International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(10), 91-101, October (2013) Int. Res. J. Biological Sci. International Science Congress Association        
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Review Paper Oxidative Stress and Heavy Metals: An Appraisal with Reference to Environmental BiologyManoj K. and Padhy P.K.Department of Environmental Studies, Institute of Science, Visva-Bharati, Santiniketan, 731235, Birbhum, West Bengal, INDIA Available online at: 
www.isca.in, www.isca.me
Received 24th June 2013, revised 1st July 2013, accepted 28th August 2013Abstract  Rapid developmental activities and other anthropogenic influences have made heavy metals (HM) ubiquitous contaminants of the environment. Investigations on pollution due to HM and their possible effects on living world have increasingly become a prime focus of the environmental biology. An increasing body of evidence shows that excessive presence of essential metals as well as minute presence of non-essential metals can cause many patho-physiological complications in the living organisms via generation of reactive species. This article discusses functional role of some HM in inducing oxidative stress along with the instruments involved viz. oxidants and antioxidative defence systems. Some recent evidences of HM induced oxidative stress in plants and animals, including human beings, have also been examined in the text.  Keywords: Ascorbate-glutathione cycle, catalase, lipid peroxidation, reactive nitrogen species, reactive oxygen species, superoxide dismutase. Introduction The advent of industrialization, urbanization, change in land use pattern, application of chemical fertilizers in agricultural fields etc. have caused massive alterations in the quality of air, water and soil. The study of environmental biology has received immense attention in recent decades because pollution emerged as a serious and challenging side effect of the increased developmental activities1-3. The release of both inorganic and organic hazardous substances is causing degradation of natural resources, making them unsuitable for use. Heavy metals (HM) form one such group of materials that are capable of causing many patho-physiological conditions in living organisms4,5. Although essential metals like iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), etc. are natural part of enzymes and other bio-molecules, and, thus, are needed for normal biochemical and physiological functioning of the body, at higher concentrations they can be detrimental. On the other hand, non-essential metals like lead (Pb), cadmium (Cd) and mercury (Hg) are toxic even at minute concentrations. These metals are widely spread in air, water and soil and find easy entrance into the living organisms. One of the most extensively investigated mechanisms via which HM can produce patho-physiological affairs is the generation of radicals, like reactive oxygen species (ROS), potent to induce oxidative stress. The accumulation of reactive species like hydroxyl radical (HO), hydrogen peroxide (H) and singlet oxygen () disturbs the oxidant-antioxidant balance bringing about a change called oxidative stress. These active species react with bio-molecules like lipids, proteins and DNA impairing their functional properties which in turn brings about alterations in the normal activities of cells, tissues, organs and ultimately organisms evident as disease symptoms, figure-1, and other pathological conditions. Involvement of ROS in metal induced cell death is widely reported. However, to counteract the menace of reactive species the antioxidant defence machinery is activated as radical scavenging system. Antioxidant defence system comprises both enzymatic biomarkers like catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD) and non-enzymatic biomarkers like -carotene, glutathione (GSH), Vitamin C (ascorbic acid), Vitamin E (-tocopherol) etc. The body tries to maintain homeostasis between the presence of oxidants and the antioxidants. Oxidative stress happens when either the amount of reactive species is enhanced significantly high or the antioxidant defence system becomes weak or both. Oxidative and antioxidative perspectivesOxidants and reactive species: Chemical reactions can be heterolytic as well as homolytic. Heterolytic reactions produce positive or negative ions as they involve transfer of electron pairs. On the other hand, homolytic reactions, which involve one electron transfer, generate radicals containing an unpaired electron. Free radicals are characterized having one or more unpaired electrons and are competent to exist independently. They are highly reactive and unstable species capable of generating new radicals via chain-reactions with consequences to cause injuries and damages to healthier cells and cellular responses6,9. Some common examples of free radicals include: alkoxyl (RO), hydroperoxyl (HOO), hydroxyl (HO), nitrogen dioxide (NO), nitric oxide (NO), superoxide (O) etc. Free radicals, primarily, can undergo four types of chemical reactions viz. hydrogen abstraction (free radical reacts with a hydrogen donor  the radical becomes stable while hydrogen donor 
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becomes a new free radical); addition (binding occurs between a free radical and a stable molecule which generates a new combined radical species); termination (reaction occurs between two radicals and a stable compound is formed); and disproportionation (reaction occurs between two identical radicals where one radical transfers its unpaired electron to the other  two different stable molecules are formed ultimately)10.     Figure-1 Outline of HM induced oxidative damage Oxidative and reductive reactions, combined as redox reactions, form the basis of the study of biological oxidative stress. Loss of electrons demonstrate oxidation, whereas, reduction is simply the gain of electrons. An oxidant is an oxidizing agent or a substance that during the reaction process gets reduced by accepting electrons and simultaneously causing other reactant to get oxidized11. In biological systems oxidants are commonly referred as pro-oxidants and as synonyms for reactive species. A pro-oxidant in general term is an oxidant of pathological significance and can be defined as a toxic substance capable of causing oxidative injury and damage to a variety of biological entities like lipids, nucleic acids and proteins resulting in alterations of their functional properties11. Reactive species comprise both free radicals (mentioned in above paragraph) as well as non-radicals. Common examples of non-radicals include hydrogen peroxide (H), peroxynitrite (ONOO), hydroperoxides (e.g. alkyl hydroperoxides, ROOH), hypochlorous acid (HOCl) etc. Though, there are many forms of reactive species, three reactive groups viz. reactive oxygen species (ROS; examples include HOO, O, H, HO), reactive nitrogen species (RNS; examples include NO, NO, ONOO) and reactive halogen species (RHS; examples include HOCl, HOBr) are the most described. A synopsis of major reactive species frequently encountered in environmental biology reports is provided in table-1.  Table-1 A list of some species involved in oxidative stressSpecies Representation 
Alkyl hydroperoxide ROOH 
Alkoxyl radical RO
•
 
Alkyl peroxynitrite ROONO 
Peroxyl radical ROO
•
 
Ferryl ion Fe
(4+)
O 
Hydrogen peroxide H
2
O
2
 
Hydroperoxyl radical HOO
•
 
Hydroxyl radical HO
•
 
Hypobromous acid HOBr 
Hypochlorous acid HOCl 
Nitric oxide radical NO
•
 
Nitrogen dioxide radical NO
2
•
 
Nitronium cation NO
2
+
 
Nitrosyl cation NO
+
 
Nitroxyl anion NO

 
Periferryl ion Fe
(5+)
O 
Peroxynitrite  ONOO

 
Peroxynitrous acid ONOOH 
Singlet oxygen 
1
O
2
 
Superoxide radical O
2
•

 
Many phenomena can explain the origin of reactive species. The electronic structure of atomic oxygen, characterized by the occurrence of two unpaired electrons in two 2p orbitals (2p and 2p) in its external shell, makes it prone to the production of radicals. The sequential and ultimate reduction of oxygen molecule to HO produces a series of ROS via stepwise addition of electrons as depicted in figure-2. Ions of metals like Fe and Cu can react with H producing extremely reactive HO• radical. Of the three major ROS species viz. O, H and HO, radical HO• is the most toxic and highly reactive. H and Oare considered as destructive ROS mainly for their involvement in HO• generation12. NO in combination with O can generate a strong oxidant ONOO, detrimental to many bio-molecules, which at acidic pH can decompose into HO, the most toxic radical. RHS like HOCl can form, figure-2, in the activated phagocytic cells like neutrophils via myeloperoxidase mediated reaction involving H and chloride ions. Formation of lipid peroxyl radicals can occur via involvement of initiator free radical like HO which abstracts hydrogen atom from lipids or polyunsaturated fatty acids. In the initiation step a lipid radical is formed which subsequently undergoes internal rearrangement and O addition in stepwise manner to generate lipid peroxyl radical13. Figure-3 illustrates the general mechanisms of formation of some radicals from unsaturated lipids.  
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Figure-2 Mechanisms of generation of some reactive species (modified from elsewhere) Oxidative stress and outcomes: Oxidative stress happens when the balance (or equilibrium) between oxidants (free radicals and non radicals both) and antioxidative machinery is disturbed causing malfunctioning of bio-molecules like proteins, lipids and nucleic acids which ultimately leads to various patho-physiological conditions and diseases to occur. In some reported literatures the term “redox stress” has been preferred over oxidative stress, since, oxidation is always accompanied by its chemical partner reduction16. As antioxidant defence machinery is a coordinated system involving many components, deficiencies in the functional properties of one component may impair the efficient working of other constituents17. Many reports suggest that ROS (particularly H) can specifically alter reactive amino acid cysteine residues within proteins, converting them from S—H (thiol) to S—OH (sulphenic) derivatives. This makes normal function of the proteins altered and corresponding damage to the signal transduction pathway. Role of RNS (for example, NO) in alteration of reactive cysteine residues has also been documented18. DNA damage because of oxidative reactions may range from injuries at abasic DNA sites, oxidation of purine and pyrimidine bases, to single and double strand breaks in DNA molecules19. HO radical is considered as the most potent agent responsible for oxidative DNA damage which can give rise to many base lesions like 8-oxo-7,8-dihydroguanine; 5,6-dihydroxy-5,6-dihydrocytosine and 5,6-dihydroxy-5,6-dihydrothymine and apart from attacking deoxyribose sugar and forming sugar radicals19,20. Loss of integrity of biological membranes can damage the structure and function of cells. Lipids are fundamental components of cellular membranes and are also targets of oxidative damage. Radicals such as hydroxyl, alkoxyl and alkylperoxyl, etc. are capable of causing lipid peroxidation (LP) and extensive damage to membrane structure21. In human beings oxidative stress phenomenon can give rise to several patho-physiological states, such as cardiovascular diseases, cancer, diabetes mellitus, neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases, atherosclerosis, inflammatory diseases, and aging etc.16,22.  
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Figure-3 Peroxidation of lipids and involvement of some HM ions (modified from elsewhere14,15) Antioxidant defence machinery: Antioxidant in its widest possible meaninghas been defined by Gutteridge, in the literature entitled ‘lipid peroxidation and antioxidants as biomarkers of tissue damage’ as “any substance that, when present at low concentrations compared with those of the oxidizable substrate, considerably delays or inhibits oxidation of the substrate”The oxidizable substrate in biological systems refers to the bio-molecules like lipids, proteins, carbohydrates and nucleic acids23. Antioxidant defence machinery can be categorized into enzymatic and non-enzymatic systems. 
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Antioxidant biological markers commonly employed to study oxidative stress are presented in table-2. Proteins, capable of binding metals, like albumin, ferritin, lactoferrin, and ceruloplasmin can sequester excess metals (like Fe and Cu) that can cause oxidative reactions when present freely in the body24. SOD is the frontline enzyme in detoxification of O radical which dismutates O into H and O. The enzyme requires specific metal(s) as cofactor for its normal function and is variously classified into Cu/Zn—SOD, Fe—SOD, Mn—SOD and most recently Ni—SOD. Another class of enzymes designated as superoxide reductase also act on O radical, but they only reduce O to hydrogen peroxide25. CAT is another class of oxidoreductase enzymes which work synergistically with SOD and converts H into HO and O. It is Fe (in heme groups) containing enzyme having four molecules of firmly bound NADPH26. GPX, a selenoenzyme and having functional similarity to CAT, also detoxifies H in the presence of GSH. In addition, selenoprotein GPX also reduces detrimental organic peroxides (hydroperoxides) using GSH and protects cellular membranes against oxidative injuries27. The basic mechanisms of working of SOD, CAT and GPX are illustrated in figure-4. Another mechanism of detoxification of H (operating in plant cells) is via the ascorbate-glutathione cycle. The cycle involves four enzymes and ascorbate as the reducing substrate for the reduction of H2 to HO. The first reaction of the cycle is catalyzed by ascorbate peroxidase (APX) where H is reduced to HO involving two molecules of ascorbate and corresponding formation of two molecules of monodehydroascorbate (MDHA). MDHA can undergoe enzymatic reduction to regenerate ascorbate as well as non-enzymatic disproportionation forming ascorbate and also dehydroascorbate (DHA). Ascorbate is again regenerated from DHA via the reaction involving DHA reductase and GSH. Glutathione disulphide (oxidized glutathione, GSSG) produced as a byproduct of this reaction is reduced back to GSH by GSH reductase28. A schematic representation of ascorbate-glutathione cycle is given in figure-4. Since, LP is an underlying reason for the patho-physiology of many diseases, scavenging of lipid peroxyl radicals is necessary to minimize oxidative damage. Vitamin E, a chain-breaking antioxidant, effectively scavenges free radical peroxyl produced during oxidation of polyunsaturated lipids and protects them from further damage14. Reduced form of Vitamin E transfers one hydrogen atom to the peroxyl radical generating lipid hydroperoxide and in the process becomes a radical itself.  Another chain breaking antioxidant Vitamin C regenerates reduced Vitamin E from its radical form. This step also generates Vitamin C radical which is converted back to the Vitamin C by dihydrolipoic acid (DHLA) forming -lipoic acid. This -lipoic acid is subsequently reduced to DHLA in the presence of NADPH. Moreover, GSH can also reduce Vitamin E radical back to its active form. The reaction generates GSSG (oxidized glutathione) which is reduced back to GSH by DHLA and becomes itself -lipoic acid. As mentioned above the -lipoic acid is subsequently converted to DHLA by NADPH29. The sequential steps showing involvement of various antioxidative biomarkers in inhibition of peroxidation of polyunsaturated lipids is shown in figure-5. Table-2 Some non-enzymatic and enzymatic biomarkers for oxidative stress study Non-enzymatic biomarkers Enzymatic biomarkers 
Albumin Ascorbate peroxidase (EC 1.11.1.11) 
Ascorbic acid Catalase (EC 1.11.1.6) 
Bilirubin Cytochrome oxidase (EC 1.9.3.1) 
-carotene Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) 
Ceruloplasmin Glutathione reductase (EC 1.8.1.7) 
Coenzyme Q Glutathione-S-transferase (EC 2.5.1.18) 
Dihydrolipoic acid Peroxidase (Guaiacol) (1.11.1.7)
Flavonoids Selenium dependent glutathione peroxidase (EC 1.11.1.9) 
Glutathione (GSH) Superoxide dismutase (EC 1.15.1.1) 
GSH/GSSG (Oxidized glutathione) 
Lactoferin 
-lipoic acid 
Lycopene 
Metallothioneins 
Transferrin 
-tocopherol 
Uric acid 
Heavy metals and oxidative stress/damageMany HM present in the environment can generate reactive species in the biological systems. Although both natural and anthropogenic activities contribute to their occurrence in the environment, the rapid and extensive developmental activities have elevated their concentrations in air, water and soil. These metals enter living organisms through inhalation, food, drinking, absorption etc. resulting in many drastic biochemical and physiological alterations in the body. A generalized scheme of release of HM in the environment and their entry into the living world (especially with reference to human beings) is illustrated in figure-6. Fe, Cu, nickel (Ni), chromium (Cr), Cd, Arsenic (As), mercury and, Pb are some of the most widely studied HM for their roles in causing oxidative stress. Redox-active metals (e.g. Cu, Cr, Fe etc.) undergo reactions of redox cycling, whereas, redox-inactive metals (e.g. Cd, Hg, Pb etc.) diminish cells’ key antioxidant defence constituents, particularly thiol-containing antioxidants and enzymes30. Redox-active metals can utilize Fenton chemistry/Haber-Weiss reaction, figure-7, to directly generate reactive species leading to oxidative damage of the structure and functions of the cellular machinery. The Haber-Weiss reaction illustrates that the interaction of less 
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reactive Oand H radicals can generate more toxic and extremely reactive HO• species.  However, to drive the reaction in biological systems a metal ion as a catalyst is needed. The most researched mechanism via which Haber-Weiss reaction produces HO• radicals utilizing a metal ion is the Fenton chemistry, which is a net combination of two reactions31 — Fe3+interacts with Oto generate Fe2+ and O; Fe2+ reacts with Hto produce HO, OH  and regenerate Fe3+ (This reaction is also called Fenton reaction). Apart from Fe2+, other metal ions like Cu2+ and Cr3+ can also produce HO• radicals via the Fenton reaction or more specifically Fenton-like reaction15. A general reaction mechanism of Fenton chemistry with examples of two metals is demonstrated below (the reaction involving Fe is displayed in figure-7). 
Figure-4 Functions of SOD, CAT, GPX and the glutathione-ascorbate cycle (lower part modified from elsewhere28) 
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Figure-5 Role of VITAMIN E in inhibition of LP (ROO = lipid peroxyl radical;modified from elsewhere29) 
Figure-6 Possible entrance routes of HM with special reference to human beings 
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HM(n)+ + O HM(n-1)+ + OHM(n-1)+ + H HM(n)+ + OH  + HOCu2+ + O Cu + OCu + H Cu2+ + OH  + HOCr3+ + O Cr2+ + OCr2+ + H Cr3+ + OH  + HODirect role of Cd in generating free radicals is absent; however, they can produce various radicals via some indirect mechanisms. One such mechanism describes Cd can displace Cu and Fe from their bound forms, like ferritin and apoferritin, and making them available to generate reactive radicals via the Fenton chemistry32. Cd is also capable of depleting Mn and Zn in SOD altering the antioxidant property of the enzyme. Ercal and co-authors30 reviewed the mechanisms of Pb induced oxidative damage through its effects on antioxidant defence constituents. Pb interferes with the disulphide bond present in the active site of the enzyme glutathione reductase (GR) and inhibits its activity. GR, an essential constituent of the antioxidant system, is responsible for catalyzing GSSG (oxidized glutathione) back to GSH (reduced form). The decrease in concentrations of GSH makes the cells more prone to oxidative injuries. Pb can also inhibit functional properties of CAT and GPX by interfering with heme synthesis and forming a complex with metal Selenium respectively. Moreover, it can also interfere with the working of SOD enzyme30. Though, at higher concentrations zinc can also be designated as a pollutant, chemically it is inert and does not engage in oxidation-reduction reactions. Presence of Zn is essential for the normal functions of many cellular constituents including antioxidant enzyme Cu/Zn—SOD. However, at elevated cellular concentrations it can be toxic causing cell death due to necrosis32. Excess Zn can exacerbate oxidative stress through enhanced production of reactive oxygen radicals33. For example, Zn2+ has been implicated in causing mitochondrial dysfunctions especially its electron transport system (ETS). The study describes Zn2+capable of binding to bc of complex III of ETS and obstructing the electron flow. The consequences of this Zn2+ induced inhibition of complex III include phenomena like decline in Oconsumption and enhanced production of ROS34. Mitochondrial origin of Zn2+ induced oxidative stress is also supported by one recent work. The study reports that intracellular Haccumulation in response to Zn2+ exposure is linked with two phenomena viz. declining mitochondrial reducing redox potential and mitochondrial membrane depolarization35.    HM induced oxidative stress study in environmental biologyThis section discusses a few studies undertaken to investigate HM exposures and oxidative stress in both plants and animals. Oxidative stress in germinating seeds due to HM exposures has been investigated36-38. In one such report, in vitro germinated seeds of Brassica juncea L. were exposed to different concentration grades of Cd (0, 50, 100 and 200 mg/l) and oxidative stress determined. GSH showed elevated content with increasing Cd concentrations which at higher concentrations accumulated significantly. Though, GST activity increased with time at 50 and 100 mg/l concentrations, significant increase in activity was noted only at the highest Cd exposure suggesting that higher concentrations are needed for enhanced induction of the enzyme. The study also demonstrated a positive correlation between accumulation of GSH and GST activity and their elevated levels explained the lower amount of LP in the metal exposed seeds. The study augmented the role of GSH and GST as non-enzymatic and enzymatic antioxidant biomarker systems respectively36. Impact assessment of trivalent (III) and hexavalent (VI) Cr species on some antioxidant enzyme activities in an aquatic bryophyte has been carried out. Fontinalis antipyretica Hedw. was exposed to different concentrations of Cr salts viz. chromium nitrate (Cr (NO), chromium chloride (CrCl) and potassium dichromate (KCr) and activities of enzymatic biomarkers namely SOD, CAT, APX, guaiacol peroxidase and GR and one non-enzymatic biomarker GSH were recorded. All studied parameters including GSSG/GSH ratio responded to Cr as its exposure disturbed the cellular redox status by generating ROS inside plant cells, substantiating that Cr could induce oxidative stress in the moss leading to impairment of cellular activities. However, nitrate salt of Cr(III) was more damaging as considerable responses were recorded even at the lowest exposure level, and was comparable to harmful effects of Cr(VI). In addition to the Ascorbate-glutathione cycle, SOD and CAT are often considered as the key metal detoxification systems in plants. The study conducted on F. antipyretica Hedw. showed increased SOD and CAT enzymes at low Cr exposure levels which decreased at high exposures. The two enzymes also displayed high correlation between them suggesting that higher Cr concentrations induced elevated generation of ROS which disturbed the cellular machinery causing cytotoxic responses. According to the authors (quoted references) two possible explanations could describe the above mentioned trend: first, disturbance of the signal transduction pathway that causes expression of genes of various antioxidative enzymes like SOD by the excess Oradical; for example, inhibition of cellular metabolism and decline of the isoforms expression of Mn—SOD; and second, excess Cr ions getting bound to the active centre of the enzymes. Similar patterns were noted for both GSH and GSSG contents and two hypotheses were suggested to explain these trends: first, increased concentrations of the two unidentified thiolic compounds (and GSH incorporation with them), possibly secondary metabolites, at high Cr exposures; and second, rise in GPX activity or over-expenditure of GSH for reduced ascorbate regeneration via glutathione-ascorbate cycle39. In other experiment same species of bryophyte was subjected to different concentrations of four HM i.e. Cd, Cu, Pb and Zn. The exposures caused enhanced LP and significant modifications in the enzymatic activities of SOD, CAT, GR, APX and guaiacol peroxidase. SOD and malondialdehyde (MDA, a product of LP) levels displayed monotonous and linear relationships suggesting their use as environmental bio-markers for bio-monitoring of pollutions involving HM40. 
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Figure-7 Haber-Weiss reaction and Fenton chemistry (modified from elsewhere31) LP and GPX activity have been suggested as suitable biomarkers with respect to multiple HM stress. A study was conducted where leaves and roots of seedlings of mangrove plant Kandelia candel and Bruguiera gymnorrhiza were exposed to different levels of multiple HM stress involving Pb, Cd and Hg. The study strengthened the view that HM toxicity to plants is via free radical generation mechanism. For example, the plants displayed production of MDA which showed enhanced free radical formation under HM induced stress conditions. Though, at medium concentrations SOD showed increased activity in both leaves and roots, the activity sharply declined at the highest concentration. This observation illustrated the phenomenon that at higher concentrations HM can impair the functioning of SOD. Furthermore, the study also indicated peroxidase as more efficient and important enzyme for scavenging of H than CAT41. Profile evaluation of oxidative insult biomarkers in the blood of directly and indirectly exposed individuals to fly ashes revealed enhanced concentrations of thiobarbitruric acid reactive substances (TBARS assay is used to quantify degree of LP) and protein carbonyls levels in both groups. Moreover, exposed groups also showed decline in blood GSH levels and noticeably increased activities of SOD and CAT. The reduction of GSH level was possibly due to its increased antioxidant activity and conjugation action with the xenobiotics present within fly ashes propelled by GST. Marked increase in SOD and CAT indicated enhanced production of Oand H on being exposed to fly ashes. The study highlighted that inhalation of particulate matter (PM) or fly ashes generated through coal combustion can produce reactive species due their high HM content capable of causing oxidative damage to both protein and lipid bio-molecules in parallel manner42. In another study impact of contaminated river water with special reference to the presence of HM on the oxidative stress of three cichlid species namely Oreochromis niloticus, Tilapia rendalliand Geophagus brasiliensis was investigated, though, other water quality parameters like nitrate, nitrite, ammonical nitrogen, silicate, conductivity etc were also taken into account. Most of the parameters including HM displayed higher concentrations in polluted site as compared to the unpolluted reference site. Species present in the polluted site showed lower haemoglobin levels, higher LP intensity and considerable differences in the activities of SOD, CAT and GPX compared to the unpolluted site. Marked increase in the activity of SOD and LP/CAT + GPX ratio were noted in response to chronic exposure to contaminants in all species exhibiting oxidative stress complications. Enhanced LP/CAT + GPX ratio confirmed ROS induced cell damage, as it suggested that higher levels of peroxide were produced in species from the polluted site and radical scavenging antioxidants like CAT and GPX were unable to neutralize the reactive species. The study implicated higher environmental presence of Zn, Mn, Fe and Cu (along with ammonia) as major culprits for inducing oxidative stress43. Bivalves are scientifically advocated as suitable bio-indicators for bio-monitoring water pollution. An assessment of HM pollution in the Saronikos Gulf of Greece was conducted through evaluation of oxidative stress in Mytilus galloprovincialis mussels. Gills and mantle of mussels from the polluted sites (Elefsis Bay present in the Saronikos Gulf) recorded two to three times concentrations of most metals involving Cd, Cr, Ni, Pb, Cu and Fe than the unpolluted reference site (Stylida in Malaikos Gulf). Moreover, Gills recorded elevated metal concentrations than the mantle. A noticeable correlation was obtained between the metal concentrations and the activities of antioxidants SOD and CAT as their activities were markedly higher in mussels collected from the Elefsis Bay than the reference site. Moreover, gills also demonstrated higher activities of the antioxidant enzymes than the mantle. However, increased antioxidative actions were unable to decrease elevated levels of LP in mussels from the pollute sites. Higher LP levels were also recorded in gills as compared to the mantle. The investigation clearly highlighted the role of metals in causing oxidative damage and also demonstrated that presence of metals at elevated concentrations overcome the actions of antioxidant enzymes like CAT44. Cu and Pb have been shown to accumulate in the mantle of the pearl oyster Pinctada fucata and altering the activities of GPX and SOD in similar way. The study also demonstrated that 
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presence of metals at lower concentrations stimulated SOD activities which gradually reduced at higher concentrations45.  The studies mentioned above clearly suggest that elevated concentrations of HM are able to generate higher levels of free radicals as well as non-radicals which antioxidant system fails to compete with resulting in pronounced oxidative damage. Conclusion The balance between oxidants and antioxidants is essential to maintain homeostasis. Environmental presence of HM and their subsequent exposure can lead to disruption of this balance leading to oxidative stress. Volumes of reported literature confirm the involvement of oxidative stress in generation of many patho-physiological conditions especially in human beings. One of the consequences of increased developmental activities is the rise in environmental presence of HM beyond their recommended or regulatory standards. Therefore, study of HM induced oxidative stress should be one of the central themes of environmental and investigational biology in order to find proper remedies to this quandary.  AcknowledgementFinancial grant provided by the Council of Scientific and Industrial Research (CSIR), India, in the form of Research Fellowship and contingency grant to the first author is deeply acknowledged. References 1.Akinyeye A.J. and Okorie T.G., Heavy Metal Studies of Industrial Effluent on Alaro Stream Sediment, Int. Res. J. Biological Sci, 1 (6), 5-9 (2012) 2.Quadros G. and Athalye R.P., Decline of Fish Diversity in the Anthropogenically Polluted Thane Creek along the Central West Coast of India, Int. Res. J. Biological Sci., 1 (4), 17-21 (2012)3.Mishra P.C., Dash A.K. and Pradhan K., Metals in Environmental segments at Hirakud of Odisha, India, ISCA J. Biological Sci., 1 (1), 7-23 (2012)4.Nwajei G.E., Obi–Iyeke G.E. and Okwagi P., Distribution of Selected Trace Metal in Fish Parts from the River Nigeria, Res. J. Recent Sci., 1 (1), 81-84 (2012)5.Nwajei G.E., Okwagi P., Nwajei R.I. and Obi-Iyeke G.E., Analytical Assessment of Trace Elements in Soils, Tomato Leaves and Fruits in the Vicinity of Paint Industry, Nigeria, Res. J. Recent Sci., 1 (4), 22-26 (2012)6.Hippeli S. and Elstner F., Transition Metal Ion-Catalyzed Oxygen Activation during Pathogenic Processes, FEBS Lett., 443, 1-7 (1999)7.Bi Y., Chen W., Zhang W., Zhou Q., Yun L. and Xing D., Production of Reactive Oxygen Species, Impairment of Photosynthetic Function and Dynamic Changes in Mitochondria are Early Events in Cadmium Induced Cell Death in Arabidopsis thaliana Biol. Cell.,101, 629–643 (2009)8.Gutteridge J.M.C., Lipid Peroxidation and Antioxidants as Biomarkers of Tissue Damage, Clin. Chem.,41 (12), 1819-1828 (1995)9.Fang Y-Z., Yang S. and Wu G., Regulation of Physiological Systems by Nutrients, Free Radicals, Antioxidants and Nutrition, Nutrition,18 (10), 872– 879 (2002)10.Wu D. and Cederbaum A.I., Alcohol, Oxidative Stress, and Free Radical Damage, Alcohol Res. Health,27 (4), 277-284 (2003)11.Prior R.L. and Cao G., In Vivo Total Antioxidant Capacity: Comparision of Different Analytical Methods, Free Radic. Biol. Med.,27 (11-12), 1173-1181 (1999)12.Karuppanapandian T., Moon J-C., Kim C., Manoharan K. and Kim W.,  Reactive Oxygen Species in Plants: Their Generation, Signal transduction, and Scavenging Mechanisms, Aust. J. Crop Sci.,5 (6), 709-725 (2011)13.Porter N.A., Caldwell S.E. and Mills K.A., Mechanisms of Free Radical Oxidation of Unsaturated Lipids, Lipids,30 (4), 277-290 (1995)14.Södergren E., Lipid Peroxidation In Vivo, Evaluation and Application of Methods for Measurement, Acta Universitatis Upsaliensis, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 949, Uppsala, ISBN 91-554-4791-0, pp. 78 (2000)15.Repetto M., Semprine J. and Boveris A., Lipid Peroxidation: Chemical Mechanism, Biological Implications and Analytical Determination, Chapter 1, Licensee InTech, 3-30 (2012)16.uraková Z., Some Current Insights into Oxidative Stress, Physiol. Res.,59, 459-469 (2010)17.Evans P. and Halliwell B., Micronutrients: Oxidant/Antioxidant Status, Br. J. Nutr.,85 (Suppl. 2), 67-74 (2001)  18.Finkel T., Oxidant Signals and Oxidative Stress, Curr. Opin. Cell Biol.,15, 247–254 (2003)19.Kryston T.B., Georgiev A.B., Pissis P and GeorgakilasA.G., Role of Oxidative Stress and DNA Damage in Human Carcinogenesis, Mutat. Res.,711, 193–201 (2011)  20.Giammarioli S., Filesi C. and Sanzini E., Oxidative Stress Markers: Specificity and Measurement Techniques, Ann. st. Super. Sanitŕ.,35 (4), 563-576 (1999)21.Schaich K.M.,Lipid Oxidation: Theoretical Aspects, Bailey’s Industrial Oil and Fat Products, Sixth Edition, Six Volume Set, Edited by Fereidoon Shahidi, Copyright John Wiley & Sons, Inc., 269-355 (2005)22.Cadenas E., Free Radicals, Oxidative Stress, and Diseases, PSC 616, http://web.mst.edu...lec01_freeradicals.pdf, 1-38, 
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