cells on 11. BAL (British Anti-Lewisite) the organic anion transport system [150]. It increases the urinary excretion of mercury in persons with an increased BAL (2,3-dimercaptopropanol) as the first clinically useful body burden from industrial exposure, dentists and dental chelating agent was developed during World War II as a technicians, persons with dental amalgams, and those specific antagonist to vesicant arsenical war gases, based on exposedtomercurouschlorideinskincreams[151, 152]. the observation that arsenic has an affinity for sulfhydryl- containing substances. BAL, a dithiol compound with two sulfuratomsonadjacentcarbonatoms,competeswiththe 13. meso-2,3-Dimercaptosuccinic Acid (DMSA) toxic effects. It was developed secretly as an antidote for Lewisite, the now-obsolete arsenic-based chemical warfare DMSA is a chemical analog of BAL. More than 90 percent of agent. Today, it is used medically in treatment of arsenic, DMSA is in the form of a mixed disulfide in which each of the mercury, lead, and other toxic metal poisoning. In addition, it sulfur atoms is in disulfide linkage with a cysteine molecule. has in the past been used for the treatment of Wilson’s disease, The drug is of current interest clinically because of its ability a genetic disorder in which the body tends to retain copper. to lower blood and lead levels. It has advantages over EDTA In humans and experimental models, BAL has been because it is given orally and has greater specificity for lead. It shown to be the most effective antidote when administered may be safer than EDTA in that it does not enhance excretion soon after exposure. Dimercaprol is itself toxic, with a of calcium and zinc to the same degree. Studies in rodents narrow therapeutic range and a tendency to concentrate showedthatasingledoseofDMSAprimarilyremoveslead arsenic in some organs like brain [147]. Other drawbacks from soft tissues [153]. DMSA possesses low toxicity upon include the need to administer it by painful intramuscular oral administration and no redistribution of metal from one injection because of its oily nature. Serious side effects include organ to another [154, 155]. Animal studies suggested that nephrotoxicity and hypertension. DMSA is an effective chelator of soft tissues, but it is unable BAL has been found to form stable chelates in vivo with tochelateleadfrombones[154]. DMSA is not known to many toxic metals including inorganic mercury, antimony, cause elevations in the excretion of calcium, zinc, or iron, bismuth, cadmium, chromium, cobalt, gold, and nickel. although zinc excretion increases to 1.8 times baseline during However, it is not necessarily the treatment of choice for treatment. 18 BioMed Research International

Table 3: Protein thiol-disulfide oxidoreductases with their substrates and functions. Enzyme Substrates Function Glutaredoxin (Grx) PSSG Antioxidant Sulfiredoxin (Srx) PSSG Reductant GSSG reductase (GR) GSSG Antioxidant, maintains GSH Thioredoxin (Trx) PSSP Reductasea, antioxidant

Trx reductase (TR) Trx-S2 Reduces Trx Protein disulfide isomerase (PDI) PSHb Protein folding Protein thiol-disulfide oxidoreductases. aCofactor for ribonucleotide reductase. bOxidizing.

14. Calcium Disodium Ethylenediaminete- Ascorbic acid behaves not only as antioxidant but also as a prooxidant. Ascorbic acid reduced transition metals, such traacetic Acid (CaNa2EDTA) 2+ 1+ as cupric ions (Cu ) to cuprous (Cu ) and ferric ions 3+ 2+ This is a colourless, water-soluble solid which is produced (Fe ) to ferrous (Fe ) during conversion from ascorbate on a large scale for many applications. Its prominence as a to dehydroxyascorbate. This reaction in vitro can generate chelating agent arises from its ability to “sequester” di- and superoxide and other ROS. However, in the body, free 2+ 3+ tricationic metal ions such as Ca and Fe .Afterbeing transitionelementsareunlikelytobepresentwhileironand bound by EDTA, metal ions remain in solution but exhibit copper are bound to diverse proteins. Recent studies show diminished reactivity. Calcium salt of ethylenediaminete- that intravenous injection of 7.5 g of ascorbate daily for 6 days traacetic acid (EDTA) must be used for clinical purposes did not increase prooxidant markers. Thus, ascorbate as a because the sodium salt has greater affinity for calcium and prooxidant is unlikely to convert metals to create ROS in vivo. will produce hypocalcemic tetany. CaNa2EDTA is a deriva- There are few side effects of vitamin C when taken in excess tive of ethylenediaminetetraacetic acid (EDTA), synthetic such as diarrhea, iron poisoning, hemolytic anemia, kidney polyaminocarboxylic acid [156]. EDTA exhibits low toxicity stone formation, and suppression of progesterone in pregnant −1 with LD50 (rat) of 2.0–2.2 g kg .Ithasbeenfoundtobe ladies. both cytotoxic and weakly genotoxic in laboratory animals. Vitamin E (𝛼-tocopherol) is the collective name for Oral exposures have been noted to cause reproductive and asetof8related𝛼-, 𝛽-, 𝛾-, and 𝛿-tocopherols and the developmental effects. CaNa2EDTA cannot pass through corresponding four tocotrienols, which are fat-soluble vita- cellular membrane so its application can help remove the mins with antioxidant properties. Vitamin E also provides metal ions present in extracellular fluid. Further, EDTA protection against silver induced lesions. When Cd was treatment can result in mobilization of lead from hard tissue coadministered with vitamin E, the results showed reduced deposit to soft organs [157, 158]. The calcium salt of EDTA accumulation of cadmium in the kidney, liver, and blood. The has been shown to cause necrosis of renal tubular cells. These antioxidants levels enhanced by Cd induction came down to drawbacks preclude application of CaNa2EDTA as a suitable near normal levels, which indicate that antioxidant properties drug to treat lead poisoning. of vitamin E may be responsible for providing protection from cadmium toxicity [160]. Vitamin E has been shown to 15. Vitamins C and E reduce lipid peroxidation and to increase the cell viability. The supplementation of vitamin E to Pb-treated erythrocytes 𝛿 Vitamin C or L-ascorbic acid is an essential nutrient and a has been shown to prevent the inhibition of -aminolevulinic potential antioxidant for humans. It is made internally by dehydratase activity and lipid oxidation [161] in vivo and in almost all organisms, with humans being a notable exception. vitro. VitaminEcouldalsobeusefultoprotectmembrane- The pharmacophore of this vitamin is an ascorbic anion. lipidsandproteinsfromoxidationduetoleadintoxication. In humans, vitamin C is essential to a healthy diet as well as being a highly effective antioxidant, acting to lessen 16. 𝛽-Carotene oxidative stress, and a substrate for ascorbate peroxidase and an enzyme cofactor for the biosynthesis of many important 𝛽-Carotene is fat-soluble organic compound, a terpenoid, biochemicals. Vitamin C acts as an electron donor for eight a red-orange pigment abundant in plants and fruits. As a different enzymes. It also acts as a scavenger of free radical carotene with 𝛽-rings at both ends, it is the most common and plays an important role in regeneration of 𝛼-tocopherol form of carotene. It is a precursor (inactive form) of vitamin [159]. The plasma ascorbate concentration in oxidative stress −1 A (retinol). The most common side effect of high doses patient (less than 45 𝜇mol L )measuredislowerthan of 𝛽-carotene consumption is carotenodermia, a harmless −1 healthyindividual(61.4–80𝜇mol L ). The increasing plasma condition that presents as a conspicuous orange skin tint ascorbate level may have therapeutic effects in oxidative arising from deposition of the carotenoid in the outermost stress individual. Individual with oxidative stress and healthy layer of the epidermis. It has also been associated with individual have different pharmacokinetics of ascorbate. increased rate of lung cancer among those who smoke [162]. BioMed Research International 19

17. N-Acetylcysteine (NAC) animals, including humans, and some other living organisms, including algae. It is produced by the pineal gland (outside N-acetylcysteine or N-acetyl-L-cysteine (NAC), a pharma- BBB) under the influence of the suprachiasmatic nuclei ceutical drug marketed with various trade names, is used (SCN) of the hypothalamus, which receives information mainly as a mucolytic agent and in the management of fromtheretinaaboutthedailypatternoflightanddarkness. paracetamol (acetaminophen) overdose. Out of the three Its circulating levels vary in a daily cycle. Despite its sulfur-containing amino acids, L-cysteine is an essential one role in regulating circadian rhythm (synchronization of as the other two may be synthesized by using it. L-cysteine the biological clock) in humans, it also acts as powerful − has been shown to act as an antioxidant and has a pivotal endogenous scavenger of ROS and RNS such as OH, O2 , role in endogenous free radicals induced intoxication in andNOradicals.Melatoninisanantioxidantthatcan the body. The exposure to heavy metals has been found to easily cross cell membranes and BBB. Melatonin is a direct reduce level of cysteine [163]. Recently it has been reported scavenger of ROS. Unlike other antioxidants, melatonin does that a high oral dose of NAC modulates inflammation in not undergo redox cycling. Redox cycling may allow other cystic fibrosis (CF) and has the potential to counter the antioxidants (such as vitamin C) to regain their antioxidant intertwined redox and inflammatory imbalances in CF164 [ ]. properties. Melatonin, on the other hand, once oxidized, The supplementation of NAC may attenuate Cd induced cannot be reduced to its former state because it forms nephrotoxicity via chelation [165]. The coadministration of several stable end-products upon reacting with free radicals. NAC and DMSO after arsenic exposure leads to removal of Therefore, it has been referred to as a terminal (or suicidal) arsenic from soft organs [45]. NAC was metabolized to S- antioxidant. nitroso-N-acetylcysteine (SNOAC), which increased blood Melatonin helps maintain membrane fluidity by prevent- pressure in mice treated with acetylcysteine. So its large doses ing LPO and scavenging ROS and RNS [169]. Its capacity to can damage the heart and lungs. absorb free radicals extends at least to the quaternary metabolites of melatonin, a process referred to as “the free radical 18. 𝛼-Lipoic Acid scavenging cascade.” Due to antioxidant potential attached to melatonin, it has been found to protect haematopoietic Lipoic acid is an organosulfur compound (yellow solid). Its cells from the lead-induced damage [170]. The indole group R-enantiomer is an essential cofactor for many enzyme com- present in the melatonin has been implicated in protection plexes. This is a carboxylic acid and features a cyclic disulfide of thiol groups of membrane proteins from lead toxicity or ditholane ring, functional group. It exists as dihydrolipoic [156, 171, 172]. acid, the reduced form of lipoic acid intracellularly. Lipoic As reported, melatonin stimulates antioxidative enzymes acidwasfirstpostulatedtobeaneffectiveendogenousthiol such as GPx and SOD and quickly eliminates H2O2 from rat antioxidant when it was found to prevent the symptoms of brain cortical cells [173, 174]. It also enhances the production vitamin C and vitamin E deficiency. Dihydrolipoic acid is of the enzymes involved in glutathione synthesis [175]. The able to regenerate (reduce) antioxidants, such as glutathione, circulating melatonin decreases with age and in recent years vitamin C, and vitamin E. It is equipped with properties to much interest has been focused on its immunomodulatory quench ROS in vitro. The relatively good scavenging activity effect. Melatonin stimulates the production of progenitor cells of lipoic acid is due to the strained conformation of the for granulocytes macrophages. It also stimulates the produc- 5-membered ring in the intramolecular disulfide. Owing tion of NK cells and CD4+ cells and inhibits CD8+ cells. The to the presence of two thiol groups, it can chelate metals production and release of various cytokines from NK cells such as iron, copper, mercury, and cadmium and remediate and T-helper lymphocytes also are enhanced by melatonin. free radical damage in biological system [166]. It is readily Melatonin presumably regulates immune function by acting available from the diet and absorbed through the gut and on the immune-opioid network by affecting G protein-cAMP can easily enter BBB in humans unlike DMSA and DMPS. signal pathway and by regulating intracellular glutathione However, its effectiveness is heavily dependent on the dosage levels [176]. Melatonin’s antioxidative property may offer and frequency of application. The exogenous supplementa- protection against carcinogenesis, neurodegeneration, and tion of LA has been reported to act as an efficient antioxidant aging [177, 178]. thereby reducing oxidative stress both in vitro and in vivo Possibly there are three different mechanisms of actions [167]. Its intake can change the redox level of tissues directly of melatonin: (1) melatonin could trigger antioxidant cellular by quenching the free radicals and indirectly by enhancing mechanisms, (2) melatonin could form a complex joining the levels of other antioxidants/antioxidant enzymes. Among thecadmiumandthusavoidtheuptakeofthemetalandits the mono- and dithiols (glutathione, cysteine, dithiothreitol, harmful effects, a fact shown at the brain and pituitary level, and lipoic acid), LA has been reported in vitro to be the most and (3) melatonin is very liposoluble so it can diffuse freely potent scavenger of free radicals generated during cadmium and cross all cellular barriers, facilitating the elimination of hepatotoxicity [168]. cadmium from tissues. In conclusion, melatonin is a natural antioxidant, with high power of scavenger of free radicals, so 19. Melatonin it can play an important role in protecting cells against the oxidative damage induced by cadmium. Thus, melatonin may Melatonin, chemically known as N-acetyl-5-methoxy be a good tool to palliate, at least in part, the toxicity of this tryptamine, is a naturally occurring hormone found in most metal [179]. 20 BioMed Research International

The physical, chemical, clinical, and treatment aspects of in animal models, still the in-depth clinical studies with pre- arsenic, lead, cadmium, and mercury have been summarised existing or newer chelating agents are required to be done to in Table 2. reap real benefit with the least side effects during combination therapy in association with those of antioxidants. In case of humans, it is also important to find out the suitable dose 20. Combination Therapy and duration of treatment to decide the optimal therapeutic index for any of the drugs to be given in isolation or in Combination therapy most often not only is used to refer different combinations as explained above. In conclusion, the to the simultaneous administration of two or more med- present stage of knowledge about the impact of heavy metals ications to treat a single disease but also is used when in biological systems indicates that enhanced formation of other types of therapy are employed at the same time. free radicals and ROS or RNS or their intermediates can be Here in chelation therapy, it refers to the administration of regarded as a common factor in determining heavy metal- two chelators differently administered with the notion that induced toxicity and carcinogenicity. various chelating agents may immobilize (trap) the toxic metals from different tissues to render better protection from their toxicity [180–184]. In combination chelation therapy, Conflict of Interests also the coadministration of dietary nutrients like vitamins, for example, thiamine [183], an essential metal such as zinc The authors declare that they do not have any conflict of [180, 185], or an amino acid like methionine with a chelating interests. agent, has been found to render better clinical recoveries as well as mobilization of lead from the tissues [186, 187]. References

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