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Heavy Metals Toxicity and the Environment

Paul B Tchounwou, Clement G Yedjou, Anita K Patlolla, Dwayne JSutton

NIH-RCMI Center for Environmental Health, College of Science, Engineering andTechnology, Jackson State University, 1400 Lynch Street, Box 18750, Jackson, MS39217, USA

Abstract: Heavy metals are naturally occurring elements that havea high atomic weight and a density at least 5 times greaterthan that of water. Their multiple industrial, domestic,agricultural, medical and technological applications have ledto their wide distribution in the environment; raisingconcerns over their potential effects on human health and theenvironment. Their toxicity depends on several factorsincluding the dose, route of exposure, and chemical species,as well as the age, gender, genetics, and nutritional statusof exposed individuals. Because of their high degree oftoxicity, arsenic, cadmium, chromium, lead, and mercury rankamong the priority metals that are of public healthsignificance. These metallic elements are considered systemictoxicants that are known to induce multiple organ damage, evenat lower levels of exposure. They are also classified as humancarcinogens (known or probable) according to the U.S.Environmental Protection Agency, and the International Agencyfor Research on Cancer. This review provides an analysis oftheir environmental occurrence, production and use, potentialfor human exposure, and molecular mechanisms of toxicity,genotoxicity, and carcinogenicity.

Keywords: heavy metals, production and use, human exposure, toxicity, genotoxicity, carcinogenicity

Introduction

Heavy metals are defined as metallic elements that have arelatively high density compared to water [1]. With theassumption that heaviness and toxicity are inter-related,heavy metals also include metalloids, such as arsenic, thatare able to induce toxicity at low level of exposure [2]. Inrecent years, there has been an increasing ecological and

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global public health concern associated with environmentalcontamination by these metals. Also, human exposure has risendramatically as a result of an exponential increase of theiruse in several industrial, agricultural, domestic andtechnological applications [3]. Reported sources of heavymetals in the environment include geogenic, industrial,agricultural, pharmaceutical, domestic effluents, andatmospheric sources [4] . Environmental pollution is veryprominent in point source areas such as mining, foundries andsmelters, and other metal-based industrial operations [1, 3,4].

Although heavy metals are naturally occurring elementsthat are found throughout the earth’s crust, mostenvironmental contamination and human exposure result fromanthropogenic activities such as mining and smeltingoperations, industrial production and use, and domestic andagricultural use of metals and metal-containing compounds [4-7]. Environmental contamination can also occur through metalcorrosion, atmospheric deposition, soil erosion of metal ionsand leaching of heavy metals, sediment re-suspension and metalevaporation from water resources to soil and ground water [8].Natural phenomena such as weathering and volcanic eruptionshave also been reported to significantly contribute to heavymetal pollution [1, 3, 4, 7, 8]. Industrial sources includemetal processing in refineries, coal burning in power plants,petroleum combustion, nuclear power stations and high tensionlines, plastics, textiles, microelectronics, wood preservationand paper processing plants [9-11].

It has been reported that metals such as cobalt (Co),copper (Cu), chromium (Cr), iron (Fe), magnesium (Mg),manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se)and zinc (Zn) are essential nutrients that are required forvarious biochemical and physiological functions [12].Inadequate supply of these micro-nutrients results in avariety of deficiency diseases or syndromes [12].

Heavy metals are also considered as trace elementsbecause of their presence in trace concentrations (ppb rangeto less than 10ppm) in various environmental matrices [13].Their bioavailability is influenced by physical factors suchas temperature, phase association, adsorption andsequestration. It is also affected by chemical factors thatinfluence speciation at thermodynamic equilibrium,

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complexation kinetics, lipid solubility and octanol/waterpartition coefficients [14]. Biological factors such asspecies characteristics, trophic interactions, andbiochemical/physiological adaptation, also play an importantrole [15].

The essential heavy metals play biochemical andphysiological functions in plants and animals. They areimportant constituents of several key enzymes and playimportant roles in various oxidation-reduction reactions [12].Copper for example serves as an essential co-factor forseveral oxidative stress-related enzymes including catalase,superoxide dismutase, peroxidase, cytochrome c oxidases,ferroxidases, monoamine oxidase, and dopamine β-monooxygenase[16-18]. Hence, it is an essential nutrient that isincorporated into a number of metalloenzymes involved inhemoglobin formation, carbohydrate metabolism, catecholaminebiosynthesis, and cross-linking of collagen, elastin, and hairkeratin. The ability of copper to cycle between an oxidizedstate, Cu(II), and reduced state, Cu(I), is used bycuproenzymes involved in redox reactions [16-18]. However, itis this property of copper that also makes it potentiallytoxic because the transitions between Cu(II) and Cu(I) canresult in the generation of superoxide and hydroxyl radicals[16-19]. Also, excessive exposure to copper has been linked tocellular damage leading to Wilson disease in humans [18, 19].Similar to copper, several other essential elements arerequired for biologic functioning, however, excess amount ofsuch metals produces cellular and tissue damage leading to avariety of adverse effects and human diseases. For someincluding chromium and copper, there is a very narrow range ofconcentrations between beneficial and toxic effects [19, 20].Other metals such as aluminium (Al), antinomy (Sb), arsenic(As), barium (Ba), beryllium (Be), bismuth (Bi), cadmium (Cd),gallium (Ga), germanium (Ge), gold (Au), indium (In), lead(Pb), lithium (Li), mercury (Hg), nickel (Ni), platinum (Pt),silver (Ag), strontium (Sr), tellurium (Te), thallium (Tl),tin (Sn), titanium (Ti), vanadium (V) and uranium (U) have noestablished biological functions and are considered as non-essential metals [20].

In biological systems, heavy metals have been reported toaffect cellular organelles and components such as cell membrane,mitochondrial, lysosome, endoplasmic reticulum, nuclei, and some

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enzymes involved in metabolism, detoxification, and damagerepair [21]. Metal ions have been found to interact with cellcomponents such as DNA and nuclear proteins, causing DNA damageand conformational changes that may lead to cell cyclemodulation, carcinogenesis or apoptosis [20-22]. Several studiesfrom our laboratory have demonstrated that reactive oxygenspecies (ROS) production and oxidative stress play a key rolein the toxicity and carcinogenicity of metals such as arsenic[23, 24, 25], cadmium [26], chromium [27, 28], lead [29, 30],and mercury [31, 32]. Because of their high degree oftoxicity, these five elements rank among the priority metalsthat are of great public health significance. They are allsystemic toxicants that are known to induce multiple organdamage, even at lower levels of exposure. According to theUnited States Environmental Protection Agency (U.S. EPA), andthe International Agency for Research on Cancer (IARC), thesemetals are also classified as known or probable humancarcinogens based on epidemiological and experimental studiesshowing an association between exposure and cancer incidencein humans and animals.

Heavy metal-induced toxicity and carcinogenicity involvesmany mechanistic aspects, some of which are not clearlyelucidated or understood. However, each metal is known to haveunique features that confer to its specific toxicologicalmechanisms of action. This review provides an analysis oftheir environmental occurrence, production and use, potentialfor human exposure, and molecular mechanisms of toxicity,genotoxicity, and carcinogenicity of arsenic, cadmium,chromium, lead and mercury.

Arsenic

Environmental Occurrence, Industrial Production and Use

Arsenic is a ubiquitous element that is detected at lowconcentrations in virtually all environmental matrices [33].The major inorganic forms of arsenic include the trivalentarsenite and the pentavalent arsenate. The organic forms arethe methylated metabolites – monomethylarsonic acid (MMA),dimethylarsinic acid (DMA) and trimethylarsine oxide.Environmental pollution by arsenic occurs as a result ofnatural phenomena such as volcanic eruptions and soil

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erosion, and anthropogenic activities [33]. Several arsenic-containing compounds are produced industrially, and have beenused to manufacture products with agricultural applicationssuch as insecticides, herbicides, fungicides, algicides, sheepdips, wood preservatives, and dye-stuffs. They have also beenused in veterinary medicine for the eradication of tapewormsin sheep and cattle [34]. Arsenic compounds have also beenused in the medical field for at least a century in thetreatment of syphilis, yaws, amoebic dysentery, andtrypanosomaiasis [34,35]. Arsenic-based drugs are still usedin treating certain tropical diseases such as African sleepingsickness and amoebic dysentery, and in veterinary medicine totreat parasitic diseases, including filariasis in dogs andblack head in turkeys and chickens [35]. Recently, arsenictrioxide has been approved by the Food and Drug Administrationas an anticancer agent in the treatment of acute promeylocyticleukemia [36]. Its therapeutic action has been attributed tothe induction of programmed cell death (apoptosis) in leukemiacells [24].

Potential for Human Exposure

It is estimated that several million people are exposed toarsenic chronically throughout the world, especially incountries like Bangladesh, India, Chile, Uruguay, Mexico,Taiwan, where the ground water is contaminated with highconcentrations of arsenic. Exposure to arsenic occurs via theoral route (ingestion), inhalation, dermal contact, and theparenteral route to some extent [33,34,37]. Arsenicconcentrations in air range from 1 to 3 ng/m3 in remotelocations (away from human releases), and from 20 to 100 ng/m3

in cities. Its water concentration is usually less than10g/L, although higher levels can occur near natural mineraldeposits or mining sites. Its concentration in various foodsranges from 20 to 140 ng/kg [38]. Natural levels of arsenic insoil usually range from 1 to 40 mg/kg, but pesticideapplication or waste disposal can produce much higher values[25].

Diet, for most individuals, is the largest source ofexposure, with an average intake of about 50 g per day.Intake from air, water and soil are usually much smaller, butexposure from these media may become significant in areas of

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arsenic contamination. Workers who produce or use arseniccompounds in such occupations as vineyards, ceramics, glass-making, smelting, refining of metallic ores, pesticidemanufacturing and application, wood preservation,semiconductor manufacturing can be exposed to substantiallyhigher levels of arsenic [39]. Arsenic has also beenidentified at 781 sites of the 1,300 hazardous waste sitesthat have been proposed by the U.S. EPA for inclusion on thenational priority list [33,39]. Human exposure at these sitesmay occur by a variety of pathways, including inhalation ofdusts in air, ingestion of contaminated water or soil, orthrough the food chain [40].

Contamination with high levels of arsenic is of concernbecause arsenic can cause a number of human health effects.Several epidemiological studies have reported a strongassociation between arsenic exposure and increased risks ofboth carcinogenic and systemic health effects [41]. Interestin the toxicity of arsenic has been heightened by recentreports of large populations in West Bengal, Bangladesh,Thailand, Inner Mongolia, Taiwan, China, Mexico, Argentina,Chile, Finland and Hungary that have been exposed to highconcentrations of arsenic in their drinking water and aredisplaying various clinico-pathological conditions includingcardiovascular and peripheral vascular disease, developmentalanomalies, neurologic and neurobehavioural disorders,diabetes, hearing loss, portal fibrosis, hematologic disorders(anemia, leukopenia and eosinophilia) and carcinoma [25, 33,35, 39]. Arsenic exposure affects virtually all organ systemsincluding the cardiovascular, dermatologic, nervous,hepatobilliary, renal, gastro-intestinal, and respiratorysystems [41]. Research has also pointed to significantlyhigher standardized mortality rates for cancers of thebladder, kidney, skin, and liver in many areas of arsenicpollution. The severity of adverse health effects is relatedto the chemical form of arsenic, and is also time- and dose-dependent [42,43]. Although the evidence of carcinogenicity ofarsenic in humans seems strong, the mechanism by which itproduces tumors in humans is not completely understood [44].

Mechanisms of Toxicity and Carcinogenicity

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Analyzing the toxic effects of arsenic is complicated becausethe toxicity is highly influenced by its oxidation state andsolubility, as well as many other intrinsic and extrinsicfactors [45]. Several studies have indicated that the toxicityof arsenic depends on the exposure dose, frequency andduration, the biological species, age, and gender, as well ason individual susceptibilities, genetic and nutritionalfactors [46]. Most cases of human toxicity from arsenic havebeen associated with exposure to inorganic arsenic. Inorganictrivalent arsenite (AsIII ) is 2-10 times more toxic thanpentavalent arsenate (AsV) [5]. By binding to thiol orsulfhydryl groups on proteins, As (III) can inactivate over200 enzymes. This is the likely mechanism responsible forarsenic’s widespread effects on different organ systems. As(V) can replace phosphate, which is involved in manybiochemical pathways [5, 47].

One of the mechanisms by which arsenic exerts its toxiceffect is through impairment of cellular respiration by theinhibition of various mitochondrial enzymes, and theuncoupling of oxidative phosphorylation. Most toxicity ofarsenic results from its ability to interact with sulfhydrylgroups of proteins and enzymes, and to substitute phosphorousin a variety of biochemical reactions [48]. Arsenic in vitroreacts with protein sulfhydryl groups to inactivate enzymes,such as dihydrolipoyl dehydrogenase and thiolase, therebyproducing inhibited oxidation of pyruvate and betaoxidation offatty acids [49]. The major metabolic pathway for inorganicarsenic in humans is methylation. Arsenic trioxide ismethylated to two major metabolites via a non-enzymaticprocess to monomethylarsonic acid (MMA), which is furthermethylated enzymatically to dimethyl arsenic acid (DMA) beforeexcretion in the urine [40, 47]. It was previously thoughtthat this methylation process is a pathway of arsenicdetoxification, however, recent studies have pointed out thatsome methylated metabolites may be more toxic than arsenite ifthey contain trivalent forms of arsenic [41].

Tests for genotoxicity have indicated that arseniccompounds inhibit DNA repair, and induce chromosomalaberrations, sister-chromatid exchanges, and micronucleiformation in both human and rodent cells in culture [50-52]and in cells of exposed humans [53]. Reversion assays withSalmonella typhimurium fail to detect mutations that are induced

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by arsenic compounds. Although arsenic compounds are generallyperceived as weak mutagens in bacterial and animal cells, theyexhibit clastogenic properties in many cell types in vivo and invitro [54]. In the absence of animal models, in vitro celltransformation studies become a useful means of obtaininginformation on the carcinogenic mechanisms of arsenictoxicity. Arsenic and arsenical compounds are toxic to andinduce morphological transformations of Syrian hamster embryo(SHE) cells as well as mouse C3H10T1/2 cells and BALB/3T3cells [55,56].

Based on the comet assay, it has been reported thatarsenic trioxide induces DNA damage in human lymphophytes [57]and also in mice leukocytes [58]. Arsenic compounds have alsobeen shown to induce gene amplification, arrest cells inmitosis, inhibit DNA repair, and induce expression of the c-fosgene and the oxidative stress protein heme oxygenase inmammalian cells [58, 59]. They have been implicated aspromoters and comutagens for a variety of toxic agents [60].Recent studies in our laboratory have demonstrated thatarsenic trioxide is cytotoxic and able to transcriptionallyinduce a significant number of stress genes and relatedproteins in human liver carcinoma cells [61].

Epidemiological investigations have indicated that long-term arsenic exposure results in promotion of carcinogenesis.Several hypotheses have been proposed to describe themechanism of arsenic-induced carcinogenesis. Zhao et al. [62]reported that arsenic may act as a carcinogen by inducing DNAhypomethylation, which in turn facilitates aberrant geneexpression. Additionally, it was found that arsenic is apotent stimulator of extracellular signal-regulated proteinkinase Erk1 and AP-1 transactivational activity, and anefficient inducer of c-fos and c-jun gene expression [63].Induction of c-jun and c-fos by arsenic is associated withactivation of JNK [64]. However, the role of JNK activation byarsenite in cell transformation or tumor promotion is unclear.

In another study, Trouba et al. [65] concluded that long-term exposure to high levels of arsenic might make cells moresusceptible to mitogenic stimulation and that alterations inmitogenic signaling proteins might contribute to thecarcinogenic action of arsenic. Collectively, several recentstudies have demonstrated that arsenic can interfere with cellsignaling pathways (e.g., the p53 signaling pathway) that are

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frequently implicated in the promotion and progression of avariety of tumor types in experimental animal models, and ofsome human tumors [66, 68]. However, the specific alterationsin signal transduction pathways or the actual targets thatcontribute to the development of arsenic-induced tumors inhumans following chronic consumption of arsenic remainsuncertain.

Recent clinical trials have found that arsenic trioxidehas therapeutic value in the treatment of acute promyelocyticleukemia, and there is interest in exploring its effectivenessin the treatment of a variety of other cancers [69,70]. Inacute promyelocytic leukemia, the specific molecular eventcritical to the formation of malignant cells is known. Thestudy by Puccetti et al. [71] found that forced overexpressionof BCR-ABL susceptibility in human lymphoblasts cells resultedin greatly enhanced sensitivity to arsenic-induced apoptosis.They also concluded that arsenic trioxide is a tumor specificagent capable of inducing apoptosis selectively in acutepromyelocytic leukemia cells. Several recent studies haveshown that arsenic can induce apoptosis through alterations inother cell signaling pathways [72,73]. In addition to acutepeomyelocytic leukemia, arsenic is thought to have therapeuticpotential for myeloma [74]. In summary, numerous cancerchemotherapy studies in cell cultures and in patients withacute promyelocytic leukemia demonstrate that arsenic trioxideadministration can lead to cell-cycle arrest and apoptosis inmalignant cells.

Previous studies have also examined p53 gene expressionand mutation in tumors obtained from subjects with a historyof arsenic ingestion. p53 participates in many cellularfunctions, cell-cycle control, DNA repair, differentiation,genomic plasticity and programmed cell death. Additionalsupport for the hypothesis that arsenic can modulate geneexpression has been provided by several different studies[75,76]. Collectively, these studies provide further evidencethat various forms of arsenic can alter gene expression andthat such changes could contribute substantially to the toxicand carcinogenic actions of arsenic treatment in humanpopulations [77].

Several in vitro studies in our laboratory havedemonstrated that arsenic modulates DNA synthesis, gene andprotein expression, genotoxicity, mitosis and/or apoptotic

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mechanisms in various cell lines including keratinocytes,melanocytes, dendritic cells, dermal fibroblasts,microvascular endothelial cells, monocytes, and T-cells [78],colon cancer cells [79], lung cancer cells [80], humanleukemia cells [81], Jurkat-T lymphocytes [82], and humanliver carcinoma cells [83]. We have also shown that oxidativestress plays a key role in arsenic induced cytotoxicity, aprocess that is modulated by pro- and/or anti-oxidants such asascorbic acid and n-acetyl cysteine [84-86]. We have furtherdemonstrated that the toxicity of arsenic depends on itschemical form, the inorganic form being more toxic than theorganic one [42].

Various hypotheses have been proposed to explain thecarcinogenicity of inorganic arsenic. Nevertheless, themolecular mechanisms by which this arsenical induces cancerare still poorly understood. Results of previous studies haveindicated that inorganic arsenic does not act through classicgenotoxic and mutagenic mechanisms, but rather may be a tumorpromoter that modifies signal transduction pathways involvedin cell growth and proliferation [68]. Although much progresshas been recently made in the area of arsenic’s possiblemode(s) of carcinogenic action, a scientific consensus has notyet reached. A recent review discusses nine different possiblemodes of action of arsenic carcinogenesis: induced chromosomalabnormalities, oxidative stress, altered DNA repair, alteredDNA methylation patterns, altered growth factors, enhancedcell proliferation, promotion/progression, suppression of p53,and gene amplification [87]. Presently, three modes(chromosomal abnormality, oxidative stress, and altered growthfactors) of arsenic carcinogenesis have shown a degree ofpositive evidence, both in experimental systems (animal andhuman cells) and in human tissues. The remaining possiblemodes of carcinogenic action (progression of carcinogenesis,altered DNA repair, p53 suppression, altered DNA methylationpatterns and gene amplification) do not have as much evidence,particularly from in vivo studies with laboratory animals, in vitrostudies with cultured human cells, or human data from case orpopulation studies. Thus, the mode-of-action studies suggestthat arsenic might be acting as a cocarcinogen, a promoter, ora progressor of carcinogenesis.

Cadmium

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Environmental Occurrence, Industrial Production and Use

Cadmium is a heavy metal of considerable environmental andoccupational concern. It is widely distributed in the earth'scrust at an average concentration of about 0.1 mg/kg. Thehighest level of cadmium compounds in the environment isaccumulated in sedimentary rocks, and marine phosphatescontain about 15 mg cadmium/kg [88].

Cadmium is frequently used in various industrialactivities. The major industrial applications of cadmiuminclude the production of alloys, pigments, and batteries[89]. Although the use of cadmium in batteries has shownconsiderable growth in recent years, its commercial use hasdeclined in developed countries in response to environmentalconcerns. In the United States for example, the daily cadmiumintake is about 0.4μg/kg/day, less than half of the U.S. EPA’soral reference dose [90]. This decline has been linked to theintroduction of stringent effluent limits from plating worksand, more recently, to the introduction of generalrestrictions on cadmium consumption in certain countries.

Potential for Human Exposure

The main route of exposure to cadmium is via inhalation orcigarette smoke or ingestion of food. Skin absorption is rare.Human exposure to cadmium is possible through a number ofseveral sources including employment in primary metalindustries, eating contaminated food, smoking cigarettes, andworking in cadmium-contaminated work places, with smokingbeing a major contributor [91, 92]. Other sources of cadmiuminclude emissions from industrial activities, includingmining, smelting, and manufacturing of batteries, pigments,stabilizers, and alloys [93]. Cadmium is also present in traceamounts in certain foods such as leafy vegetables, potatoes,grains and seeds, liver and kidney, and crustaceans andmollusks [94]. In addition, foodstuffs that are rich incadmium can greatly increase the cadmium concentration inhuman bodies. Examples are liver, mushrooms, shellfish,mussels, cocoa powder and dried seaweed. An important route ofexposure is the circulatory system whereas blood vessels areconsidered to be main stream organs of cadmium toxicity.

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Chronic inhalation exposure to cadmium particulates isgenerally associated with changes in pulmonary function andchest radiographs that are consistent with emphysema [95].Workplace exposure to airborne cadmium particulates has beenassociated with decreases in olfactory function [96]. Severalepidemiologic studies have documented an association ofchronic low-level cadmium exposure with decreases in bonemineral density and osteoporosis [97-99].

Exposure to cadmium is commonly determined by measuringcadmium levels in blood or urine. Blood cadmium reflectsrecent cadmium exposure (from smoking, for example). Cadmiumin urine (usually adjusted for dilution by calculating thecadmium/creatinine ratio) indicates accumulation, or kidneyburden of cadmium [100, 101]. It is estimated that about 2.3%of the U.S. population has elevated levels of urine cadmium(>2µg/g creatinine), a marker of chronic exposure and bodyburden [102]. Blood and urine cadmium levels are typicallyhigher in cigarette smokers, intermediate in former smokersand lower in nonsmokers [102, 103]. Because of continuing useof cadmium in industrial applications, the environmentalcontamination and human exposure to cadmium have dramaticallyincreased during the past century [104].

Molecular Mechanisms of Toxicity and Carcinogenicity

Cadmium is a severe pulmonary and gastrointestinal irritant,which can be fatal if inhaled or ingested. After acuteingestion, symptoms such as abdominal pain, burning sensation,nausea, vomiting, salivation, muscle cramps, vertigo, shock,loss of consciousness and convulsions usually appear within 15to 30 min [105]. Acute cadmium ingestion can also causegastrointestinal tract erosion, pulmonary, hepatic or renalinjury and coma, depending on the route of poisoning [105,106]. Chronic exposure to cadmium has a depressive effect onlevels of norepinephrine, serotonin, and acetylcholine [107].Rodent studies have shown that chronic inhalation of cadmiumcauses pulmonary adenocarcinomas [108, 109]. It can alsocause prostatic proliferative lesions includingadenocarcinomas, after systemic or direct exposure [110].

Although the mechanism of cadmium induced toxicity ispoorly understood, it has been speculated that cadmium causesdamage to cells primarily through the generation of reactive

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oxygen species [111], which causes single-strand DNA damageand disrupts the synthesis of nucleic acids and proteins [112].Studies using two-dimensional gel electrophoresis have shownthat several stress response systems are expressed in responseto cadmium exposure, including those for heat shock, oxidativestress, stringent response, cold shock, and SOS [113- 115]. Invitro studies indicate that cadmium induces cytotoxic effects atthe concentrations 0.1 to 10mM and free radical-dependent DNAdamage [116, 117]. In vivo studies have shown that cadmiummodulates male reproduction in mice model at a concentrationof 1 mg/kg body weight [118]. However, cadmium is a weakmutagen when compared with other carcinogenic metals [119].Previous reports have indicated cadmium affects signaltransduction pathways; inducing inositol polyphosphateformation, increasing cytosolic free calcium levels in variouscell types [120], and blocking calcium channels [121, 122]. Atlower concentrations (1-100 μM), cadmium binds to proteins,decreases DNA repair [123], activates protein degradation, up-regulates cytokines and proto-oncogenes such as c-fos, c-jun,and c-myc [124], and induces expression of several genesincluding metallothioneins [125], heme oxygenases, glutathionetransferases, heat-shock proteins, acute-phase reactants, andDNA polymerase β [126].

Cadmium compounds are classified as human carcinogens byseveral regulatory agencies. The International Agency forResearch on Cancer [91] and the US National Toxicology Programhave concluded that there is adequate evidence that cadmium isa human carcinogen. This designation as a human carcinogen isbased primarily on repeated findings of an association betweenoccupational cadmium exposure and lung cancer, as well as onvery strong rodent data showing the pulmonary system as atarget site [91]. Thus, the lung is the most definitivelyestablished site of human carcinogenesis from cadmiumexposure. Other target tissues of cadmium carcinogenesis inanimals include injection sites, adrenals, testes, and thehemopoietic system [91, 108, 109]. In some studies,occupational or environmental cadmium exposure has also beenassociated with development of cancers of the prostate,kidney, liver, hematopoietic system and stomach [108, 109].Carcinogenic metals including arsenic, cadmium, chromium, andnickel have all been associated with DNA damage through basepair mutation, deletion, or oxygen radical attack on DNA

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[126]. Animal studies have demonstrated reproductive andteratogenic effects. Small epidemiologic studies have noted aninverse relationship between cadmium in cord blood, maternalblood or maternal urine and birth weight, and length at birth[127, 128].

Chromium

Environmental Occurrence, Industrial Production and Use

Chromium (Cr) is a naturally occurring element present in theearth’s crust, with oxidation states (or valence states)ranging from chromium (II) to chromium (VI) [129]. ElementalChromium [Cr(0)] does not occur naturally. Chromium compoundsare stable in the trivalent [Cr(III)] form and occur in naturein this state in ores, such as ferrochromite. The hexavalent[Cr(VI)] form is the second-most stable state [28]. Chromiumenters into various environmental matrices (air, water, andsoil) from a wide variety of natural and anthropogenic sourceswith the largest release occurring from industrialestablishments. Industries with the largest contribution tochromium release include metal processing, tannery facilities,chromate production, stainless steel welding, and ferrochromeand chrome pigment production. In humans and animals,[Cr(III)] is an essential nutrient that plays a role inglucose, fat and protein metabolism by potentiating the actionof insulin [5]. Hexavalent chromium [Cr(VI)] is a toxicindustrial pollutant that is classified as human carcinogen byseveral regulatory and non-regulatory agencies [130-132]. Thehealth hazard associated with exposure to chromium depends onits oxidation state, ranging from the low toxicity of themetal form to the high toxicity of the hexavalent form. AllCr(VI)-containing compounds were once thought to be man-made,with only Cr(III) naturally ubiquitous in air, water, soil andbiological materials. Recently, however, naturally occurringCr(VI) has been found in ground and surface waters at valuesexceeding the World Health Organization limit for drinkingwater of 50 µg of Cr(VI) per liter [133]. Chromium is widelyused in numerous industrial processes and as a result, is acontaminant of many environmental systems [134]. Commerciallychromium compounds are used in industrial welding, chromeplating, dyes and pigments, leather tanning and wood

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preservation. Chromium is also used as anticorrosive incooking systems and boilers [135, 136].

Potential for Human Exposure

It is estimated that more than 300,000 workers are exposedannually to chromium and chromium-containing compounds in theworkplace. The increase in the environmental concentrations ofchromium has been linked to air and wastewater release ofchromium, mainly from metallurgical, refractory, and chemicalindustries. Chromium released into the environment fromanthropogenic activity occurs mainly in the hexavalent form[Cr(VI)] [130]. Occupational Cr(VI) exposure has been a majorconcern, as workers in industries that use Cr(VI) compoundsare at higher risk of developing Cr-induced diseases [137].However, the general human population and some wildlife mayalso be at risk. It is estimated that 33 tons of total Cr arereleased annually into the environment [130]. The U.S.Occupational Safety and Health Administration (OSHA) recentlyset a ‘‘safe’’ level of 5µg/m3, for an 8-hr time-weightedaverage, even though this revised level may still pose acarcinogenic risk [138]. For the general human population,atmospheric levels range from 1 to 100 ng/cm3 [139], but canexceed this range in areas that are close to Cr manufacturing.

Non-occupational exposure to the metal occurs viaingestion of chromium containing food and water whereasoccupational exposure occurs via inhalation [140]. Chromiumconcentrations range between 1 and 3000 mg/kg in soil, 5 to800 µg/L in sea water, and 26 µg/L to 5.2 mg/L in rivers andlakes [129]. Chromium content in foods varies greatly anddepends on the processing and preparation. In general, mostfresh foods typically contain chromium levels ranging from <10to 1,300 µg/kg. Present day workers in chromium-relatedindustries can be exposed to chromium concentrations twoorders of magnitude higher than the general population [141].Even though the principal route of human exposure to chromiumis through inhalation, and the lung is the primary targetorgan, significant human exposure to chromium has also beenreported to take place through the skin [142, 143]. Forexample, the widespread incidence of dermatitis noticed amongconstruction workers is attributed to their exposure tochromium present in cement [143]. Occupational and

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environmental exposure to Cr(VI)-containing compounds is knownto cause multiorgan toxicity such as renal damage, allergy andasthma, and cancer of the respiratory tract in humans [5, 144].

Breathing high levels of chromium (VI) can causeirritation to the lining of the nose, and nose ulcers. Themain health problems seen in animals following ingestion ofchromium (VI) compounds are irritation and ulcers in thestomach and small intestine, anemia, sperm damage and malereproductive system damage. Chromium (III) compounds are muchless toxic and do not appear to cause these problems. Someindividuals are extremely sensitive to chromium(VI) orchromium(III), allergic reactions consisting of severe rednessand swelling of the skin have been noted. An increase instomach tumors was observed in humans and animals exposed tochromium(VI) in drinking water. Accidental or intentionalingestion of extremely high doses of chromium (VI) compoundsby humans has resulted in severe respiratory, cardiovascular,gastrointestinal, hematological, hepatic, renal, andneurological effects as part of the sequelae leading to deathor in patients who survived because of medical treatment[141]. Although the evidence of carcinogenicity of chromium inhumans and terrestrial mammals seems strong, the mechanism bywhich it causes cancer is not completely understood [145].

Mechanisms of Toxicity and Carcinogenicity

Major factors governing the toxicity of chromium compounds areoxidation state and solubility. Cr(VI) compounds, which arepowerful oxidizing agents and thus tend to be irritating andcorrosive, appear to be much more toxic systemically thanCr(III) compounds, given similar amount and solubility [146,147]. Although the mechanisms of biological interaction areuncertain, the variation in toxicity may be related to theease with which Cr(VI) can pass through cell membranes and itssubsequent intracellular reduction to reactive intermediates.Since Cr(III) is poorly absorbed by any route, the toxicity ofchromium is mainly attributable to the Cr(VI) form. It can beabsorbed by the lung and gastrointestinal tract, and even to acertain extent by intact skin. The reduction of Cr(VI) isconsidered to serve as a detoxification process when it occursat a distance from the target site for toxic or genotoxiceffect while reduction of Cr(VI) may serve to activate

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chromium toxicity if it takes place in or near the cellnucleus of target organs [148]. If Cr(VI) is reduced toCr(III) extracellularly, this form of the metal is not readilytransported into cells and so toxicity is not observed. Thebalance that exists between extracellular Cr(VI) andintracellular Cr(III) is what ultimately dictates the amountsand rates at which Cr(VI) can enter cells and impart its toxiceffects [134].

Cr(VI) enters many types of cells and under physiologicalconditions can be reduced by hydrogen peroxide (H2O2),glutathione (GSH) reductase, ascorbic acid, and GSH to producereactive intermediates, including Cr(V), Cr(IV),thiylradicals, hydroxyl radicals, and ultimately, Cr(III). Anyof these species could attack DNA, proteins, and membranelipids, thereby disrupting cellular integrity and functions[149, 150].

Studies with animal models also found many harmfuleffects of Cr (VI) on mammals. Subcutaneous administration ofCr (VI) to rats caused severe progressive proteinuria, ureanitrogen and creatinine, as well as elevation in serum alanineaminotransferase activity and hepatic lipid peroxide formation[151]. Similar studies reported by Gumbleton and Nicholls[152] found that Cr (VI) induced renal damage in rats whenadministered by single sc injections. Bagchi et aldemonstrated that rats received Cr (VI) orally in waterinduced hepatic mitochondrial and microsomal lipidperoxidation, as well as enhanced excretion of urinary lipidmetabolites including malondialdehyde [153, 154]. Moreover,some adverse health effects induced by Cr (VI) have beenreported in humans. Epidemiological investigations havereported respiratory cancers in workers occupationally exposedto Cr (VI)-containing compounds [142, 148]. DNA strand breaksin peripheral lymphocytes and lipid peroxidation products inurine observed in chromium-exposed workers also support theevidence of Cr (VI)-induced toxicity to humans [155, 156].Oxidative damage is considered to be the underlying cause ofthese genotoxic effects including chromosomal abnormalities[157, 158], and DNA strand breaks [159]. Nevertheless, recentstudies indicate a biological relevance of non-oxidativemechanisms in Cr(VI) carcinogenesis [160].

Carcinogenicity appears to be associated with theinhalation of the less soluble/insoluble Cr(VI) compounds. The

18

toxicology of Cr(VI) does not reside with the elemental form.It varies greatly among a wide variety of very differentCr(VI) compounds [161]. Epidemiological evidence stronglypoints to Cr(VI) as the agent in carcinogenesis. Solubilityand other characteristics of chromium, such as size, crystalmodification, surface charge, and the ability to bephagocytized might be important in determining cancer risk[135].

Studies in our laboratory have indicated that chromium(VI) is cytotoxic and able to induce DNA damaging effects suchas chromosomal abnormalities [162], DNA strand breaks, DNAfragmentation and oxidative stress in Sprague-Dawley rats andhuman liver carcinoma cells [27, 28]. Recently, our laboratoryhas also demonstrated that chromium (VI) induces biochemical,genotoxic and histopathologic effects in liver and kidney ofgoldfish, carassius auratus [163].

Various hypothesis have been proposed to explain thecarcinogenicity of chromium and its salts, however someinherent difficulties exist when discussing metalcarcinogenesis. A metal cannot be classified as carcinogenicper se since its different compounds may have differentpotencies. Because of the multiple chemical exposure inindustrial establishments, it is difficult from anepidemiological standpoint to relate the carcinogenic effectto a single compound. Thus, the carcinogenic risk must oftenbe related to a process or to a group of metal compoundsrather than to a single substance. Differences in carcinogenicpotential are related not only to different chemical forms ofthe same metal but also to the particle size of the inhaledaerosol and to physical characteristics of the particle suchas surface charge and crystal modification [164].

Lead

Environmental Occurrence, Industrial Production and Use

Lead is a naturally occurring bluish-gray metal present insmall amounts in the earth’s crust. Although lead occursnaturally in the environment, anthropogenic activities such asfossil fuels burning, mining, and manufacturing contribute tothe release of high concentrations. Lead has many differentindustrial, agricultural and domestic applications. It is

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currently used in the production of lead-acid batteries,ammunitions, metal products (solder and pipes), and devices toshield X-rays. An estimated 1.52 million metric tons of leadwere used for various industrial applications in the UnitedStated in 2004. Of that amount, lead-acid batteries productionaccounted for 83 percent, and the remaining usage covered arange of products such as ammunitions (3.5 percent), oxidesfor paint, glass, pigments and chemicals (2.6 percent), andsheet lead (1.7 percent) [165, 166].

In recent years, the industrial use of lead has beensignificantly reduced from paints and ceramic products,caulking, and pipe solder [167]. Despite this progress, it hasbeen reported that among 16.4 million United States homes withmore than one child younger than 6 years per household, 25% ofhomes still had significant amounts of lead-contaminateddeteriorated paint, dust, or adjacent bare soil [168]. Lead indust and soil often re-contaminate cleaned houses [169] andcontributes to elevating blood lead concentrations in childrenwho play on bare, contaminated soil [170]. Today, the largestsource of lead poisoning in children comes from dust and chipsfrom deteriorating lead paint on interior surfaces [171].Children who live in homes with deteriorating lead paint canachieve blood lead concentrations of 20µg/dL or greater [172].

Potential for Human Exposure

Exposure to lead occurs mainly via inhalation of lead-contaminated dust particles or aerosols, and ingestion oflead-contaminated food, water, and paints [173, 174]. Adultsabsorb 35 to 50% of lead through drinking water and theabsorption rate for children may be greater than 50%. Leadabsorption is influenced by factors such as age andphysiological status. In the human body, the greatestpercentage of lead is taken into the kidney, followed by theliver and the other soft tissues such as heart and brain butthe lead in the skeleton represents the major body fraction[175]. The nervous system is the most vulnerable target oflead poisoning. Headache, poor attention spam, irritability,loss of memory and dullness are the early symptoms of theeffects of lead exposure on the central nervous system (CNS).

Since the late 1970’s, lead exposure has decreasedsignificantly as a result of multiple efforts including the

20

elimination of lead in gasoline, and the reduction of leadlevels in residential paints, food and drink cans, andplumbing systems [173, 174]. Several federal programsimplemented by state and local health governments have notonly focused on banning lead in gasoline, paint and solderedcans, but have also supported screening programs for leadpoisoning in children and lead abatement in housing [167].Despite the progress in these programs, human exposure to leadremains a serious health problem [176, 177]. Lead is the mostsystemic toxicant that affects several organs in the bodyincluding the kidneys, liver, central nervous system,hematopoetic system, endocrine system, and reproductive system[173].

Lead exposure usually results from lead in deterioratinghousehold paints, lead in the work place, lead in crystals andceramic containers that leaches into water and food, lead usein hobbies, and lead use in some traditional medicines andcosmetics [167, 174]. Several studies conducted by theNational Health and Nutrition Examination surveys (NHANES)have measured blood lead levels in the U.S. populations andhave assessed the magnitude of lead exposure by age, gender,race, income and degree of urbanization [176]. Although theresults of these surveys have demonstrated a general declinein blood lead levels since the 1970s, they have also shownthat large populations of children continue to have elevatedblood lead levels (> 10g/dL). Hence, lead poisoning remainsone of the most common pediatric health problems in the UnitedStates today [167, 173, 174, 176-179]. Exposure to lead is ofspecial concern among women particularly during pregnancy.Lead absorbed by the pregnant mother is readily transferred tothe developing fetus [180]. Human evidence corroborates animalfindings [181], linking prenatal exposure to Pb with reducedbirth weight and preterm delivery [182], and with neuro-developmental abnormalities in offspring [183].

Molecular Mechanisms of Toxicity and CarcinogenicityThere are many published studies that have documented theadverse effects of lead in children and the adult population.In children, these studies have shown an association betweenblood level poisoning and diminished intelligence, lowerintelligence quotient-IQ, delayed or impaired neurobehavioraldevelopment, decreased hearing acuity, speech and language

21

handicaps, growth retardation, poor attention span, and antisocial and diligent behaviors [178, 179, 184, 185]. In theadult population, reproductive effects, such as decreasedsperm count in men and spontaneous abortions in women havebeen associated with high lead exposure [186, 187]. Acuteexposure to lead induces brain damage, kidney damage, andgastrointestinal diseases, while chronic exposure may causeadverse effects on the blood, central nervous system, bloodpressure, kidneys, and vitamin D metabolism [173, 174, 178,179, 184-187].

One of the major mechanisms by which lead exerts itstoxic effect is through biochemical processes that includelead's ability to inhibit or mimic the actions of calcium andto interact with proteins [173]. Within the skeleton, lead isincorporated into the mineral in place of calcium. Lead bindsto biological molecules and thereby interfering with theirfunction by a number of mechanisms. Lead binds to sulfhydryland amide groups of enzymes, altering their configuration anddiminishing their activities. Lead may also compete withessential metallic cations for binding sites, inhibitingenzyme activity, or altering the transport of essentialcations such as calcium [188]. Many investigators havedemonstrated that lead intoxication induces a cellular damagemediated by the formation of reactive oxygen species (ROS)[189]. In addition, Jiun and Hseien [190] demonstrated thatthe levels of malondialdehyde (MDA) in blood strongly correlatewith lead concentration in the blood of exposed workers. Otherstudies showed that the activities of antioxidant enzymes,including superoxide dismutase (SOD), and glutathioneperoxidase in erythrocytes of workers exposed to lead areremarkably higher than that in non-exposed workers [191]. Aseries of recent studies in our laboratory demonstrated thatlead-induced toxicity and apoptosis in human cancer cellsinvolved several cellular and molecular processes includinginduction of cell death and oxidative stress [29, 192],transcriptional activation of stress genes [30], DNA damage[29], externalization of phosphatidylserine and activation ofcaspase-3 [193].

A large body of research has indicated that lead acts byinterfering with calcium-dependent processes related toneuronal signaling and intracellular signal transduction. Leadperturbs intracellular calcium cycling, altering releasability

22

of organelle stores, such as endoplasmic reticulum andmitochondria [194, 195]. In some cases lead inhibits calcium-dependent events, including calcium-dependent release ofseveral neurotransmitters and receptor-coupled ionophores inglutamatergic neurons [196]. In other cases lead appears toaugment calcium-dependent events, such as protein kinase C andcalmodulin [194, 197].

Experimental studies have indicated that lead ispotentially carcinogenic, inducing renal tumors in rats andmice [198, 199], and is therefore considered by the IARC as aprobable human carcinogen [200]. Lead exposure is also knownto induce gene mutations and sister chromatid exchanges [201,202], morphological transformations in cultured rodent cells[203], and to enhance anchorage independence in diploid humanfibroblasts [204]. In vitro and in vivo studies indicated that leadcompounds cause genetic damage through various indirectmechanisms that include inhibition of DNA synthesis andrepair, oxidative damage, and interaction with DNA-bindingproteins and tumor suppressor proteins. Studies by Roy and hisgroup showed that lead acetate induced mutagenicity at a toxicdose at the E. coli gpt locus transfected to V79 cells [205]. Theyalso reported that toxic doses of lead acetate and leadnitrate induced DNA breaks at the E. coli gpt locus transfected toV79 cells [205]. Another study by Wise and his collaboratorsfound no evidence for direct genotoxic or DNA-damaging effectsof lead except for lead chromate. They pointed out that thegenotoxicity may be due to hexavalent chromate rather thanlead [206].

Mercury

Environmental Occurrence, Industrial Production and Use

Mercury is heavy metal belonging to the transition elementseries of the periodic table. It is unique in that it existsor is found in nature in three forms (elemental, inorganic,and organic), with each having its own profile of toxicity[207]. At room temperature elemental mercury exists as aliquid which has a high vapor pressure and is released intothe environment as mercury vapor. Mercury also exists as acation with oxidation states of +1 (mercurous) or +2(mercuric) [208]. Methylmercury is the most frequently

23

encountered compound of the organic form found in theenvironment, and is formed as a result of the methylation ofinorganic (mercuric) forms of mercury by microorganisms foundin soil and water [209].

Mercury is a widespread environmental toxicant andpollutant which induces severe alterations in the body tissuesand causes a wide range of adverse health effects [210]. Bothhumans and animals are exposed to various chemical forms ofmercury in the environment. These include elemental mercuryvapor (Hg0), inorganic mercurous (Hg+1), mercuric (Hg+2), andthe organic mercury compounds [211]. Because mercury isubiquitous in the environment, humans, plants and animals areall unable to avoid exposure to some form of mercury [212].

Mercury is utilized in the electrical industry (switches,thermostats, batteries), dentistry (dental amalgams), andnumerous industrial processes including the production ofcaustic soda, in nuclear reactors, as antifungal agents forwood processing, as a solvent for reactive and precious metal,and as a preservative of pharmaceutical products [213]. Theindustrial demand for mercury peaked in 1964 and began tosharply decline between 1980 and 1994 as a result of federalbans on mercury additives in paints, pesticides, and thereduction of its use in batteries [214].

Potential for Human Exposure

Humans are exposed to all forms of mercury through accidents,environmental pollution, food contamination, dental care,preventive medical practices, industrial and agriculturaloperations, and occupational operations [215]. The majorsources of chronic, low level mercury exposure are dentalamalgams and fish consumption. Mercury enters water as anatural process of off-gassing from the earth’s crust and alsothrough industrial pollution [216]. Algae and bacteriamethylate the mercury entering the waterways. Methyl mercurythen makes its way through the food chain into fish,shellfish, and eventually into humans [217].

The two most highly absorbed species are elementalmercury (Hg0) and methyl mercury (MeHg). Dental amalgamscontain over 50% elemental mercury [218]. The elemental vaporis highly lipophilic and is effectively absorbed through thelungs and tissues lining the mouth. After it enters the blood,

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it rapidly passes through cell membranes, which include boththe blood-brain barrier and the placental barrier [219]. Onceit gains entry into the cell, Hg0 is oxidized and becomeshighly reactive Hg2+. Methyl mercury derived from eating fishis readily absorbed in the gastrointestinal tract and becauseof its lipid solubility, can easily cross both the placentaland blood-brain barriers. Once mercury is absorbed it has avery low excretion rate. A major proportion of what isabsorbed accumulates in the kidneys, neurological tissue andthe liver. All forms of mercury are toxic and their effectsinclude gastrointestinal toxicity, neurotoxicity, andnephrotoxicity [213].

Molecular Mechanisms of Mercury Toxicity

The molecular mechanisms of toxicity of mercury are based onits chemical activity and biological features which suggestthat oxidative stress is involved in its toxicity [220].Through oxidative stress mercury has shown mechanisms ofsulfhydryl reactivity. Once in the cell both Hg2+ and MeHgform covalent bonds with cysteine residues of proteins anddeplete cellular antioxidants. Antioxidants enzymes serve as aline of cellular defense against mercury compounds [221]. Theinteraction of mercury compounds suggests the production ofoxidative damage through the accumulation of reactive oxygenspecies which would normally be eliminated by cellularantioxidants.

In eukaryotic organisms the primary site for theproduction of reactive oxygen species occurs in themitochondria through normal metabolism [222]. Inorganicmercury has been reported to increase the production of thesereactive oxygen species through causing defects in oxidativephosphorylation and electron transport at the ubiquinone-cytochrome b5 step [223]. Through the acceleration of the rateof electron transfer in the electron transport chain in themitochondria, mercury induces the premature shedding ofelectrons to molecular oxygen which causes an increase in thegeneration of reactive oxygen species [224].

Oxidative stress appears to also have an effect oncalcium homeostasis. The role of calcium in the activation ofproteases, endonucleases and phospholipases is wellestablished. The activation of phospholipase A2 has been shown

25

to result in an increase in reactive oxygen species throughthe increase generation of arachidonic acid. Arachidonic acidhas also been shown to be an important target of reactiveoxygen species [225]. Both organic and inorganic mercury havebeen shown to alter calcium homeostasis but through differentmechanisms. Organic mercury compounds (MeHg) are believed toincrease intracellular calcium through accelerating the influxof calcium from the extracellular medium and mobilizingintracellular stores, while inorganic mercury (Hg2+) compoundsincrease intracellular calcium stores only through the influxof calcium from the extracellular medium [226]. Mercurycompounds have also been shown to induce increased levels ofMDA in both the livers, kidneys, lungs and testes of ratstreated with HgCl2 [227]. This increase in concentration wasshown to correlate with the severity of hepatotoxicity andnephrotoxicity [228]. HgCl2-induced lipid peroxidation wasshown to be significantly reduced by antioxidant pretreatmentwith selenium. Selenium has been shown to achieve thisprotective effect through direct binding to mercury or servingas a cofactor for glutathione peroxidase and facilitating itsability to scavenge reactive oxygen species [229]. Vitamin Ewas also shown to protect against HgCl2-induced lipidperoxidation in the liver [230].

Molecular Mechanisms of Mercury Carcinogenicity

Metal-induced carcinogenicity has been a research subject ofgreat public health interest. Generally, carcinogenesis isconsidered to have three stages including initiation,promotion, and progression and metastasis. Although mutationsof DNA, which can activate oncogenesis or inhibit tumorsuppression, were traditionally thought to be crucial factorsfor the initiation of carcinogenesis, recent studies havedemonstrated that other molecular events such as transcriptionactivation, signal transduction, oncogene amplification, andrecombination, also constitute significant contributingfactors [231, 232]. Studies have shown that mercury and othertoxic metals effect cellular organelles and adversely affecttheir biologic functions [231, 233]. Accumulating evidencealso suggests that reactive oxygen species play a major rolein the mediation of metal-induced cellular responses andcarcinogenesis [234-236].

26

The connection between mercury exposure andcarcinogenesis is very controversial. While some studies haveconfirmed its genotoxic potential, others have not shown anassociation between mercury exposure and genotoxic damage[237]. In studies implicating mercury as a genotoxic agent,oxidative stress has been described has the molecularmechanism of toxicity. Hence, mercury has been shown to inducethe formation of reactive oxygen species known to cause DNAdamage in cells, a process which can lead to the initiation ofcarcinogenic processes [238, 239]. The direct action of thesefree radicals on nucleic acids may generate genetic mutations.Although mercury-containing compounds are not mutagenic inbacterial assays, inorganic mercury has been shown to inducemutational events in eukaryotic cell lines with doses as lowas 0.5 µM [240]. These free radicals may also induceconformational changes in proteins that are responsible forDNA repair, mitotic spindle, and chromosomal segregation[241]. To combat these effects, cells have antioxidantmechanisms that work to correct and avoid the formation ofreactive oxygen species (free radicals) in excess. Theseantioxidant mechanisms involve low molecular molecules such asvitamins C and E, melatonin, glutathione, superoxidedismutase, catalase, glutathione peroxidase and glutathionereductase. Cells respond to mercury exposures by increasingthe levels of these low molecular weight molecules whichprotect them through their antioxidant capacity and thechelation of mercury [242].

Glutathione levels in human populations exposed tomethylmercury intoxication by eating contaminated fish havebeen shown to be higher than normal. These studies were alsoable to confirm a direct and positive correlation betweenmercury and glutathione levels in blood. They also confirmedan increased mitotic index and polyploidal aberrationsassociated with mercury exposure [243]. Epidemiologicalstudies have demonstrated that enzymatic activity was alteredin populations exposed to mercury; producing genotoxicalterations, and suggesting that both chronic and relativelylow level mercury exposures may inhibit enzyme activity andinduce oxidative stress in the cells [244]. There is no doubtthat the connection between mercury exposure andcarcinogenesis is very controversial. However, in-vitro studiessuggest that the susceptibility to DNA damage exists as a

27

result of cellular exposure to mercury. These studies alsoindicate that metal-induced toxicity and carcinogenicity maybe cell-, organ- and/or species- specific.

Prospects

A comprehensive analysis of published data indicates thatheavy metals such as arsenic cadmium, chromium, lead, andmercury, occur naturally. However, anthropogenic activitiescontribute significantly to environmental contamination. Thesemetals are systemic toxicants known to induce adverse healtheffects in humans, including cardiovascular diseases,developmental abnormalities, neurologic and neurobehavioraldisorders, diabetes, hearing loss, hematologic and immunologicdisorders, and various types of cancer. The main pathways ofexposure include ingestion, inhalation, and dermal contact.The severity of adverse health effects is related to the typeof heavy metal and its chemical form, and is also time- anddose-dependent. Among many other factors, speciation plays akey role in metal toxicokinetics and toxicodynamics, and ishighly influenced by factors such as valence state, particlesize, solubility, biotransformation, and chemical form.Several studies have shown that toxic metals exposure causeslong term health problems in human populations. Although theacute and chronic effects are known for some metals, little isknown about the health impact of mixtures of toxic elements.Recent reports have pointed out that these toxic elements mayinterfere metabolically with nutritionally essential metalssuch as iron, calcium, copper, and zinc [245, 246]. However,the literature is scarce regarding the combined toxicity ofheavy metals. Simultaneous exposure to multiple heavy metalsmay produce a toxic effect that is either additive,antagonistic or synergistic.

A recent review of a number of individual studies thataddressed metals interactions reported that co-exposure tometal/metalloid mixtures of arsenic, lead and cadmium producedmore severe effects at both relatively high dose and low doselevels in a biomarker-specific manner [247]. These effectswere found to be mediated by dose, duration of exposure andgenetic factors. Also, human co-exposure to cadmium andinorganic arsenic resulted in a more pronounced renal damagethan exposure to each of the elements alone [248]. In many

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areas of metal pollution, chronic low dose exposure tomultiple elements is a major public health concern.Elucidating the mechanistic basis of heavy metal interactionsis essential for health risk assessment and management ofchemical mixtures. Hence, research is needed to furtherelucidate the molecular mechanisms and public health impactassociated with human exposure to mixtures of toxic metals.

Acknowledgement

This research was supported in by the National Institutes ofHealth RCMI Grant No. 2G12RR013459, and in part by theNational Oceanic and Atmospheric Administration ECSC Grant No.NA06OAR4810164 & Subcontract No. 000953.

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