Review Article Synergistic Effects of Toxic Elements …downloads.hindawi.com/journals/bmri/2014/564136.pdfDepartment of Environmental Sciences, COMSATS Institute of Information Technology,

Review ArticleSynergistic Effects of Toxic Elements on Heat Shock Proteins

Khalid Mahmood1 Saima Jadoon2 Qaisar Mahmood3

Muhammad Irshad3 and Jamshaid Hussain3

1 Department of Biology Government Post-Graduate College Asghar Mall Rawalpindi Pakistan2Department of Natural Resource Engineering and Management University of Kurdistan-Hawler Erbil Kurdistan Region Iraq3 Department of Environmental Sciences COMSATS Institute of Information Technology Abbottabad 22060 Pakistan

Correspondence should be addressed to Qaisar Mahmood mahmoodzjugmailcom

Received 23 November 2013 Revised 24 May 2014 Accepted 17 June 2014 Published 20 July 2014

Academic Editor Abhik Gupta

Copyright copy 2014 Khalid Mahmood et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Heat shock proteins show remarkable variations in their expression levels under a variety of toxic conditions A research spanexpanded over five decades has revealed their molecular characterization gene regulation expression patterns vast similarityin diverse groups and broad range of functional capabilities Their functions include protection and tolerance against cytotoxicconditions through theirmolecular chaperoning activitymaintaining cytoskeleton stability and assisting in cell signalingHowevertheir role as biomarkers formonitoring the environmental risk assessment is controversial due to a number of conflicting validatingand nonvalidating reports The current knowledge regarding the interpretation of HSPs expression levels has been discussed in thepresent review The candidature of heat shock proteins as biomarkers of toxicity is thus far unreliable due to synergistic effects oftoxicants and other environmental factorsThe adoption of heat shock proteins as ldquosuit of biomarkers in a set of organismsrdquo requiresfurther investigation

1 Introduction

Human population explosion has led to an era of rapidand heavy industrialization which threatens life in almostall possible habitats The environment is being increasinglypolluted with the addition of a large number of heavy metalschemicals and radiation which are dangerous not only forhumanbut also for other living beings In these circumstancesbiologists are playing a pivotal role in creating awarenessregarding the effects of hazardous materials along otherissues towards the restoration and conservation of a healthyenvironment Therefore one key area of focus is the use ofbiomarkers as indicators of biochemical change providingan early warning of environmental risk and its assessmentAs such biochemical markers often parallel changes in thephysiochemical characteristics of the environment the useof biomarkers as a detective measure can enable adoptingsome timely preventivemeasures to avoid certain hazards Toachieve these objectives certain investigations have suggested

the linking of stress genes such as HSP70 and metalloth-ionein with a reporter gene for farmed vegetation that couldbe employed to enable the use of satellite images to gaugecrop status for environmental health Furthermore these bio-markers may be utilized as tools to gain better understandingof the links existing among the environmental quality foodchains and human health [1]

Until present various biomarkers proposed for environ-mental risk assessment have been presented in Table 1

Thebiomarkers presented inTable 1 have been consideredas important tools for the identification and quantificationof exposure effects or susceptibility in individuals of apopulation under adverse conditions Therefore biomarkerselection should be primarily based on the ability of abiomarker being sufficiently sensitive to provide an accuratemeasurement in a sample

Heat shock proteins (HSPs) are specifically producedwhen cells are exposed for a while to temperatures higherthan their normal growth requirementThe synthesis of HSPs

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 564136 17 pageshttpdxdoiorg1011552014564136

2 BioMed Research International

Table 1 Biomarkers proposed for environmental risk assessment

Name of biomarker ReferenceEsterases [130]Polytene chromosomes of chironomids (Diptera) [131]HSP60 HSP70 alpha B-crystallin homologue lipidperoxide total glutathione level ubiquitinmitochondrial manganese superoxide dismutasemetallothionein and cytochrome P450 2E homologue

[93 122123]

Numbers of macrophages in liver tissue changes invarious blood parameters [105]

Histological and ultrastructural markers lysosomalmembrane stability of coelomocytes histidine [51 132]

Catalase activity [78]Metabonomics analysis using NMR techniques [133]P-glycoprotein major vault protein topoisomerase-II [103]Acetylcholinesterase inhibition and imposex [134]Digestive enzymes glycolytic enzymes and cellularenergy allocation [124]

Apoptosis in marine sponges and spiders [135 136]

is a universal phenomenon in all studied plant and animalspecies including humans Because HSPs can also be inducedby oxidants toxins heavy metals free radicals viruses andother stressors they are sometimes called the ldquostress proteinsrdquo[2] Heat shock proteins were initially discovered in 1962 inDrosophila melanogaster larvae in response to heat shock [3]and the term ldquoheat shock proteinrdquo was coined by Tissieres etal [4] Heat shock proteins show characteristically markedvariations in their expression in most of organisms under arange of temperatures [5 6] heavy metals [7ndash10] chemicals[11ndash14] radiations [15ndash19] metabolites and hormones [20ndash24] clinical situations [16 25ndash30] or pathogens that may beabove or beyond their optimal limits [31ndash34] and drugs [35ndash37]

Heat shock proteins are cosmopolitan in all living organ-isms and are usually classified as different families accordingto their molecular sizemdashHSP27 HSP47 HSP60 HSP70HSP90 and HSP110 These families of heat shock proteinsplay crucial roles in physiological processes such as proteinchaperoning activity protection against apoptosis steroido-genesis and stress tolerance In addition heat shock proteinsespecially HSP70 and HSP60 have also been proposed asbiomarkers of exposure levels and toxicity Their candidatureas biomarkers is based on many observations showing thatthe HSP60 induction in certain organisms (eg musselMytilus edulis and nematode Plectus acuminatus) results ina several fold sensitive response than the use of other com-parable parameters such as quantifying adverse effects onbiomass or reproduction [38 39] Such an observation inmussels (M edulis) led to their recommendation as one ofthemost suitable organisms for biomonitoring of aquatic eco-systems

The role ofHSP70HSP60 as a biomarker is highly topicalCertain studies have produced contradictory results someindicate a high HSP70 sensitivity to pollutants while otherssuggest otherwise The confounding issue in many such

studies seems to be the use of varying concentrations oftoxicity exposure some authors have realistically used lowconcentrations somehave used high concentrations but onlyvery few tested real-world contamination exposures modelsFurthermore the existing validated bioassays mostly basedon lethality or reproduction have somewhat limited appli-cability due to their crude sensitivity long exposure or theoverall expenses of the test In contrast changes at bio-chemical level are usually the first detectable response toenvironmental disturbance Therefore analysis of toxicity-induced changes in gene expression (ie alterations in pat-terns of protein synthesis) and resultant cell injury may bequite handy factors to be considered as biomarkers of toxicityexposure As these changes underline all effects at higherorganizational level therefore these have been regarded ashighly sensitive indicators of toxicity

2 Favoring Evidences HSPs as a Biomarker

21 Aquatic Environment The first favorable report on theuse of HSPs as a toxicity biomarker came from the study ofCochrane et al [40] It was reported that exposure of rotiferBrachionus plicatilis to sublethal doses of CuSO

4resulted in

a 4-5 fold increase in HSP58 with the maximum increaseoccurring at approximately 5 of the LC50 for the species Asimilar response was seen with tributyltin Kinetics of induc-tionwas sigmoidwith induction occurring in the range of 20ndash30 120583gL However no response was observed when exposedtoAlHg Zn sodiumarsenite sodiumazide sodiumdodecylsulfate or pentachlorophenol It was suggested that HSP58abundance might act as a biomarker of toxicity exposure

A number of studies conducted on mollusks mostlyon mussels for example Sanders and Martin [41] reportedelevated levels of HSP60 and HSP70 in mussels and fishtissue collected from polluted areas The collection of dataon sediment and water chemistry from the sampling sitesand on contaminant body burdens indicated their exposureto contaminants was long-term This study suggested thatHSP accumulation might provide a method of quantifyingadverse biological impacts of exposure to toxicants whenexamining wild populations from contaminated sites Simi-larly another study conducted by Porte et al [42] illustratedstatistically insignificant differences in the total levels ofcytochrome P450 and benzo(a) pyrene hydroxylase activityand significantly induced HSP70 which correlated with thequantities of the PAHs accumulated in mussels Mytilusgalloprovincialis (collected from sites polluted with aliphaticand polycyclic aromatic hydrocarbonsmdashPAH) Again it wassuggested that HSP70 could be used as a biomarker inMytilus galloprovincialis against PAH toxicity Another studyin mollusks considered HSP70 as biomarkers (in gill mantleand digestive gland) and the reason of survival of Macomanasuta (clam) in response to heavy metals (Ni Cr and Cu)and trace organic pollutants (like PAH and organochlorinepesticidesmdashaldrin and DDT and its metabolites DDD andDDE) exposure Pearson and Spearman correlation analysisrevealed that mortality and HSP70 in gills were significantlycorrelated with tissue concentrations of DDT andor itsmetabolites [43]

BioMed Research International 3

Schroder et al [44] validated hepatic HSP70 as a poten-tial biomarker of cellular stress responses in fish Limandalimanda at spawning stage 2 Varying levels of HSP70 (con-sisting of two forms 75 and 73 kDa) among individuals wereobserved at different locations of North Sea (Germany) buteach was correlated with intensity of DNA damage (single-strand breaks and alkaline labile sites) It was suggested that Llimandamight serve as a useful bioindicator and heat-shockproteins as a useful biomarker for the monitoring of environ-mental pollution The fish epidermis is highly susceptible asit is the interface between the fish and its surrounding aquaticenvironment Heresztyn and Nicholson [45] investigated theuse of HSP70 as ameasure of sublethal ecotoxicity in culturedskin epidermal cells of rainbow trout Oncorhynchus mykissexposed to chemical stress (24-dichloroaniline) A positivetoxicant concentration-dependent increase was noted in theconcentrations of HSP70 (quantified by immunocytochem-istry) In addition to skin epidermal cells the erythrocytes ofcertain fishes (eg silver sea bream Sparus sarba) have theability to synthesize HSPs due to presence of a nucleus andtherefore are considered as an interesting cellularmodel for invitro toxicological studies In one such study their exposureto sublethal concentrations of Cd Pb or Cr-VI as low as01 120583M(a value which represented threshold concentration invitro) for 1 to 2 hours has shown significant overexpressionof HSP70 [46] Guizani et al [47] also provided favoringevidence in support of HSP70 to act as biomarkers ofenvironmental stress

In addition to rotifers mollusks and fishes some authorsexplored the potential of HSP70 as a biomarker of stressin algae and other plants For example Bierkens et al [48]suggested that HSP70 in alga R subcapitata is a sensitivebiomarker as it displayed a dose-dependent increase inresponse to a wide range of pollutants (ZnCl

2 SeO2 lindane

carbaryl and SDSmdashbut not pentachlorophenol) at concen-trations below the range of classical cytotoxicity (ie growthinhibition lethality) However ZnCl

2and SeO

2were found

to be the strongest inducers of HSP70 In another studyIreland et al [49] reported elevated concentrations of HSP70in toothed wrack Fucus serratus and common duckweedLemna minor exposed for 24 hours to osmotic and cadmiumstresses In both stresses the production of HSP70 increasedto the maximum and subsequently decreased as the stressorlevels increased They suggested that HSP70 tested by anindirect competitive enzyme-linked immunosorbent assaycould potentially be applied for the stress detection in theseaquatic species In addition toHSP70 the biomarker responseof MitosHSP to heat ChlsHSP to H

2O2 and antioxidant

enzymes (Mn-SOD and Fe-SOD) and HSP60 to heat H2O2

and Pb in the dinoflagellate Karenia brevis has also beenreported by Miller-Morey and Van Dolah [50]

22 Terrestrial Environment Various studies have also sup-ported the use of HSPs as biomarkers in monitoring of soilpollution In this context most attention has been paid tosoil invertebrates especially Plectus acuminatus (nematodes)Lumbricus terrestris (annelids) and gastropods (mollusks)with the emphasis on HSP70 andor HSP60 as a biomarkerof toxicity For example Kammenga et al [39] reported the

induction of HSP60 related to increased concentrations ofCu and Cd (4ndash400 120583gL and 7ndash700 120583gL resp) For copperthe induction of HSP60 was three orders of magnitude moresensitive than was the EC

20for reproduction For cadmium

HSP60 induction was one order ofmagnitudemore sensitiveTheir results pointed out that HSP60 induction occurred atconcentrations that were realistic for the field situation (2to 48 120583g of Cu for loamy sand and clayey soil) ThereforeHSP60 was suggested to be suitable as a potential biomarkerto toxicant stress in P acuminatus About two years later theypresented a review on the potential and limitations of inver-tebrate biomarkers (including HSPs metallothioneins andmetal-binding proteins esterases histological and ultrastruc-tural markers lysosomal integrity and the novel biomarkerhistidine) for soil risk assessment purposes They concludedthat the HSP response in soil invertebrates was especiallysuitable to indicate the effects of exposure to comparativelylow concentrations for a range of toxicants and could beregarded as a biomarker of general stress [51] However inanother study they suggested that HSP60 response in thenematode alone was not a suitable biomarker for heavilycontaminated soils It had indicative value related to theHSP70 response in the isopods (Oniscus asellus and Porcellioscaber) and could be a suitable biomarker for moderatelycontaminated soils Furthermore HSP70 concentrations innontolerant individuals of both these isopods species wereconsidered to be suitable for use as potential biomarkers formonitoring environmental pollution [52]

Nadeau et al [53] reported that HSP70i analysis bywestern blot in intestinal tissues of L terrestris was a suitableand sensitive bioassay for the assessment of adverse effectsin earthworms when exposed to chemicals and heavy metals(chloroacetamide pentachlorophenol Pb Cd Cu and Hgfor 1ndash16 days) Their data also showed a good level ofreproducibility despite some individual variations In addi-tion they suggested that the use of animals from pristinehabitats transposed into contaminated environments is ofhigh ecological relevance Induction ofHSP70 in earthwormsshould represent not only a good wide-spectrum biomarkerof exposure but also a biomarker of toxicity effects sinceknown toxicants altered gene expression in these animalsData gathered therefore is not simply a measure of accu-mulation of HSP The detection of HSP70 in earthwormscould represent an early-warning system for the presenceof potentially deleterious agents in soils particularly in Lterrestris and earthworms in general acting as potentialsentinel animal species In other studies HSP70 has alsobeen suggested as a sensitive biomarker in coelomocytes ofthe earthworm Eisenia fetida exposed to metals like Zn CuPb and Cd (at concentration of 132 120583gcm2 of filter paper)[54] and in McCoy cells exposed to low Hg Cd and CuCl

2

concentrations (07 1 and 3 120583M resp) [55]

23 HSP in Transgenic Systems Some authors have empha-sized the use of HSPs in transgenic cells and organisms forenvironmental risk assessment Examples include transfectedHeLa cells with firefly luciferase having the HSP22 promotersequence of Drosophila melanogaster for CdCl

2 Cd (NO

3)2

NaAsO2 alachlor fentin acetate thiram and maneb in


the concentration range of 005ndash50 120583M [56] Other exam-ples include the use of transgenic Drosophila melanogaster(HSP70-lacZ) for the phthalimide group of chemicals captancaptafol and folpet [57] for cypermethrin (0002 02 05and 500 ppm) and for effluents of chrome plating industrycontainingCr Zn FeNiMn Pb andCu [58 59] Embryos ofa stable transgenic zebrafish with HSP70eGFP reporter genesystem in which HSP70 expression was activated in a tissue-specific manner following exposure to a number of differenttoxins including Cd (data not given by author) served as areliable and extremely quick indicator of cell-specific toxicity[60] In transgenic zebrafish reporter gene under humanHSP70 promoter showed sensitivity to detect CuSO

4at doses

as 12m120583M [61]Thus HSP70 has often been highlighted as a particularly

sensitive biomarker of exposure to different pollutants inearthworms mussels clamsDrosophila certain fishes algaeand some aquatic plants Quite a few studies have suggestedit as a biomarker of other adverse effects Widely acceptedmodels such as Drosophila made transgenic for differentstress genes namelyHSP70HSP83 andHSP26 tagged withreporter genes like 120573-galactosidase or GFP have been used todetect cellular stress caused by environmental chemicals ortheir mixtures [62] The designed assays allowed the quan-tification of stress gene expression following chemical pollu-tion exposure suggesting the magnitude of cellular toxicityinflicted by the chemicals [63ndash68] Nisamedtinov et al [69]studied the response of the yeast Saccharomyces cerevisiae todifferent stress conditions employing transgenic technologyThe group used HSP12p-Gfp2p fusion protein construct anddemonstrated that the abundance of HSP12p under differentenvironmental conditions depended on the specific stressfactor A rapid shift in stress factors gave higher rates ofHSP12p synthesis compared to gradually changing stressconditions Similarly for developmental toxicity studieszebrafish transgenic for HSP70 or HSP27 tagged with a GFPreporter were exposed to heavy metals to examine the effectof the xenobiotics on different stages of development [70]

3 Contradictory Evidences

A number of researchers have criticized the use of HSP70or HSP60 as biomarkers Wieganta et al [71] for examplereported that different stressors (arsenite cadmium dinitro-phenol and ethanol) recognized as well-known HSP-inducers failed to stimulate specific HSPs in rat hepatomacells to a degree that is comparable to the induction of theseHSPs by thermal shockTherefore validation of stressor-spe-cific risk assessment was considered through further researchwith larger groups of proteins Mirkes et al [72] reportedthat the heat shock response characterized by the synthesisand accumulation of HSP72 was not a general biomarkerin rat embryos for chemical teratogens such as N-acetoxy-2-acetylaminofluorene CdCl

2 cyclophosphamide sodium

arsenite (AS) and sodium salicylate (SAL) Last two chem-icals induced the synthesis and accumulation of HSP72 andboth have different accumulation kinetics otherwise thesechemicals caused embryotoxicity characterized by abnormaldevelopment and growth retardation Overexpression of

HSP72 after short-term exposure (2ndash6 hr) of pulmonary cellline (A549) to acute Cd concentrations (higher than 50 120583M)was considered an early biomarker for occupational exposureto Cd but long-term (1month) chronic exposure in vivomadeit doubtful because the expression of HSP72 decreased dueto cellular adaptation to chronic Cd exposure [73] Similarlyin juvenile rainbow trout exposed to Cd (15120583gL) and Zn(150 120583gL) for 21 days an adaptive response to a lesserextent in the liver was shown by an increase in antioxidantdefenses (total glutathione superoxide dismutase and Troloxequivalent antioxidant capacity) without any impairmentof GSH redox status or induction of HSP70 and HSP60[74]

Efremova et al [75] reported that Pb and Zn causeda strong induction of HSP organochlorines and pen-tachlorophenol also caused induction but did not enhanceconsistently Wastewater from the Pulp and Paper Plantcaused a concentration-dependent increase inHSP70 expres-sion in freshwater sponges endemic in Lake Baikal Howeverthere was no difference in the basal concentrations of HSP70between sponges collected from polluted (with wastewater ofPulp and Paper Plant) and unpolluted sites Another study onthe sponge Crambe crambe reported that the accumulationand response of HSP54 were higher and quicker than HSP72for Cu exposure However HSP72 was significantly inducedonly in the individuals transplanted to the contaminated siteUnder experimental conditions both heat shock proteinswere induced by copper at 30 120583gL and inhibited at 100 120583gLThe highest mean values of HSP54 and HSP72 correspondedto the sponges that showed the lowest mean values of toxicityThus toxicity and production of HSP displayed antagonistictrends [76] In ascidian Pseudodistoma crucigaster HSPswere induced only where Cu concentrations were underhalf of the actual concentration in their polluted harborThe author suggested that HSP was useful only as an earlywarning system for sublethal Cu pollution in the ascidianas the response was inhibited above a threshold value ofthe stressing agent which was variable among species [77]Furthermore the effect of Cu (50 100 and 960120583gL for 2ndash24 h at 19∘C) on the levels of HSP60 analyzed by westernblotting analysis of the planarians Dugesia schubarti revealedno changes in HSP60 expression However catalase activitywas significantly affected Therefore it was concluded thatHSP60 should not be used as biomarker for Cu pollution inplanarians [78]

Insect Tetrix tenuicornis was collected from polluted andunpolluted sites for studying heavy metals accumulation andits impact on stress proteins level Insects collected from apolluted area had higher concentrations (ranging from 15- to42-fold) of Cu Zn Pb andCd than control insectsMoreoverheavy metals accumulations caused only minor variations inthe accumulation of Hsc70 and HSP70i [9] Furthermore aninhibition of HSP70 synthesis has also been observed in theearthworm Lumbricus terrestris in response to a variety ofmetals such as Pb Cd and Cu [53] While evaluating thepower ofHSP70 as an environmental biomarker of fish healthduring field conditionsWebb and Gagnon [79] reported thatHSP70 measurement alone was insufficient to evaluate fishhealth conditions


4 Discussion Critical Analysis

A general conclusion regarding the validity of HSP70 andorHSP60 as biomarkers of toxicity exposure or effect is difficultto reach in part due to the conflicting nature of many ofthe above research reports However certain research reportsenable us to draw conclusions regarding the strength of spe-cific biomarker candidates In analyzing the research to datewe have encountered 9 basic concerns regarding the limi-tations of the use of HSP as biomarkers These are explainedin detail in the following discussion The following concernsshould be addressed by the researchers in order to reach someconcrete conclusions

(1) Can these HSPs as biomarkers reveal both the con-centration and nature of a specific toxicant in anenvironment

(2) Are uptake threshold concentration and inducingability of all metalstoxicants the same

(3) Do variability of HSP70 concentrations among vari-ous organisms and the acquisition of tolerance signif-icantly affect the results

(4) Do age and gender based differences have no effect onexpression of HSP70

(5) Are physical properties of soil and variable detox-ification mechanisms among species not acting asconfounding factors

(6) Does a synergistic effect of different toxicants alongenvironmental factors modify the expression of heatshock proteins

(7) Do different techniques for HSP detection producesimilar results for a particular toxicant in the sameorganism

(8) Are the studies validating HSP as biomarkers broadenough

(9) Is application of biomarker sets a useful strategy forassessment of toxicity

41 Concentration and Nature of a Specific Toxicant TheseHSPs did not reveal the concentration or the nature of aspecific stressor that is type of heavy metal or chemical asthese showed a marked variation in their concentrations inresponse to a variety of stressed conditions Moreover theseconcentrations did not give a true picture of different toxi-cants in an environment that is how many toxicants werepresent in a specific locality or habitat under investigation

42 UptakeThreshold Concentration and Induction Thresh-old metal concentrations for the HSP70 induction varyamong differentmetals For example in zebramussels (Dreis-sena polymorpha) metal uptake analysis for Pt Pd Rh Cdand Pb indicated the highest uptake for Cd followed by Pt PbPd and Rh The highest HSP70 values were observed in thecase of exposure to Pd followed by Pt Rh Pb and CdThere-fore Pd seems to be a particularly potent inducer of HSP70despite its relatively low threshold concentration [8] There-fore simple conclusions on the basis of body burdens should

be avoided and results should be interpreted on the basisof strength of toxicant Furthermore the potency of varioustoxicants significantly differs in inducing HSP70 promoterFor example metals like Cd Zn and Hg and organic com-pounds like chlorophenol derivatives 3 4-dichloroanilineethyl parathion benzo(a)pyrene 2 4-dichlorophenoxyaceticacid endosulfan diuron and 4-nonylphenol are effective atnoncytotoxic doses while tetrachlorohydroquinone and 1-chloro-24-dinitrobenzene induce the promoter at cytotoxicdoses [13] Occasionally one metal enhances the accumu-lation of others This is the case with a combination of Cuand Zn which resulted in higher accumulation of Zn inHepG2 cells [80] Occasions were also noted where HSP70concentrations did not correlate with the degree of metalexposure (as in crabs Carcinus maenas for Cu and Zn) [81]Furthermore a deficiency of certain metals for example Cureduced the expression of HSP70 in certain tissues (cardiac)and their mitochondria [82] and the exact effect of suchdietary deficiency on other organscases is still in question

43 Interindividual Variability of HSP70 Concentrations andAcquisition of Tolerance Individual differences exist evenat molecular levels a factor for instance giving rise toindividual variability inHSP70 expression in intestinal tissuesof L terrestris [82] Some authors have reported a weak cor-relation between toxicant and HSP70 as biomarkers due tointerindividual variability of HSP70 concentrationsThis wasthe case in the bay musselMytilus trossulus where the inter-individual variability tended to mask inductions of HSP70at low concentrations of As(III) thus making it a less effi-cient biomarker of toxicity To avoid such problems pre- orpoststress specimens are required to be analyzed to allow forgreater HSP70 sensitivity and reliability Other markers suchas the use of actin are required to be used as controls the useofHSP70-reporter gene constructs is suggested and detectionwith HSP60 heme oxygenase-1 metallothionein CYP450MXR or GPx has been recommended by La Porte [83]

Another difficulty in validating HSPs as biomarker forERA is the enhancement of stress response (in form ofincreased synthesis of HSP70HSP60) in self-tolerance aswell as in cross-tolerance by nonlethal doses of toxicant insensitized cellstissues (the cells or tissues already exposed tosuch toxicants in self-tolerance or to other toxicants in cross-tolerance) An example is amphetamine and Cd in hepaticcells [84 85] Hg [86] and uranium [87ndash89] in the kidneyare able to induce self-tolerance while Zn against Cd in ratproximal tubule cells [90] Pb against CdC

2H2in astroglial

cells [91] As Cd Hg Pb Cu menadione and diethyldi-thiocarbamate against lethal temperature in Reuber H35hepatoma cells [92] were also able to induce cross-toleranceldquoHow many timesrdquo and ldquoTo what extent has an organismbeen exposedrdquo are questions to be addressed under the natu-ral environmentsTherefore acquired resistance against cyto-toxicity as well as cross-tolerance in a large number of org-anisms also adds uncertainty in the role of HSP as biomarkerin ERA Likewise biomarker responses vary in relation to theduration and level of exposure under laboratory conditionsand are also dependent on the population [93 94] Acquisi-tion of tolerance by certain organisms not onlymasks the true


picture but also seems as a dubious factor when applying theissue to the establishment of water quality criteria [95]

44 Age and Gender Based Differences Certain studies haverevealed that not merely the functional ability of HSP70against stress conditions that decreases with aging [96] butalso aging is associated with an actual decreased concentra-tion of HSP70 and subsequently a decreased ability to res-pond to stressed conditions [97] Certain studies have alsorevealed gender based differences regarding expression ofHSPs For example HSP70 gene assay in the reproductiveorgans of adult flies showed its expression restricted to maleflies [59] Considering these studies such gender and agebased differences are also expected in other organisms withrespect to HSP70 in response to toxicants The age and sexof an organism should also be considered especially whenstudying the role of HSPs as a risk assessment of environ-mental pollutionrsquos effects in sex organs

45 Confounding Factors In case of soil pollution Filzeket al [98] emphasized the consideration of the underlyinggeology the nature of the soil and the land use as essentialprerequisites to understand the significance of any observedbiological effectsThe authors also provided extensive discus-sion on how the availability and mobility of various heavymetals at the selected field sites were influenced by a widerange of factors such as pH organic matter and clay contentThe significant differences in toxic responses measured inthe laboratory exposed versus field exposed nematodes byArts et al [52] were explained due to confounding factorssuch as food availability and differences in contaminantuptake routes under the different exposure regimes It wasalso suggested that differences existed between native ani-mals transplanted to the field and field collected animalspartly attributable to the increased and probably inheritedtolerance of the field population Furthermore physiologicaldifferences in the way an individual species handles theuptake detoxification assimilation and eventual excretionof accumulated metal also influenced the HSP70 responsein isopods Such physiological differences exist not only interrestrial organisms but also in aquatic [99]

46 Synergistic Effects among Toxicants as well as betweenToxicant and Environmental Factors No data exist regardingthe synergistic effects of different toxicants andor with theother aspects of environmental stresses (temperature pHsalinity etc) For instance the toxic effect is significantlyaltered under the additive effects of many heavy metals ascompared to cases of isolated single metal toxicity where twoor more metals are found in a combination Individual doseof 20120583M of As Cd and Hg induced only a modest HSP70increase whereas their combination at the lowest levels oftoxicity still induced a greater accumulation of these proteins(Figure 1) [100] Furthermore a number of stress genes whichrespond to heavy metals (such as HSPs and MT) containmetal-response elements (MREs) in their promoterenhancerregion which is activated by ametal-responsive transcriptionfactor-1 (MTF-1) The response to heat shock is mediatedby heat shock transcription factor-1 (HSF-1) which activates

a battery of heat shock genes Synergistic activation hasalso demonstrated the metal-responsive promoters by heavymetals (Zn or Cd) and heat shock in combination Heat alsostimulates the intracellular accumulation of Zn and Cd whenprovided exogenously during a heat shock (in HEK293-mammalian cells) and thus results in a hyperactivationof the metal response pathway Interestingly relatively lowconcentrations of these heavy metals alone hardly inducedtranscription at all and served as sufficient trigger for suchsynergistic activation of mammalian HSP70 promoter (Fig-ures 2(a) and 2(b)) [101] Similarly water-soluble fractions ofdifferent sludge containing varying concentrations of heavymetals (Cd Cr Cu Ni Pb and Zn) when given separatelyto human cultured cells (HT29 cell line from gut mucosa)failed to trigger significant expression of HSP72 When givenin combination they exerted a strong synergistic effect bycausing significant overexpression of HSP72 (Figure 3) [102]Increasing concentrations of HSP70 have also been observedin HepG2 cells under the synergistic effects of Cu and Znas compared with each metal (Figure 4) [80] Another studyconducted by Aıt-Aıssa et al [74] reported that 331015840441015840-tetrachlorobiphenyl (1mgkg) strongly inducedHSP70 whileits coexposure with metals did not modulate significantlyits effects However 17-beta-oestradiol in combination withCdZn had shown a synergistic effect

Apart from the synergistic effects of toxicants environ-mental factors such as temperature (Figure 5) [103] salinity[104] and oxygen supply [105] also influence the expressionof HSPs andmay also have a synergistic effect in combinationwith toxicants thus making their consideration as biomark-ers doubtful For example Cd exposure increased the HSP70concentrations in marine clams whereas salinity markedlylowered the same level in that species A laboratory studyregarding the effects of salinity on HSP70 concentrationsindicated that exposure to 01 ppt salinity markedly loweredHSP70 concentrations in clams Potamocorbula amurensiscompared with those exposed to higher salinities (Figure 6and Table 2) [106] Increasing the salinity from 5 to 25 perthousand resulted in lowering the toxicity and concentrationsof the freemetal ions (Figure 7)This effect has been regardedas the strongest for Cd and Pb while such smaller effectswere observed for Ni Cu and Zn [104] The rate of uraniumaccumulation in the gill tissues of clam Corbicula flumineawas higher under hypoxia than normoxia At the cellularlevel uranium instead of hypoxia induced the expression ofmultixenobiotic resistance protein On the contrary HSP60was induced by hypoxia instead of uranium [107]

Some authors have also regarded HSP70 as a biomarkerin the algae Raphidocelis subcapitata in response to changesin pH temperature humic acids nitrates and phosphatesAlgae responded to these changes by a transient increasein HSP70 concentration Temperature and pH were foundto induce acquired tolerance that is algae grown at a pHor at a temperature different from control conditions wereshown to have acquired resistance to a subsequent challengewith Zn (105M)These results qualify HSP70 as a biomonitorfor environmental pollution provided that essential environ-mental parameters such as pH and temperature are keptconstant [108] Apart from temperature andpHmuch similar


10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00Cd Hg As

(a)

10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00

Aslowastlowast

Aslowast

Cd + Hg Cd + As Hg + As Cd + Hg + As

(b)

Figure 1 Toxic metals induce HSP70i accumulation in podocytes in a dose-dependent manner Results of a quantitative western blot analysisof HSP70i accumulation in podocytes treated with various concentrations of individual toxic metals (a) or combinations of two and threetoxic metals totaling 10 20 or 40mM (b) for 3 days Values are expressed as ng of HSP70i per mg total protein Basal HSP70i levels werebelow the limits of detection [100]

0

100

200

300

400

500

120583g

g pr

otei

n

ZnCd

Non

treat

ed

Zn (1

00120583

M1

h)

Cd

(60120583

M1

h)

Hea

t (42

C1h

)

Hea

t+Zn

Hea

t+C

d

(a)

Fold

activ

atio

n

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12

Zinc (100120583M)Cadmium (60120583M)

Heat shock (43C 1h)mHSF-1 (2120583g)

+ +

+ + +

++++++

+++

+ + + +

+ +minus

minus

minus minus minus minus minus minus minus minus

minus minus

minus minus minus minus minus

minus minus minus

minus

minus minus minus minus minus minus minus

(b)

Figure 2 (a) Cellular accumulation of Zn and Cd is boosted by heat shock After addition of Zn and Cd to final concentration of 100and 60 uM respectively with or without heat shock (42∘C for 1 h) HEK293 cells were harvested and analyzed by ICP-MS The data fromthree independent determinations has been shown [101] (b) Expression of HSP70 promoter by Cd and heat in presence or absence ofHSF-1 HEK293 cells were transfected with HSP70-Luc promoter-reporter construct the CMV-LacZ reference construct and mouse HSF-1expression vector 36 h after transfection cells were treated with 100120583M ZnCl

2or 60120583M CdCl

2with or without heat shock at 43∘C for 1 h

The cells were collected and reporter gene activities were determined by luciferase assay The basal level was taken as 1 to calculate the foldactivation [101]


Table 2 HSP70 levels in clams during adaptation to various salinitieslowast

Salinity (ppt) HSP70 (relative density times103)After 24 h After 24 h After 24 h

01 1791 1741 14593 1772 2392 38086 2734 3925 358210 (ambient) 3105 2620 178014 1956 3610 32627 3753 3108 3556lowastClams collected from Martinez marina (salinity 10 ppt) Values represent relative density arbitrary units of bands detected by western blotting of pooledsamples of five clams Potamocorbula amurensis with no significant differences in temperature but salinity increasing in gradient manner in the sampling sites(Werner and Hinton 2000 [106])

06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM

06 12 06 12SE SE + HMHMSE SE + HM HM

CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15

Figure 3 Immunoblots showing HSP72 levels in HT29 cellsafter a 24 h exposure to the different sludge extracts at differentconcentrations Cells were subjected to soluble sludge extracts alone(SE) to soluble sludge extracts + heavymetal solution (SE +HM) orto heavy metal solution alone (HM) at the indicated concentrationsexpressed as grams per kilogram of dry material Untreated cells arecontrol (C) S1 S2 S3 different sludge [102]

emphasis has also been placed on the analysis of nutrientselectrolytes and dissolved oxygen content [105]

Another environmental factor influencing the HSPexpression is seasonal variation which acts partially accord-ing to the corresponding temperature regimes Seasonalvariations in HSP70 as noted in the mussel (Mytilus gallo-provincialis) at two sites ofMediterranean SeaCarteau (nativesite) and La Fourcade (transplantation site) in a two-yearstudy may be more likely a result of combined environmentalfactors (temperature salinity and turbidity) and chemicalcontamination levels [109] Bodin et al [109] conducted acomprehensive study they did not suggest any correlationof variations in biomarkers level with other parametersTheyalso reported that mussels of both sites have specific chemicalcontamination profiles but having a similar range of valuesFor example both sites were highly contaminated by heavymetals (201 and 2584mgkg dw resp) and considered asmoderately impacted for polychlorinated biphenyls and poly-cyclic aromatic hydrocarbons nevertheless contaminationlevels at Carteau were twice as high for PAHs (1015mgg dw)

0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)

Figure 4 Scanning densitometry analysis of HSP70 in Cu(30 120583gmL) and Zn (50120583gmL) alone or in combination treatedHepG2 cells expressed as percent of control at the time described[80]

C NA 27 C NA + 27C

Figure 5 Effect of 2 h heat shock (27∘C) and sodium arsenate(10120583m) in A6 cells of Xenopus laevis with control 22∘C [120]

and PCBs (902mgg dw) as La Fourcade The seasonalcontamination trend at Carteau showed a sixfold higher levelof pyrolytic pollutants in winter It showed that seasonalvariation in contamination levels was a man-made activitywhich correlatedwell with their daily life needs [110] Anotherstudy by Hamer et al [111] investigated the concentrationsof HSP70 in the gills of the mussel Mytilus galloprovincialis


41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)

Figure 6 HSP70 levels in Potamocorbula amurensis (means plusmn SD119899 = 90) measured each month (119899 = 6ndash8 per station) from 071996to 011998 (except 1096 1ndash397 and 1097) One-way ANOVA andTukey analysis revealed twomajor groups of sites group Awith sites41 and 61 and group B with sites 81 and 125 (119875 lt 0001) [106]

Exposed toxicity0 1 2 3 4 5 6 7 8 9 10

0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25

Figure 7The concentration-response curve ofNeomysis integer fora mixture of six metals at different salinities (5 10 15 and 25permil)The toxicity decreases with increasing salinity and higher salinitiesabove 25permilhad no further influence on the 96 h LC50 of themixtureof six metals (Hg Cd Cu Zn Ni Pb) which is situated at a valuebetween 44 and 46 TU [104]

seasonally collected from different sites of the Rovinj coastalarea (Croatia) They observed maximal levels of HSP72 andHSP70 in summer (September) and minimal concentrationsin winter (December) HSP70 showed significant correlationwith the sea temperature (119903 = +0822 119875 lt 005) only Simi-larly a significant seasonal (March and September) differencein HSP70 content has also been found in centipedes collectedfrom unpolluted areas [112] Female fishes collected from twodifferent localities during spring (265∘C) and winter (48∘C)also displayed a similar trend in the ovarian and liver tissue ofthe black bullhead Lepomis macrochirus in head and kidneyof the bluegill sunfish Ameiurus melas and in the gill tissueof both species [113]

Thus seasonal variations act through temperature andnutritional regimes as well as through quantity and quality ofpollutants dumped into environments according to seasonalactivities of human beings As a result complex molecularinteractions in actual environmental habitats are operatingon biological structures and in the case of chronic pollutionthe action of the toxic substances may not be predominantbut is associated with many other environmental stressors In

combinationwith other environmental factors pollutants cancontribute to the weakening of defense and regulatory mech-anisms of studied organisms Hence the biomarkers of expo-sure related to these mechanisms of early physiological regu-lation are subject to variations that make it difficult to detectthe specific effects of chemical pollutants The interference ofnatural environmental factors in the expression of biomark-ers is an important issue with respect to the use of biomarkersin monitoring the biological effects of pollutants in theirnatural environments making field interpretations difficultTherefore the effects of environmental factors should also beconsidered in sampling strategies formonitoring programs toprevent false interpretation of results Furthermore certainfield studies have also shown that stress response can occureven at minute pollutant concentrations that are usuallyprevalent in the environment Increasing knowledge on thekinetics and persistence of the stress response to complexenvironmental mixtures (the influence of both physiologicaland environmental parameters) the constitutive levels ofHSPs and the acquisition of tolerance are required before safeapplication of HSPs to assess onsite pollution

47 Detection of HSPs through Northern and Western BlotNormally both techniques are used for HSPs detection toquantify HSPs as biomarkers of toxicity Some researchersemphasized that northern blot is a highly sensitive andinitial step in the detection of environmental stress on geneexpression Other scientists emphasized on western blot bythe fact that changes in mRNA expression do not necessarilycorrespond to changes in protein levels [7] or that polyribo-some may be involved in protein synthesis under certain cir-cumstances A simplemeasure ofmRNAmay yield a doubtfulreliabilityWhich is reallymore reliable remains questionableFor some HSPs a correlation was noted between mRNAinduction and its proteins (HSP60 HSP68 and HSP84)[114] Some others did not find any correlation betweenmRNA levels and protein synthesis for example HSP68[115] Some cases have also been seen where mRNA levelsremained constant after exposure to heavy metals (ZnCl

2

0ndash330 120583M) while protein levels significantly increased in adose-dependent manner [116] Hence conclusions cannot bedrawn only on the basis of either one and both aspects shouldbe explored in proposed model organisms

We do not intend to imply a lack of quality of the workin the studies validating HSPs as biomarkers In fact all suchstudies are valid and we acknowledge them for their valuablefindings However in our opinion there are some logicalquestions that must be addressed with reference to certainstudies For example Arts et al [52] narrated that HSP60response in the nematode Plectus acuminatus had an indica-tive value related to HSP70 response in isopods and couldbe a suitable biomarker for less heavily contaminated soilsSuch reasoning is ambiguous If we need to check the extentof soil contamination prior to concluding the significanceof a biomarker response then what is the advantage of suchbiomarkers

Most of the studies validated HSPs as biomarker of toxic-ity on the basis of 2ndash5 toxicants and even some on the basis ofonly a single toxicant [58 59 78] Just a very few investigators


considered synergistic effects Aıt-Aıssa et al [13] investigatedthe maximum number of toxicants (3 metals and 15 organicchemicals) in this context We were unable to locate anyreference for the studies with analysis of significantly broadrange of toxicants or studies containing all possible heavymetals or all organic pollutants If an initial study used CuCd andHg for gill and hepatopancreas in fishes for examplea more useful continuation of such research would be tocontinue testing different metals on the same organs of sameanimal rather than using the same metals for a differentanimal In this latter case it is very difficult if not impossibleto validate HSPs as a biomarker of toxicity exposure or ofadverse effects For example if we are testing the organism forenvironmental risk assessment in which expression of HSP70or HSP60 was considered as biomarker in response to CuCd Zn and Hg the environment may also have Cr andororganic toxicant in addition to electromagnetic waves thenhow can we interpret the results as HSPs respond to a varietyof toxicants in suppressive as well as in overexpressive waysdepending on the concentration of toxicant and durationof exposure To broaden the testing in a range of potentialtoxicants and environmental factors for a single specieswouldadvance the field to a far greater extent

48 Application of Multiple Biomarkers as a Set The rela-tionship of one biomarker to other potential biomarkerson exposure to particular contaminants in different speciesand in different organs or tissues of the same species is akey consideration prior to their widespread application inenvironmental management Though more complex such atechnique may enable the use of biomarkers to give a muchclearer picture of the environmental situation Certain studieshave already begun to consider this area Their systematicapproach is being considered here as follows

(a) Certain studies have used various xenobiotics as a toolto study stress protein synthesis in target organs inorder to evaluate the target tissue-specificity of thetoxicant For example the kidney is a target tissuefor chronic Cd exposure so HSP expression in it canbe used as biomarker [117] and even some metalslikeHg induce regional and cell-specific stress proteinexpressions in rat kidneys [86] Similarly tissue-specific differences in the accumulation of HSP70 andHSP60 inMytilus edulis exposed to a range of copperconcentrations have also been reported by Sanders etal [38] Such studies could help in the selection of acombination of target tissuesorgans for evaluation ofHSPs as a biomarker for ERA

(b) Some stressors have quite opposite effects on theexpression of HSP70 in different cases For exampleNi concentration of 600M has no effect on HSP70expression at the transcriptional level in HeLa cells[118] whereas it has shown sufficient expression inblack sea bream fibroblast cell line at concentrationof 001M [10] Arsenite is a much lesser inducerof MT but a more effective inducer of HSPs whilenickel is a good inducer of MT but poor inducer of

HSPs [119] These studies suggest the application ofsuch biomarkers in combination as a set for ERA

(c) Toxicity responses broadly vary among individuals ofdifferent species Some examples exist where toxicitystress alters theHSP levels in some organs (eg in gillsand livers in case of trout) and in some cases whole ofthe organism as in gammarids [105] Such studies andthose that have contradicted the validity of HSPs asbiomarker for ERA can still help us in the selection oforganisms

(d) The expression pattern of HSPs is not only tissue-specific [34 120] but also species-specific as revealedby decrease moderately increase and overexpressionof HSP73 in COS-7 cells (African green monkeykidney cells) A549 cells (human lung tumor cells)and rats kidney cells in exposure to 100 200 or 400MNiCl2 respectively for 4 days [121] Such studies

suggest the selection of organisms as a combinationset for ERA

To determine the variability of sublethal effects of pol-lutants only a few studies have been conducted up to thethird point For example Downs et al [122 123] developeda molecular biomarker system (MBS) based on 9 specificcellular parameters to assess the physiological status of thegrass shrimp Palaemonetes pugio (exposed to Cd atrazineand bunker fuel) and mud snails Ilyanassa obsoleta (exposedto Cd atrazine bunker fuel endosulfan and heat stresses)They assayed HSP60 HSP70 alpha B-crystallin homologuelipid peroxide total glutathione level ubiquitin mitochon-drial manganese superoxide dismutase metallothionein andcytochrome P-450 2E homologue They reported that MBSwas distinguishable among responses to each stressor andto nonstressed control conditions that is the biomarkersmetallothionein and cytochrome P450 2E homologue distin-guished betweenmetal and nonmetal stresses Aıt-Aıssa et al[74] confirmed bymultivariate analyses that some correlationexists between these biomarkers and concluded the useof complementary biomarkers as necessary to discriminatebetween different treatments and to highlight interactiveeffects In addition to animals HSP70 could potentially beapplied to the detection of stress in aquatic plants like Fucusserratus and Lemna minor But it would be most effectivewhen used in conjunction with other measurements to pro-vide a stressor-specific biomarker profile or fingerprint [49]

In the case of soil Arts et al [52] reported that HSP60response in the nematode Plectus acuminatus had an indica-tive value when related to HSP70 response in isopods andcould be a suitable biomarker for less heavily contaminatedsoils Similarly for analysis of HSP70 in coelomocytes Homaet al [54] also reinforced the notion given by earlier studiesthat the value of biomarkers is higherwhen they are employedin combination as suite rather than individually [93 124]

Previous researchers tested only a limited number of toxi-cants and in some cases only heavymetals were under inves-tigationThe application of these suits is possible in an alreadytested environmentlocality with similar levels of toxi-cants and conditions In any other region with a different


nature or extent of contamination results may remain con-founded However such an approach can yield better resultsif applied like a taxonomic key

Gupta et al [62] considered HSPs as suitable earlywarning bioindicator of cellular hazard It was further arguedthat despite having enormous use in toxicology the currentstate of knowledge in defining a mechanism of action oraccurately predicting toxicity based on stress gene expressionwarrants further investigation The properties of heat shockproteins as (i) part of the cellular protective machinery (ii)inducible nature against a wide range of chemicals and (iii)higher conservation across the taxa have proven them to bevaluable as a first tier biomarker in risk assessment Althoughstress gene expression has proved promising to understandthe toxicity of chemicals literature linking activation of stressgenes to mechanism of toxicity is limited [62]

In a recent study HSP expression profile was used asbiomarker in the fish for monitoring the water quality ofa river [125] Four of HSP genes namely HSP30 HSP60HSP70 and HSP90 were amplified and sequenced by usingdegenerate primers Later on gene specific primers weredeveloped and subsequently used to monitor the expressionof above mentioned four genes Compared to the fish atreference site up to 10-fold difference in expression of HSP70 was observed in the liver More profound differenceswere observed in expression of HSP30 in the kidneys of fishHowever as far as HSP60 and HSP90 are concerned nodifference in expression level was observedThese differencesin HSP expression correlated well with the quality of watermore profound differences were observed in downstreamwater as compared to upstream Authors were of the opinionthat HSP30 and HSP70 expression can be used as biomarkerfor evaluation of water quality [125]

Transcription profiles of two of HSP genes that isHSP70 and HSP90 were used as biomarkers against metalsand organic compoundsrsquo stress in marine diatom Ditylumbrightwellii (Db) [126] D brightwellii cultures were exposedto various metal compounds namely CuSO

4 NiSO

4 CuCl

2

and NiCl2 and HSPs gene expression was monitored by

using real-time PCR It was observed that all tested metalcompounds induced the expression of HSP90 gene howeverinduction pattern was different according to the tested metalcompound All concentrations of CuSO

4effectively induced

the transcription of HSP90 while only higher concentrationsof CuCl

2and NiSO

4were able to cause a significant increase

in expression NiCl2initially increased the expression of

HSP90 in a concentration-dependent manner However atmuch elevated level opposite effect was observed that isexpression decreased with further increase in NiCl

2level

HSP70 expression followed a different pattern expressionwas induced by CuSO

4and NiSO

4but not by CuCl

2and

NiCl2 Moreover the expression of former two genes was not

concentration dependent In the same study effect of thermalstress and organic pollutants was also analyzed Thermalstress induced expression of both genes however testedorganic pollutants had no significant effect on expressionof HSP genes This data shows that HSPs are differentiallyinvolved in defense against various stressors Based on find-ings of above mentioned study an important point needs to

be considered it seems that anionic conjugates of metal (SO4

Cl2 etc) may be responsible for specific induction of HSP

genes This can be observed from the fact that metal ionsconjugated with SO

4were able to modulate the transcription

ofHSP70 however this was not the case formetals conjugatedwith Cl

2[126]

Transcription profile ofHSP70was also studied inMytiluscoruscus in response to fuel and heavy metals stress [127] Inall the treatments HSP expression was induced with varyingdegree of magnitude HSP70 expression steadily increasedwith the passage of time and reached to the maximum (about6-fold increase) after 25 days of the treatment Howeverexpression started decreasing after gaining the peak leveland at day 30 expression was about twofold of the controlSimilar pattern was observed for heavymetals Cu2+ andCd2+but with more pronounced increase in the expression levelof HSP70 Both Cu2+ and Cd2+ enhanced expression to 10-fold and 11-fold respectively However time of maximumexpression was different in the case of Cu2+ peak expressionwas achieved at day 15 while in the case of Cd2+ it was at day9 [127] Although authors have advocated HSP70 as potentialbiomarker for heavy metals and fuels this notion is ques-tionable due to the fact that HSPs expression is responsiveto other environmental factors like increase in temperatureMoreover it seems at least in this study by Liu et al [127]that HSPs expression showed a delayed response to heavymetals stress as compared to hydrocarbons In the latter caseexpression was modulated in matter of hours while for heavymetals it happened in days In real environmental conditionsit will become almost impossible to correlate the change inexpression of HSPs to a particular stressor

HSP70 expression was taken as one of the biomarkers forstudying the effects of heavy metal accumulation in milk fish(Chanos chanos) collected from polluted sites of KattupalliIsland India [128] Through immune fluorescence scanningand western blotting HSP70 accumulation was observed ingills and liver of the milk fish Expression of HSP70 was morein the gills of fish collected from polluted site as comparedto that from less polluted site Similar expression pattern ofHSP70 was observed in the liver tissue of the fish [128] Theauthors have shown more realistic approach in drawing theconclusion based on this study In spite of showing optimismin taking HSPs as a biomarker they have stressed on moreintegrated approach for assessment ofmetal contamination inecosystem To summarize HSP70 can be taken as biomarkerhowever not in isolation Instead it could be more logical touse it as biomarker along with other parameters like oxidativestress biomarkers and ultrastructural changes

Few reports have also shown the downregulation ofHSP70 gene in response to heavy metal stress In a recentstudy Luo et al [129] have observed the effect of long-termheavy metals stress on Crassostrea hongkongensis through aproteomic approach Differentially expressed proteins wereidentified in oyster exposed to heavy metals such as zinccopper manganese and lead One of differentially regulatedproteins was identified to be HSP70 In contrast to usu-ally reported upregulation HSP70 was found to be down-regulated in Crassostrea hongkongensis [129] This unusual


transcription pattern was attributed to prolonged exposureto heavy metals This raises further question mark on thevalidity of HSPs as a universal biomarker of stress

5 Closing Remarks

All studies whether supporting or contradicting the validityof HSPs as biomarker of effect or exposure are useful forestablishing the biological exposure limits of toxicants andin creating awareness about their biological effects Thoughsome contradictory studies reject the application of HSPs asbiomarkers in various fields and situations these actuallyfrom one perspective also aid the more appropriate appli-cation of HSPs as biomarkers elsewhere by suggesting thetypes or organisms or conditions where HSPs may be lesssuitable or useful Furthermore these studies also suggestcertain new fields of research in proposed model organismsfor ERA For example the search for self- and cross-toleranceand seasonal individual sex and gender based variations inthe levels of HSP70HSP60 alone as well as in combinationwith various environmental factors (temperature salinitypH oxygenhypoxia etc) with emphasis on toxicant uptakeaccumulation detoxification synergistic effects thresholdlevels and induction kinetics of HSPs in proposed modelsare all areas of valid further examination Further ldquosuit ofbiomarkers in a set of organismsrdquo should also be investigatedunder these guide lines before suggestion of the applicationof such for environmental risk assessment Furthermore therelative sensitivity of northern or western blotting shouldalso be examined to authenticate the either techniques forbiomarkers studies in proposed model organisms Thoughthe levels of HSPs increase in dose- and time-dependentmanner this is only up to a particular limit of each toxicantafter which their expression decreases This aspect has a highpotential for the confounding of resultsTherefore evaluatingstudies should be initially conducted for different time inter-vals along with the running of controls In conclusion twomain objections remainThe first is that the synergistic effectsof toxicants with each other and also with environmentalfactors are strong enough to confound the validity of HSPsas a biomarker of toxicity exposure or effect This is as theenvironment operates as a ldquowholerdquo as a dynamic and fluctuat-ing system and as such its factors fail to operate in isolation orin neat sequences The second difficulty is in their expressionin response to variety of stress conditions that are not relatedto toxicity and therefore in how to isolate and ascertain thecause from the response

From recent studies it becomes evident that HSPs showvariable response in different organisms and even to differentstressors Hence before application as biomarker their res-ponse should be carefully checked against different stressorsMoreover false interpretations could be drawn if solely HSPsare used as biomarkers However more integrated approachcould be more conclusive

Thus it is clear that at the present studies of heat shockproteins remain so far unable to give more than an overallgeneral picture of the environment instead of more exactinformation regarding a particular toxicant or pollutant Amuch more systematic study is required we suggest with

the focus upon broadening the testing of a range of potentialtoxicants and environmental factors for a limited number ofkey target species Furthermore there is need of search fortarget organs specific for a particular toxicant With such asystematic and focused approach such biomarkers could yetpossibly be elevated when applied in sets from the currentgeneral indications they provide to becoming techniqueswhich yield much more specific useful and accurate datareadily applicable for environmental management

Conflict of Interests

The authors do not have any conflict of interests or financialgain from this paper

Acknowledgments

The authors thankfully acknowledge the permission grantedby Elsevier Publisher Oxford University Press Allen PressInc andThe American Society for Biochemistry andMolec-ular Biology for the use of certain illustrations and a table forthe compilation of present paper

References

[1] SWicherek and J P Oudinet ldquoEnvironment and health-studiesusing biomarkersrdquo Folia Medica Cracoviensia vol 38 no 3-4pp 133ndash144 1997

[2] M Ponomarenko I Stepanenko and N Kolchanov ldquoHeatshock proteinsrdquo in Brennerrsquos Encyclopedia of Genetics pp 402ndash405 2nd edition 2013

[3] F A Ritossa ldquoA new puffing pattern induced by temperatureshock and DNP in drosophilardquo Experientia vol 18 no 12 pp571ndash573 1962

[4] A Tissieres H K Mitchell and U M Tracy ldquoProtein synthesisin salivary glands of Drosophila melanogaster relation to chro-mosome puffsrdquo Journal of Molecular Biology vol 84 no 3 pp389ndash398 1974

[5] S P Place and G E Hofmann ldquoTemperature interactions of themolecular chaperone Hsc70 from the eurythermal marine gobyGillichthys mirabilisrdquo Journal of Experimental Biology vol 204no 15 pp 2675ndash2682 2001

[6] S Franzellitti and E Fabbri ldquoDifferential HSP70 gene expres-sion in the Mediterranean mussel exposed to various stressorsrdquoBiochemical and Biophysical Research Communications vol 336no 4 pp 1157ndash1163 2005

[7] A N Boone and M M Vijayan ldquoConstitutive heat shock pro-tein 70 (HSC70) expression in rainbow trout hepatocytes effectof heat shock and heavy metal exposurerdquo Comparative Bio-chemistry and Physiology C Toxicology and Pharmacology vol132 no 2 pp 223ndash233 2002

[8] C Singer S Zimmermann and B Sures ldquoInduction of heatshock proteins (hsp70) in the zebra mussel (Dreissena polymor-pha) following exposure to platinum group metals (platinumpalladium and rhodium) comparison with lead and cadmiumexposuresrdquo Aquatic Toxicology vol 75 no 1 pp 65ndash75 2005

[9] E Warchałowska-Sliwa M Niklinska A Gorlich P Mich-ailova and E Pyza ldquoHeavy metal accumulation heat shockprotein expression and cytogenetic changes inTetrix tenuicornis


(L) (Tetrigidae Orthoptera) from polluted areasrdquo Environmen-tal Pollution vol 133 no 2 pp 373ndash381 2005

[10] E E Deane and N Y S Woo ldquoImpact of heavy metals andorganochlorines on hsp70 and hsc70 gene expression in blacksea bream fibroblastsrdquo Aquatic Toxicology vol 79 no 1 pp 9ndash15 2006

[11] I Low-Friedrich and W Schoeppe ldquoEffects of calcium channelblockers on stress protein synthesis in cardiac myocytesrdquoJournal of Cardiovascular Pharmacology vol 17 no 5 pp 800ndash806 1991

[12] J Liu K S Squibb M Akkerman G F Nordberg M Lipskyand B A Fowler ldquoCytotoxicity zinc protection and stress pro-tein induction in rat proximal tubule cells exposed to cadmiumchloride in primary cell culturerdquo Renal Failure vol 18 no 6 pp867ndash882 1996

[13] S Aıt-Aıssa J-M Porcher A-P Arrigo and C Lambre ldquoActi-vation of the hsp70 promoter by environmental inorganic andorganic chemicals relationships with cytotoxicity and lipophi-licityrdquo Toxicology vol 145 no 2-3 pp 147ndash157 2000

[14] D Wirth E Christians C Munaut C Dessy J M Foidart andP Gustin ldquoDifferential heat shock gene HSP70-1 response totoxicants revealed by in vivo study of lungs in transgenic micerdquoCell Stress Chaperones vol 7 pp 387ndash395 2002

[15] F Trautinger I Kindas-Mugge R M Knobler and H Honi-gsmann ldquoStress proteins in the cellular response to ultravioletradiationrdquo Journal of Photochemistry and Photobiology B Biol-ogy vol 35 no 3 pp 141ndash148 1996

[16] H Lin M Opler M Head M Blank and R GoodmanldquoElectromagnetic field exposure induces rapid transitory heatshock factor activation in human cellsrdquo Journal of CellularBiochemistry vol 66 pp 482ndash488 1997

[17] H Yamada and S Koizumi ldquoEffect of ultraviolet irradiation onthe protein synthesis of human skin cells a study with a mono-chromatic ultraviolet irradiation apparatusrdquo Industrial Healthvol 41 no 2 pp 88ndash93 2003

[18] H Yamada M Murata K Suzuki and S Koizumi ldquoUltravioletirradiation increases the sensitivity of cultured human skin cellsto cadmiumprobably through the inhibition ofmetallothioneingene expressionrdquo Toxicology and Applied Pharmacology vol200 no 3 pp 251ndash257 2004

[19] C G Cranfield A Dawe V Karloukovski R E Dunin-Borko-wski D de Pomerai and J Dobson ldquoBiogenic magnetite inthe nematode Caenorhabditis elegansrdquo Proceedings of the RoyalSociety vol 271 supplement 6 pp S436ndashS439 2004

[20] T Abe T Konishi T Katoh et al ldquoInduction of heat shock 70mRNA by cadmium is mediated by glutathione suppressive andnon-suppressive triggersrdquo Biochimica et Biophysica Acta vol1201 no 1 pp 29ndash36 1994

[21] T Abe K Yamamura S Gotoh M Kashimura and K HigashildquoConcentration-dependent differential effects of N-acetyl-L-cysteine on the expression of HSP70 andmetallothionein genesinduced by cadmium in human amniotic cellsrdquo Biochimica etBiophysica Acta General Subjects vol 1380 no 1 pp 123ndash1321998

[22] Z Guo A Ersoz D A Butterfield and M P Mattson ldquoBene-ficial effects of dietary restriction on cerebral cortical synapticterminals Preservation of glucose and glutamate transport andmitochondrial function after exposure to amyloid 120573-peptideiron and 3-nitropropionic acidrdquo Journal of Neurochemistry vol75 no 1 pp 314ndash320 2000

[23] G de Boeck B de Wachter A Vlaeminck and R BlustldquoEffect of cortisol treatment andor sublethal copper exposure

on copper uptake andheat shock protein levels in common carpCyprinus carpiordquo Environmental Toxicology and Chemistry vol22 pp 1122ndash1126 2003

[24] H B Chen Y Chan A C Hung Y Tsai and S H Sun ldquoElu-cidation of ATP-stimulated stress protein expression of RBA-2 type-2 astrocytes ATP potentiate HSP60 and CuZn SODexpression and stimulates pI shift of peroxiredoxin IIrdquo Journalof Cellular Biochemistry vol 97 no 2 pp 314ndash326 2006

[25] J RNevins ldquoInduction of the synthesis of a 70000 daltonmam-malian heat shock protein by the adenovirus E1A gene productrdquoCell vol 29 no 3 pp 913ndash919 1982

[26] J L Zimmerman W Petri and M Meselson ldquoAccumulationof a specific subset of D melanogaster heat shock mRNAs innormal development without heat shockrdquo Cell vol 32 no 4pp 1161ndash1170 1983

[27] B J Wu and R I Morimoto ldquoTranscription of the humanhsp70 gene is induced by serum stimulationrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 82 no 18 pp 6070ndash6074 1985

[28] P M Filipe and A C Fernandes ldquoStress proteinsrdquo Acta medicaportuguesa vol 7 no 12 pp 711ndash715 1994

[29] E Mocchegiani L Costarelli R Giacconi et al ldquoNutrient-geneinteraction in ageing and successful ageing A single nutrient(zinc) and some target genes related to inflammatoryimmuneresponserdquoMechanisms of Ageing and Development vol 127 no6 pp 517ndash525 2006

[30] R I Morimoto andM Gabriella Santoro ldquoStress-inducible res-ponses and heat shock proteins new pharmacologic targets forcytoprotectionrdquo Nature Biotechnology vol 16 no 9 pp 833ndash838 1998

[31] S Kantengwa and B S Polla ldquoPhagocytosis of Staphylococcusaureus induces a selective stress response in humanmonocytes-macrophages (M120593) modulation by M120593 differentiation and byironrdquo Infection and Immunity vol 61 no 4 pp 1281ndash1287 1993

[32] ANeuer S D Spandorfer P Giraldo S Dieterle Z Rosenwaksand S S Witkin ldquoThe role of heat shock proteins in reproduc-tionrdquo Human Reproduction Update vol 6 no 2 pp 149ndash1592000

[33] L Pospısil and J Canderle ldquoHeat shock protein (hsp60) ofchlamydial origin and fertility disturbancesrdquo Ceska Gynekolo-gie vol 68 no 3 pp 186ndash190 2003

[34] V Ramaglia G M Harapa NWhite and L T Buck ldquoBacterialinfection and tissue-specific Hsp72 -73 and -90 expression inwestern painted turtlesrdquo Comparative Biochemistry and Physio-logy C Toxicology and Pharmacology vol 138 no 2 pp 139ndash1482004

[35] R A Kroes K Abravaya J Seidenfeld and R I MorimotoldquoSelective activation of human heat shock gene transcription bynitrosourea antitumor drugs mediated by isocyanate-induceddamage and activation of heat shock transcription factorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 88 no 11 pp 4825ndash4829 1991

[36] W Hu W Wu C E Verschraegen et al ldquoProteomic identifica-tion of heat shock protein 70 as a candidate target for enhancingapoptosis induced by farnesyl transferase inhibitorrdquo Proteomicsvol 3 no 10 pp 1904ndash1911 2003

[37] C Brochu A Halmeur and M Ouellette ldquoThe heat shock pro-teinHSP70 andheat shock cognate proteinHSC70 contribute toantimony tolerance in the protozoan parasite Leishmaniardquo CellStress and Chaperones vol 9 no 3 pp 294ndash303 2004


[38] B M Sanders L S Martin S R Howe W G Nelson E SHegre and D K Phelps ldquoTissue-specific differences in accu-mulation of stress proteins in Mytilus edulis exposed to a rangeof copper concentrationsrdquo Toxicology and Applied Pharmaco-logy vol 125 no 2 pp 206ndash213 1994

[39] J E Kammenga M S J Arts and W J M Oude-BreuilldquoHSP60 as a potential biomarker of toxic stress in the nematodePlectus acuminatusrdquo Archives of Environmental Contaminationand Toxicology vol 34 no 3 pp 253ndash258 1998

[40] B J Cochrane R B Irby and T W Snell ldquoEffects of copperand tributyltin on stress protein abundance in the rotiferBrachionus plicatilisrdquo Comparative Biochemistry and PhysiologyC Comparative vol 98 no 2-3 pp 385ndash390 1991

[41] B M Sanders and L S Martin ldquoStress proteins as biomarkersof contaminant exposure in archived environmental samplesrdquoScience of the Total Environment vol 139-140 pp 459ndash470 1993

[42] C Porte X Biosca M Sole and J Albaiges ldquoThe integrateduse of chemical analysis cytochrome P450 and stress proteinsin mussels to assess pollution along the Galician coast (NWSpain)rdquo Environmental Pollution vol 112 no 2 pp 261ndash2682001

[43] IWerner S J Teh S Datta X Lu and TM Young ldquoBiomarkerresponses in Macoma nasuta (Bivalvia) exposed to sedimentsfrom northern San Francisco Bayrdquo Marine EnvironmentalResearch vol 58 no 2ndash5 pp 299ndash304 2004

[44] H C Schroder R Batel H M A Hassanein et al ldquoCorrela-tion between the level of the potential biomarker heat-shockprotein and the occurrence of DNA damage in the dabLimanda limanda a field study in the North Sea and the EnglishChannelrdquo Marine Environmental Research vol 49 no 3 pp201ndash215 2000

[45] T Heresztyn and B C Nicholson ldquoHeat shock protein 70 levelsin rainbow trout primary epidermal cultures in response to24-dichloroaniline exposure a novel in vitro aquatic toxicitymarkerrdquo Environmental Toxicology vol 16 no 3 pp 253ndash2592001

[46] E Fulladosa EDeaneAHYNgNY SWoo J CMurat andI Villaescusa ldquoStress proteins induced by exposure to sublethallevels of heavy metals in sea bream (Sparus sarba) blood cellsrdquoToxicology in Vitro vol 20 no 1 pp 96ndash100 2006

[47] M Guizani Y Nogoshi F Ben Fredj J Han H Isoda and NFunamizu ldquoHeat shock protein 47 stress responses in Chinesehamster ovary cells exposed to raw and reclaimed wastewaterrdquoJournal of EnvironmentalMonitoring vol 14 no 2 pp 492ndash4982012

[48] J Bierkens J Maes and F Vander Plaetse ldquoDose-dependentinduction of heat shock protein 70 synthesis in Raphido-celis subcapitata following exposure environmental pollutantsrdquoEnvironmental Pollution vol 101 no 1 pp 91ndash97 1998

[49] H E Ireland S J Harding G A BonwickM Jones C J Smithand J HWilliams ldquoEvaluation of heat shock protein 70 as a bio-marker of environmental stress in Fucus serratus and Lemnaminorrdquo Biomarkers vol 9 no 2 pp 139ndash155 2004

[50] J S Miller-Morey and F M Van Dolah ldquoDifferential responsesof stress proteins antioxidant enzymes and photosynthetic effi-ciency to physiological stresses in the Florida red tide dinoflag-ellate Karenia brevisrdquoComparative Biochemistry andPhysiologyC Toxicology and Pharmacology vol 138 no 4 pp 493ndash5052004

[51] J E Kammenga R Dallinger M H Donker et al ldquoBiomarkersin terrestrial invertebrates for ecotoxicological soil risk assess-mentrdquoReviews of Environmental Contamination andToxicologyvol 164 pp 93ndash147 2000

[52] M S J Arts RO Schill T KniggeH Eckwert J E Kammengaand H Kohler ldquoStress proteins (hsp70 hsp60) induced inisopods and nematodes by field exposure tometals in a gradientnear Avonmouth UKrdquo Ecotoxicology vol 13 no 8 pp 739ndash7552004

[53] DNadeau S Corneau I Plante GMorrow andRMTanguayldquoEvaluation for HSP70 as a biomarker of effect of pollutantson the earthworm Lumbricus terrestrisrdquo Cell Stress Chaperonesvol 6 pp 153ndash163 2001

[54] J Homa E Olchawa S R Sturzenbaum A John Morgan andB Plytycz ldquoEarly-phase immunodetection of metallothioneinand heat shock proteins in extruded earthworm coelomocytesafter dermal exposure to metal ionsrdquo Environmental Pollutionvol 135 no 2 pp 275ndash280 2005

[55] L H Damelin ldquoHormesis a stress response in cells exposedto low levels of heavy metalsrdquo Human and Experimental Toxi-cology vol 19 no 7 pp 420ndash430 2000

[56] C A Mandon C Diaz A-P Arrigo and L J Blum ldquoChemicalstress sensitive luminescent human cells molecular biologyapproach using inducible Drosophila melanogaster hsp22 pro-moterrdquo Biochemical and Biophysical Research Communicationsvol 335 no 2 pp 536ndash544 2005

[57] A Nazir D K Saxena and D Kar Chowdhuri ldquoInduction ofhsp70 in transgenic Drosophila biomarker of exposure againstphthalimide group of chemicalsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1621 no 2 pp 218ndash225 2003

[58] IMukhopadhyay A Nazir D K Saxena andD K ChowdhurildquoToxicity of cypermethrin Hsp70 as a biomarker of response intransgenic Drosophilardquo Biomarkers vol 7 no 6 pp 501ndash5102002

[59] I Mukhopadhyay D K Saxena and D K Chowdhuri ldquoHaz-ardous effects of effluent from the chrome plating industry70 kDa heat shock protein expression as a marker of cellulardamage in transgenic Drosophila melanogaster (hsp70-lacZ)rdquoEnvironmentalHealth Perspectives vol 111 no 16 pp 1926ndash19322003

[60] P H Krone S R Blechinger T G Evans J A Ryan E JNoonan and L E Hightower ldquoUse of fish liver PLHC-1 cellsand zebrafish embryos in cytotoxicity assaysrdquoMethods vol 35no 2 pp 176ndash187 2005

[61] SH Seok JH ParkMWBaek et al ldquoSpecific activation of thehumanHSP70 promoter by copper sulfate in mosaic transgeniczebrafishrdquo Journal of Biotechnology vol 126 no 3 pp 406ndash4132006

[62] S C Gupta A Sharma M Mishra R K Mishra and D KChowdhuri ldquoHeat shock proteins in toxicology how close andhow farrdquo Life Sciences vol 86 no 11-12 pp 377ndash384 2010

[63] S C Gupta H R Siddique N Mathur R K Mishra D K Sax-ena and D K Chowdhuri ldquoAdverse effect of organophosphatecompounds dichlorvos and chlorpyrifos in the reproductivetissues of transgenic Drosophila melanogaster 70 kDa heatshock protein as a marker of cellular damagerdquo Toxicology vol238 no 1 pp 1ndash14 2007

[64] S C Gupta H R Siddique N Mathur et al ldquoInductionof hsp70 alterations in oxidative stress markers and apop-tosis against dichlorvos exposure in transgenic Drosophila


melanogaster modulation by reactive oxygen speciesrdquo Biochim-ica et Biophysica ActamdashGeneral Subjects vol 1770 no 9 pp1382ndash1394 2007

[65] HR Siddique S CGupta KMitra et al ldquoAdverse effect of tan-nery waste leachates in transgenic Drosophila melanogasterrole of ROS inmodulation of HSP70 oxidative stress and apop-tosisrdquo Journal of Applied Toxicology vol 28 no 6 pp 734ndash7482008

[66] H R Siddique K Mitra V K Bajpai K Ravi Ram D K Sax-ena and D K Chowdhuri ldquoHazardous effect of tannery solidwaste leachates on development and reproduction in Droso-phila melanogaster 70 kDa heat shock protein as a marker ofcellular damagerdquo Ecotoxicology and Environmental Safety vol72 no 6 pp 1652ndash1662 2009

[67] D Bhargav M Pratap Singh R C Murthy et al ldquoToxic poten-tial of municipal solid waste leachates in transgenic Drosophilamelanogaster (hsp70-lacZ) hsp70 as a marker of cellular dam-agerdquo Ecotoxicology and Environmental Safety vol 69 no 2 pp233ndash245 2008

[68] M P Singh M M K Reddy N Mathur D K Saxena and DK Chowdhuri ldquoInduction of hsp70 hsp60 hsp83 and hsp26 andoxidative stressmarkers in benzene toluene and xylene exposedDrosophila melanogaster role of ROS generationrdquo Toxicologyand Applied Pharmacology vol 235 no 2 pp 226ndash243 2009

[69] I Nisamedtinov G G Lindsey R Karreman et al ldquoThe res-ponse of the yeast Saccharomyces cerevisiae to sudden vs grad-ual changes in environmental stress monitored by expression ofthe stress response protein Hsp12prdquo FEMS Yeast Research vol8 no 6 pp 829ndash838 2008

[70] Y L Wu X Pan S P Mudumana H Wang P W Kee and ZGong ldquoDevelopment of a heat shock inducible gfp transgeniczebrafish line by using the zebrafish hsp27 promoterrdquoGene vol408 no 1-2 pp 85ndash94 2008

[71] F A C Wieganta J E M Souren J van Rijn and R van WijkldquoStressor-specific induction of heat shock proteins in rat hepa-toma cellsrdquo Toxicology vol 94 no 1ndash3 pp 143ndash159 1994

[72] P E Mirkes B Doggett and L Cornel ldquoInduction of a heatshock response (HSP 72) in rat embryos exposed to selectedchemical teratogensrdquoTeratology vol 49 no 2 pp 135ndash142 1994

[73] F Croute B Beau C Arrabit et al ldquoPattern of stress proteinexpression in human lung cell-line A549 after short- or long-term exposure to cadmiumrdquo Environmental Health Perspectivesvol 108 no 1 pp 55ndash60 2000

[74] S Aıt-Aıssa O Ausseil O Palluel E Vindimian J Garnier-Laplace and J Porcher ldquoBiomarker responses in juvenile rain-bow trout (Oncorhynchus mykiss) after single and combinedexposure to low doses of cadmium zinc PCB77 and 17120573-oestradiolrdquo Biomarkers vol 8 no 6 pp 491ndash508 2003

[75] S M Efremova B A Margulis I V Guzhova et al ldquoHeatshock protein Hsp70 expression and DNA damage in Baikaliansponges exposed to model pollutants and wastewater fromBaikalsk Pulp and Paper Plantrdquo Aquatic Toxicology vol 57 no4 pp 267ndash280 2002

[76] G Agell M Uriz E Cebrian and RMartı ldquoDoes stress proteininduction by copper modify natural toxicity in spongesrdquoEnvironmental Toxicology and Chemistry vol 20 no 11 pp2588ndash2593 2001

[77] G Agell X Turon S De Caralt S Lopez-Legentil and M JUriz ldquoMolecular and organism biomarkers of copper pollutionin the ascidian Pseudodistoma crucigasterrdquo Marine PollutionBulletin vol 48 no 7-8 pp 759ndash767 2004

[78] T N Guecheva B Erdtmann M S Benfato and J A P Hen-riques ldquoStress protein response and catalase activity in freshwa-ter planarian Dugesia (Girardia) schubarti exposed to copperrdquoEcotoxicology and Environmental Safety vol 56 no 3 pp 351ndash357 2003

[79] D Webb and M M Gagnon ldquoThe value of stress protein 70as an environmental biomarker of fish health under field con-ditionsrdquo Environmental Toxicology vol 24 no 3 pp 287ndash2952009

[80] C Urani P Melchioretto F Morazzoni C Canevali and MCamatini ldquoCopper and zinc uptake and HSP70 expression inHepG2 cellsrdquo Toxicology in Vitro vol 15 no 4-5 pp 497ndash5022001

[81] S N Pedersen A-K Lundebye and M H Depledge ldquoFieldapplication of metallothionein and stress protein biomarkersin the shore crab (Carcinus maenas) exposed to trace metalsrdquoAquatic Toxicology vol 37 no 2-3 pp 183ndash200 1997

[82] J M Matz M J Blake J T Saari and A M Bode ldquoDietarycopper deficiency reduces heat shock protein expression incardiovascular tissuesrdquo FASEB Journal vol 8 no 1 pp 97ndash1021994

[83] P F La Porte ldquoMytilus trossulus hsp70 as a biomarker forarsenic exposure in the marine environment laboratory andreal-world resultsrdquo Biomarkers vol 10 no 6 pp 417ndash428 2005

[84] W F Salminen Jr R Voellmy and S M Roberts ldquoProtectionagainst hepatotoxicity by a single dose of amphetamine thepotential role of heat shock protein inductionrdquo Toxicology andApplied Pharmacology vol 147 no 2 pp 247ndash258 1997

[85] F A CWiegant J van Rijn and R vanWijk ldquoEnhancement ofthe stress response byminute amounts of cadmium in sensitizedReuberH35 hepatoma cellsrdquoToxicology vol 116 no 1ndash3 pp 27ndash37 1997

[86] P L Goering B R Fisher B T Noren A Papaconstantinou JL Rojko and R J Marler ldquoMercury induces regional and cell-specific stress protein expression in rat kidneyrdquo ToxicologicalSciences vol 53 no 2 pp 447ndash457 2000

[87] R Furuya H Kumagai and A Hishida ldquoAcquired resistance torechallenge injurywith uranyl acetate in LLC-PK1 cellsrdquo Journalof Laboratory and Clinical Medicine vol 129 no 3 pp 347ndash3551997

[88] N Honda and M Sudo ldquoResistance to uranyl acetate-inducedacute renal failure in rabbits renal function and morphologyrdquoin Acute Renal Failure H E Eliahou Ed p 105 John LibbeyLondon UK 1982

[89] S Mizuno K Fujita R Furuy et al ldquoAssociation of HSP73 withthe acquired resistance to uranyl acetate-induced acute renalfailurerdquo Toxicology vol 117 no 2-3 pp 183ndash191 1997

[90] H Liu R Lightfoot and J L Stevens ldquoActivation of heat shockfactor by alkylating agents is triggered by glutathione depletionand oxidation of protein thiolsrdquo Journal of Biological Chemistryvol 271 no 9 pp 4805ndash4812 1996

[91] L A Opanashuk and J N Finkelstein ldquoRelationship of lead-induced proteins to stress response proteins in astroglial cellsrdquoJournal of Neuroscience Research vol 42 no 5 pp 623ndash6321995

[92] F A C Wiegant I Y Malyshev A L Kleschyov E VanFaassen and A F Vanin ldquoDinitrosyl iron complexes with thiol-containing ligands and S-nitroso-DL-penicillamine as induc-tors of heat shock protein synthesis in H35 hepatoma cellsrdquoFEBS Letters vol 455 no 1-2 pp 179ndash182 1999

[93] T Lukkari M Taavitsainen M Soimasuo A Oikari and JHaimi ldquoBiomarker responses of the earthworm Aporrectodea


tuberculata to copper and zinc exposure differences betweenpopulations with and without earlier metal exposurerdquo Environ-mental Pollution vol 129 no 3 pp 377ndash386 2004

[94] E Scofield R T Bowyer and L K Duffy ldquoBaseline levels of Hsp70 a stress protein and biomarker in halibut from the CookInlet region of Alaskardquo Science of the Total Environment vol226 no 1 pp 85ndash88 1999

[95] P L Klerks and J S Weis ldquoGenetic adaptation to heavy metalsin aquatic organisms a reviewrdquoEnvironmental Pollution vol 45no 3 pp 173ndash205 1987

[96] S Shpund and D Gershon ldquoAlterations in the chaperone acti-vity of HSP70 in aging organismsrdquo Archives of Gerontology andGeriatrics vol 24 no 2 pp 125ndash131 1997

[97] K A Hoekstra D V Godin J Kurtu and KM Cheng ldquoEffectsof oxidant-induced injury on heme oxygenase and glutathionein cultured aortic endothelial cells from atherosclerosis-susceptible and -resistant Japanese quailrdquoMolecular and Cellu-lar Biochemistry vol 254 no 1-2 pp 61ndash71 2003

[98] P D B Filzek D J Spurgeon G Broll et al ldquoPedological cha-racterisation of sites along a transect from a primary cad-miumleadzinc smelting worksrdquo Ecotoxicology vol 13 no 8pp 725ndash737 2004

[99] T Ikemoto T Kunito H Tanaka N Baba N Miyazaki and STanabe ldquoDetoxification mechanism of heavy metals in marinemammals and seabirds interaction of selenium with mercurysilver copper zinc and cadmium in liverrdquo Archives of Environ-mental Contamination and Toxicology vol 47 no 3 pp 402ndash413 2004

[100] T E Eichler R F Ransom and W E Smoyer ldquoDifferentialinduction of podocyte heat shock proteins by prolonged singleand combination toxic metal exposurerdquo Toxicological Sciencesvol 84 no 1 pp 120ndash128 2005

[101] N Saydam F Steiner O Georgiev andW Schaffner ldquoHeat andheavy metal stress synergize to mediate transcriptional hyper-activation bymetal-responsive transcription factorMTF-1rdquoTheJournal of Biological Chemistry vol 278 no 34 pp 31879ndash318832003

[102] E Fulladosa F Delmas L Jun I Villaescusa and J C MuratldquoCellular stress induced in cultured human cells by exposure tosludge extracts from water treatment plantsrdquo Ecotoxicology andEnvironmental Safety vol 53 no 1 pp 134ndash140 2002

[103] A Luedeking and A Koehler ldquoRegulation of expression ofmultixenobiotic resistance (MXR) genes by environmentalfactors in the blue mussel Mytilus edulisrdquo Aquatic Toxicologyvol 69 no 1 pp 1ndash10 2004

[104] T Verslycke M Vangheluwe D Heijerick K de Schamphe-laere P van Sprang and C R Janssen ldquoThe toxicity of metalmixtures to the estuarine mysid Neomysis integer (CrustaceaMysidacea) under changing salinityrdquoAquatic Toxicology vol 64no 3 pp 307ndash315 2003

[105] R Triebskorn S Adam H Casper et al ldquoBiomarkers as diag-nostic tools for evaluating effects of unknown past water qualityconditions on stream organismsrdquo Ecotoxicology vol 11 pp 451ndash465 2002

[106] IWerner andD EHinton ldquoSpatial profiles of hsp70 proteins inAsian clam (Potamocorbula amurensis) in Northern San Fran-cisco Bay may be linked to natural rather than anthropogenicstressorsrdquo Marine Environmental Research vol 50 no 1ndash5 pp379ndash384 2000

[107] D Tran J-P Bourdineaud J-C Massabuau and J Garnier-Laplace ldquoModulation of uranium bioaccumulation by hypoxia

in the freshwater clam Corbicula fluminea induction of multi-xenobiotic resistance protein and heat shock protein 60 in gilltissuesrdquo Environmental Toxicology and Chemistry vol 24 no 9pp 2278ndash2284 2005

[108] J Bierkens W van de Perre and J Maes ldquoEffect of differentenvironmental variables on the synthesis of Hsp70 in Raphi-docelis subcapitatardquo Comparative Biochemistry and PhysiologyA Molecular amp Integrative Physiology vol 120 no 1 pp 29ndash341998

[109] N Bodin T Burgeot J Y Stanisiere et al ldquoSeasonal variationsof a battery of biomarkers and physiological indices for themusselMytilus galloprovincialis transplanted into the northwestMediterranean Seardquo Comparative Biochemistry and PhysiologyC Toxicology amp Pharmacology vol 138 no 4 pp 411ndash427 2004

[110] M S Hossain and Y S A Khan ldquoTrace metals in Penaeidshrimp and Spiny lobster from the Bay of Bengalrdquo ScienceAsiavol 27 pp 165ndash168 2001

[111] B Hamer D P Hamer W E G Muller and R Batel ldquoStress-70proteins in marine musselMytilus galloprovincialis as biomark-ers of environmental pollution a field studyrdquo EnvironmentInternational vol 30 no 7 pp 873ndash882 2004

[112] E Pyza P Mak P Kramarz and R Laskowski ldquoHeat shockproteins (HSP70) as biomarkers in ecotoxicological studiesrdquoEcotoxicology and Environmental Safety vol 38 no 3 pp 244ndash251 1997

[113] J L Yoo and D M Janz ldquoTissue-specific HSP70 levels andreproductive physiological responses in fishes inhabiting ametal-contaminated creekrdquoArchives of Environmental Contam-ination and Toxicology vol 45 no 1 pp 110ndash120 2003

[114] H Ovelgonne M Bitorina and R van Wijk ldquoStressor-specificactivation of heat shock genes in H35 rat hepatoma cellsrdquo Toxi-cology and Applied Pharmacology vol 135 no 1 pp 100ndash1091995

[115] J H Ovelgonne J E M Souren F A C Wiegant and R vanWijk ldquoRelationship between cadmium-induced expression ofheatshock genes inhibition of protein synthesis and cell deathrdquoToxicology vol 99 no 1-2 pp 19ndash30 1995

[116] S R Sturzenbaum M S J Arts and J E Kammenga ldquoMolec-ular cloning and characterization of Cpn60 in the free-livingnematode Plectus acuminatusrdquo Cell Stress and Chaperones vol10 no 2 pp 79ndash85 2005

[117] P L Goering C L Kish and B R Fisher ldquoStress protein syn-thesis induced by cadmium-cysteine in rat kidneyrdquo Toxicologyvol 85 no 1 pp 25ndash39 1993

[118] M Murata P Gong K Suzuki and S Koizumi ldquoDifferentialmetal response and regulation of human heavymetal-induciblegenesrdquo Journal of Cellular Physiology vol 180 pp 105ndash113 1999

[119] J W Bauman J Liu and C D Klaassen ldquoProduction of metal-lothionein and heat-shock proteins in response to metalsrdquo Fun-damental and Applied Toxicology vol 21 no 1 pp 15ndash22 1993

[120] K S Ali L Dorgai M Abraham and E Hermesz ldquoTissue-and stressor-specific differential expression of two hsc70 genesin carprdquoBiochemical and Biophysical Research Communicationsvol 307 no 3 pp 503ndash509 2003

[121] N Hfaiedh M S Allagui A El Feki et al ldquoEffects of nickelpoisoning on expression pattern of the 7273 and 94 kDa stressproteins in rat organs and in the COS-7 HepG2 and A549 celllinesrdquo Journal of Biochemical and Molecular Toxicology vol 19no 1 pp 12ndash18 2005

[122] C A Downs J E Fauth and C M Woodley ldquoAssessing thehealth of grass shrimp (Palaeomonetes pugio) exposed to natural


and anthropogenic stressors a molecular biomarker systemrdquoMarine Biotechnology vol 3 no 4 pp 380ndash397 2001

[123] C A Downs R T Dillon Jr J E Fauth and C MWoodley ldquoAmolecular biomarker system for assessing the health of gastro-pods (Ilyanassa obsoleta) exposed to natural and anthropogenicstressorsrdquo Journal of Experimental Marine Biology and Ecologyvol 259 no 2 pp 189ndash214 2001

[124] W M de Coen and C R Janssen ldquoA multivariate biomarker-based model predicting population-level responses of Daphniamagnardquo Environmental Toxicology and Chemistry vol 22 no 9pp 2195ndash2201 2003

[125] L H An K Lei and B H Zheng ldquoUse of heat shock pro-tein mRNA expressions as biomarkers in wild crucian carp formonitoring water qualityrdquo Environmental Toxicology and Phar-macology vol 37 no 1 pp 248ndash255 2014

[126] R Guo M A Lee and J S Ki ldquoDifferent transcrip-tional responses of heat shock protein 7090 in the marinediatom Ditylum brightwellii exposed to metal compounds andendocrine-disrupting chemicalsrdquo Chemosphere vol 92 no 5pp 535ndash543 2013

[127] H H Liu J Y He C F Chi and J Shao ldquoDifferential HSP70expression in Mytilus coruscus under various stressorsrdquo Genevol 543 no 1 pp 166ndash117 2014

[128] S Rajeshkumar J Mini and N Munuswamy ldquoEffects of heavymetals on antioxidants and expression of HSP70 in differenttissues of Milk fish (Chanos chanos) of Kaattuppalli IslandChennai Indiardquo Ecotoxicology and Environmental Safety vol98 pp 8ndash18 2013

[129] L Luo C Ke X Guo B Shi and M Huang ldquoMetal accumula-tion and differentially expressed proteins in gill of oyster (Crass-ostrea hongkongensis) exposed to long-term heavy metal-con-taminated estuaryrdquo Fish amp Shellfish Immunology vol 38 no 2pp 318ndash329 2014

[130] B D Moffat and T W Snell ldquoRapid toxicity assessment usingan in vivo enzyme test for Brachionus plicatilis (rotifera)rdquo Eco-toxicology and Environmental Safety vol 30 no 1 pp 47ndash531995

[131] P Michailova N Petrova S Bovero G Sella and L RamellaldquoStructural and functional rearrangements in polytene chro-mosomes of Chironomids (Diptera) as biomarkers for heavymetal pollution in aquatic ecosystemsrdquo in Proceedings of theInteranational Conference on Heavy Metals in the Environmentpp 70ndash79 University of Michigan Ann Arbor Mich USA2000

[132] P Li X Xiong G Yang W Liu H Xu and P Tai ldquoApplicationof terrestrial invertebrates biomarkers in soil pollution ecologystudyrdquo Chinese Journal of Applied Ecology vol 14 no 12 pp2347ndash2350 2003

[133] J G Bundy D J Spurgeon C Svendsen et al ldquoEnvironmentalmetabonomics applying combination biomarker analysis inearthworms at ametal contaminated siterdquo Ecotoxicology vol 13no 8 pp 797ndash806 2004

[134] M P Cajaraville M J Bebianno J Blasco C Porte C Sara-squete and A Viarengo ldquoThe use of biomarkers to assess theimpact of pollution in coastal environments of the IberianPeninsula a practical approachrdquo Science of the Total Environ-ment vol 247 no 2-3 pp 295ndash311 2000

[135] C Wagner R Steffen C Koziol et al ldquoApoptosis in marinesponges a biomarker for environmental stress (cadmium andbacteria)rdquoMarine Biology vol 131 no 3 pp 411ndash421 1998

[136] G Wilczek ldquoApoptosis and biochemical biomarkers of stressin spiders from industrially polluted areas exposed to high

temperature and dimethoaterdquo Comparative Biochemistry andPhysiology C Toxicology and Pharmacology vol 141 no 2 pp194ndash206 2005

Submit your manuscripts athttpwwwhindawicom

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MEDIATORSINFLAMMATION

of


Table 1 Biomarkers proposed for environmental risk assessment

Name of biomarker ReferenceEsterases [130]Polytene chromosomes of chironomids (Diptera) [131]HSP60 HSP70 alpha B-crystallin homologue lipidperoxide total glutathione level ubiquitinmitochondrial manganese superoxide dismutasemetallothionein and cytochrome P450 2E homologue

[93 122123]

Numbers of macrophages in liver tissue changes invarious blood parameters [105]

Histological and ultrastructural markers lysosomalmembrane stability of coelomocytes histidine [51 132]

Catalase activity [78]Metabonomics analysis using NMR techniques [133]P-glycoprotein major vault protein topoisomerase-II [103]Acetylcholinesterase inhibition and imposex [134]Digestive enzymes glycolytic enzymes and cellularenergy allocation [124]

Apoptosis in marine sponges and spiders [135 136]

is a universal phenomenon in all studied plant and animalspecies including humans Because HSPs can also be inducedby oxidants toxins heavy metals free radicals viruses andother stressors they are sometimes called the ldquostress proteinsrdquo[2] Heat shock proteins were initially discovered in 1962 inDrosophila melanogaster larvae in response to heat shock [3]and the term ldquoheat shock proteinrdquo was coined by Tissieres etal [4] Heat shock proteins show characteristically markedvariations in their expression in most of organisms under arange of temperatures [5 6] heavy metals [7ndash10] chemicals[11ndash14] radiations [15ndash19] metabolites and hormones [20ndash24] clinical situations [16 25ndash30] or pathogens that may beabove or beyond their optimal limits [31ndash34] and drugs [35ndash37]

Heat shock proteins are cosmopolitan in all living organ-isms and are usually classified as different families accordingto their molecular sizemdashHSP27 HSP47 HSP60 HSP70HSP90 and HSP110 These families of heat shock proteinsplay crucial roles in physiological processes such as proteinchaperoning activity protection against apoptosis steroido-genesis and stress tolerance In addition heat shock proteinsespecially HSP70 and HSP60 have also been proposed asbiomarkers of exposure levels and toxicity Their candidatureas biomarkers is based on many observations showing thatthe HSP60 induction in certain organisms (eg musselMytilus edulis and nematode Plectus acuminatus) results ina several fold sensitive response than the use of other com-parable parameters such as quantifying adverse effects onbiomass or reproduction [38 39] Such an observation inmussels (M edulis) led to their recommendation as one ofthemost suitable organisms for biomonitoring of aquatic eco-systems

The role ofHSP70HSP60 as a biomarker is highly topicalCertain studies have produced contradictory results someindicate a high HSP70 sensitivity to pollutants while otherssuggest otherwise The confounding issue in many such

studies seems to be the use of varying concentrations oftoxicity exposure some authors have realistically used lowconcentrations somehave used high concentrations but onlyvery few tested real-world contamination exposures modelsFurthermore the existing validated bioassays mostly basedon lethality or reproduction have somewhat limited appli-cability due to their crude sensitivity long exposure or theoverall expenses of the test In contrast changes at bio-chemical level are usually the first detectable response toenvironmental disturbance Therefore analysis of toxicity-induced changes in gene expression (ie alterations in pat-terns of protein synthesis) and resultant cell injury may bequite handy factors to be considered as biomarkers of toxicityexposure As these changes underline all effects at higherorganizational level therefore these have been regarded ashighly sensitive indicators of toxicity

2 Favoring Evidences HSPs as a Biomarker

21 Aquatic Environment The first favorable report on theuse of HSPs as a toxicity biomarker came from the study ofCochrane et al [40] It was reported that exposure of rotiferBrachionus plicatilis to sublethal doses of CuSO

4resulted in

a 4-5 fold increase in HSP58 with the maximum increaseoccurring at approximately 5 of the LC50 for the species Asimilar response was seen with tributyltin Kinetics of induc-tionwas sigmoidwith induction occurring in the range of 20ndash30 120583gL However no response was observed when exposedtoAlHg Zn sodiumarsenite sodiumazide sodiumdodecylsulfate or pentachlorophenol It was suggested that HSP58abundance might act as a biomarker of toxicity exposure

A number of studies conducted on mollusks mostlyon mussels for example Sanders and Martin [41] reportedelevated levels of HSP60 and HSP70 in mussels and fishtissue collected from polluted areas The collection of dataon sediment and water chemistry from the sampling sitesand on contaminant body burdens indicated their exposureto contaminants was long-term This study suggested thatHSP accumulation might provide a method of quantifyingadverse biological impacts of exposure to toxicants whenexamining wild populations from contaminated sites Simi-larly another study conducted by Porte et al [42] illustratedstatistically insignificant differences in the total levels ofcytochrome P450 and benzo(a) pyrene hydroxylase activityand significantly induced HSP70 which correlated with thequantities of the PAHs accumulated in mussels Mytilusgalloprovincialis (collected from sites polluted with aliphaticand polycyclic aromatic hydrocarbonsmdashPAH) Again it wassuggested that HSP70 could be used as a biomarker inMytilus galloprovincialis against PAH toxicity Another studyin mollusks considered HSP70 as biomarkers (in gill mantleand digestive gland) and the reason of survival of Macomanasuta (clam) in response to heavy metals (Ni Cr and Cu)and trace organic pollutants (like PAH and organochlorinepesticidesmdashaldrin and DDT and its metabolites DDD andDDE) exposure Pearson and Spearman correlation analysisrevealed that mortality and HSP70 in gills were significantlycorrelated with tissue concentrations of DDT andor itsmetabolites [43]




2 SeO2 lindane


2and SeO

2were found


2O2 and antioxidant








2



2 Cd (NO

3)2




4at doses



























2H2in astroglial











10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00Cd Hg As

(a)

10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00

Aslowastlowast

Aslowast


(b)


0

100

200

300

400

500

120583g

g pr

otei

n

ZnCd

Non

treat

ed

Zn (1

00120583

M1

h)

Cd

(60120583

M1

h)

Hea

t (42

C1h

)

Hea

t+Zn

Hea

t+C

d

(a)

Fold

activ

atio

n

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12

Zinc (100120583M)Cadmium (60120583M)


+ +

+ + +

++++++

+++

+ + + +

+ +minus

minus


minus minus


minus minus minus

minus


(b)


2or 60120583M CdCl







06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM


CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15




0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)


C NA 27 C NA + 27C




41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




2 SeO2 lindane


2and SeO

2were found


2O2 and antioxidant








2



2 Cd (NO

3)2




4at doses



























2H2in astroglial











10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00Cd Hg As

(a)

10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00

Aslowastlowast

Aslowast


(b)


0

100

200

300

400

500

120583g

g pr

otei

n

ZnCd

Non

treat

ed

Zn (1

00120583

M1

h)

Cd

(60120583

M1

h)

Hea

t (42

C1h

)

Hea

t+Zn

Hea

t+C

d

(a)

Fold

activ

atio

n

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12

Zinc (100120583M)Cadmium (60120583M)


+ +

+ + +

++++++

+++

+ + + +

+ +minus

minus


minus minus


minus minus minus

minus


(b)


2or 60120583M CdCl







06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM


CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15




0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)


C NA 27 C NA + 27C




41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



4at doses



























2H2in astroglial











10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00Cd Hg As

(a)

10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00

Aslowastlowast

Aslowast


(b)


0

100

200

300

400

500

120583g

g pr

otei

n

ZnCd

Non

treat

ed

Zn (1

00120583

M1

h)

Cd

(60120583

M1

h)

Hea

t (42

C1h

)

Hea

t+Zn

Hea

t+C

d

(a)

Fold

activ

atio

n

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12

Zinc (100120583M)Cadmium (60120583M)


+ +

+ + +

++++++

+++

+ + + +

+ +minus

minus


minus minus


minus minus minus

minus


(b)


2or 60120583M CdCl







06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM


CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15




0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)


C NA 27 C NA + 27C




41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















2H2in astroglial











10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00Cd Hg As

(a)

10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00

Aslowastlowast

Aslowast


(b)


0

100

200

300

400

500

120583g

g pr

otei

n

ZnCd

Non

treat

ed

Zn (1

00120583

M1

h)

Cd

(60120583

M1

h)

Hea

t (42

C1h

)

Hea

t+Zn

Hea

t+C

d

(a)

Fold

activ

atio

n

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12

Zinc (100120583M)Cadmium (60120583M)


+ +

+ + +

++++++

+++

+ + + +

+ +minus

minus


minus minus


minus minus minus

minus


(b)


2or 60120583M CdCl







06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM


CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15




0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)


C NA 27 C NA + 27C




41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00Cd Hg As

(a)

10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00

Aslowastlowast

Aslowast


(b)


0

100

200

300

400

500

120583g

g pr

otei

n

ZnCd

Non

treat

ed

Zn (1

00120583

M1

h)

Cd

(60120583

M1

h)

Hea

t (42

C1h

)

Hea

t+Zn

Hea

t+C

d

(a)

Fold

activ

atio

n

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12

Zinc (100120583M)Cadmium (60120583M)


+ +

+ + +

++++++

+++

+ + + +

+ +minus

minus


minus minus


minus minus minus

minus


(b)


2or 60120583M CdCl







06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM


CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15




0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)


C NA 27 C NA + 27C




41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00Cd Hg As

(a)

10120583m20120583m40120583m

Hsp70

i(ng

120583g

prot

ein)

10

05

00

Aslowastlowast

Aslowast


(b)


0

100

200

300

400

500

120583g

g pr

otei

n

ZnCd

Non

treat

ed

Zn (1

00120583

M1

h)

Cd

(60120583

M1

h)

Hea

t (42

C1h

)

Hea

t+Zn

Hea

t+C

d

(a)

Fold

activ

atio

n

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12

Zinc (100120583M)Cadmium (60120583M)


+ +

+ + +

++++++

+++

+ + + +

+ +minus

minus


minus minus


minus minus minus

minus


(b)


2or 60120583M CdCl







06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM


CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15




0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)


C NA 27 C NA + 27C




41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





06 12 06 12 06 12

SE SE + HM HM

SE SE + HM HM

06 12 06 12 06 12SE SE + HM


CC

C C

CC

C

25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

SE SE + HM HM25 5 25 5 25 5

S1

S2

S3

07 15 07 15 07 15SE SE + HM HM07 15 07 15 07 15

SE SE + HM HM07 15 07 15 07 15




0

25

50

75

100

125

150

2 2 + 1 2 + 24 4 4 + 1 4 + 24

Time (hr)

CuZnCu + Zn

Con

trol (

)


C NA 27 C NA + 27C




41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


41 61 81 125

Station

0

20000

40000

60000

80000

100000

Hsp70

(rel

ativ

e den

sity)



0

20

40

60

80

100

Mor

talit

y (

)

5

10

15

25






2





















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






4 NiSO

4 CuCl

2





2and NiSO




2level


4and NiSO

4but not by CuCl

2and








2[126]






5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



5 Closing Remarks







Acknowledgments


References

























































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Review Article Synergistic Effects of Toxic Elements …downloads.hindawi.com/journals/bmri/2014/564136.pdfDepartment of Environmental Sciences, COMSATS Institute of Information Technology,