the accumulation of metals in the environment , stemming from their origination in the earth 's crust , as well as from anthropogenic sources , creates the potential for significant human exposures and subsequent health hazards .
deleterious metal - induced health effects , including carcinogenesis and neurodegeneration , have been reported in all body systems , with exposure stemming from multiple sources , including contact with contaminated food , water , air , or soil .
the particular metals considered in this review , antimony , arsenic , cadmium , manganese , mercury , silver , and uranium , are among the classes of essential nutrients , as is the case with copper and manganese , as well as the nonessential , naturally occurring metals , such as arsenic , cadmium , and mercury , all of which can induce toxicity depending on the concentration level and exposure duration . over time
, organisms have developed protective mechanisms to deal with metal exposure , most of which function in one of three ways : ( 1 ) decreasing the uptake of the metal , ( 2 ) stimulating the expulsion of the metal , or ( 3 ) activating the organism 's general stress response mechanisms .
metals can disrupt homeostasis by generating oxidative stress , inhibiting enzyme activity , impairing dna repair , and disrupting protein binding and normal cellular function , including proliferation , cell cycle progression , and apoptosis     [ 14 ] . elucidating the mechanism(s ) of metal detoxification
has been difficult due to the complexity of mammalian systems and the reductionist approach inherent to cell culture systems .
the model organism , caenorhabditis elegans ( c. elegans ) , offers the advantage of an in vivo system that is less complex than the mammalian system while still sharing high homology . c. elegans possess ~60%80% of human genes   and contain conserved regulatory proteins     [ 68 ] .  
this soil nematode has been used in a number of toxicity studies due to its well - characterized genetic , physiological , molecular , and developmental stages .
some of the advantages afforded by the c. elegans model system are small size ( ~1.5  mm adult ) , short lifespan ( ~3 weeks ) , and rapid lifecycle ( ~3 days )     [ 810 ] . at adulthood ,
a single c. elegans hermaphrodite is capable of producing ~300 progeny . c. elegans are hermaphrodites , but approximately 1% of c. elegans are male , allowing for genetic experimentation .
the nematode 's small genome and relative anatomical simplicity ( less than 1000 cells ) contribute to the appeal of this model system for genetic manipulation .
in addition , the use of rna interference ( rnai ) and chromosomal deletion in worms has provided valuable information regarding the increased sensitivity of mutant strains to metal toxicity     [ 1214 ] .
maintenance of nematode strains is relatively simple ; they grow on bacteria - seeded plates and can be maintained at 20c .
strains can also be frozen indefinitely , easily allowing for the accumulation of large stocks of worms . c. elegans provide the researcher with a uniquely powerful model , as the worm 's translucent body allows for the in vivo visualization of fluorescently labeled individual cells and proteins .
the in vivo   c. elegans model system is especially valuable for the investigation of metal detoxification and is particularly amenable for examining gene - environment interactions , albeit with a few considerations to take into account ( table 1 ) .
several toxicity endpoints are readily detected and well documented in the nematode , including mortality , lifespan , reproduction , and feeding     [ 1719 ] .
acute toxicity can also be assessed in the nematode through behavioral endpoints , such as locomotive behavior , head thrashing , body bending , and other basic movements     [ 2024 ] .
recently , the role of c. elegans as a biomonitor in environmental risk assessment has also been explored     [ 2528 ] .    
several cellular systems such as the glutathione ( gsh ) , metallothioneins ( mts ) , heat shock proteins ( hsps ) , as well as various pumps and transporters work in concert to detoxify and excrete metals .
it remains to be established whether the knockdown or overload of one detoxifying system upregulates other compensatory mechanisms . in this review ,
we offer a brief summary of the ways in which the c. elegans model has shed novel insights on the various mechanisms of metal detoxification .
metals considered herein are antimony ( sb ) , arsenic ( as ) , cadmium ( cd ) , lead ( pb ) , mercury ( hg ) , silver ( ag ) , and uranium ( u ) .
glutathione ( gsh ) is a cysteine - containing tripeptide , consisting of glutamic acid , cysteine , and glycine and is found in most life forms .
gsh possesses antioxidant properties , since the thiol group of cysteine is a reducing agent and can be reversibly oxidized ( gssg ) and reduced ( gsh ) .
gsh is maintained in the reduced form by the enzyme , glutathione reductase ( gr ) , and functions by reducing other metabolites and enzyme systems .
glutathione peroxidase ( gpx ) catalyzes the oxidation of gsh to gssg in the presence of ros ( figure 1 ) .
gssg can be converted back into gsh via gr and the conversion of nadph to nadp+     .
proton - translocating mitochondrial nicotinamide nucleotide transhydrogenase ( nnt ) catalyzes the reduction of nadp+ by nadh and is an important source of nadph , as has been demonstrated using nnt-1 deletion mutants     .
glutathione s - transferases ( gsts ) can catalyze the conversion of gsh to gs , which can then form complexes with various xenobiotics to facilitate excretion .
these enzymes are found at particularly high levels in the liver , and gsh is typically the most abundant sulfhydryl - containing compound in cells .
additionally , gsh is known to offer protection against metal - generated ros by binding free radicals .
gshs have been reported to increase , decrease , or remain constant after exposure to metals     [ 5557 ] . the gsh system is found in animals , plants , and microorganisms . c. elegans express approximately 50 gsts .
additionally , phytochelatins ( pcs ) , a family of metal - inducible peptides synthesized enzymatically from gsh by pc synthase ( pcs ) in the presence of heavy metal ions , have been identified in c. elegans     .
although pcs are synthesized from gsh , they are broadly classified as class iii metallothioneins and have been shown to be important in the detoxification of heavy metals     .  
liao and yu   investigated the involvement of the gsh system in response to inorganic arsenic exposure .
results confirmed that oxidative stress plays a role in arsenic - induced toxicity by mutating glutamylcysteine synthetase ( gcs ) ( gcs-1 ) , the rate - limiting enzyme in gsh synthesis , in worms .
the gcs-1 loss - of - function strain demonstrated hypersensitivity to arsenic exposure in lethality testing , as compared to wild - type animals , an effect that was rescued by the addition of gsh to the medium , indicating that these enzymes are crucial for mediating arsenic - induced toxicity .  
furthermore , helmcke and aschner   reported a significant increase in fluorescence in a gst-4::gfp strain following both acute and chronic exposure to mehg .
it was also demonstrated that knockout gst-4 worms did not display greater sensitivity to mehg than did n2 wild - type worms .
gsh levels were found to be increased in worms subjected to acute exposure , whereas worms subjected to chronic exposure exhibited depleted levels of gsh . in their hormetic model , helmcke and aschner demonstrated an increase in gst-4::gfp expression after low concentration , acute exposure , a finding which indicates that gst-4 may be involved in the hormetic response to mehg . taken together , the data from these studies suggest that gst-4 contributes to the response to mehg exposure , but that knockdown of this gene does not affect with overall lethality .   in a study examining the role of pcs in the elimination of cadmium , vatamaniuk and colleagues      reported that the c. elegans pcs-1 gene encodes a functional pc synthase critical for heavy metal tolerance . using double stranded rnai against pcs-1 , this group showed that , although the progeny of worms injected with the dsrnai and exposed to cadmium ( 525  m ) managed to reach adulthood , these worms were small , necrotic , sterile , and had a much shorter lifespan than did the wild - type controls     . after being exposed to 50 and 100  m concentrations of cadmium , the pcs-1 worms arrested at the l2l4 stage were necrotic and died by day 6 .
the results were definitively dependent on the presence of cadmium , because pcs-1-deficient worms , in the absence of cadmium exposure , responded identically to the wild - type controls     .  
hughes and colleagues      examined metabolic profiles following cadmium exposure in phytochelation synthase-1 ( pcs-1 ) mutants .
results from these studies showed that the primary response to low levels of cadmium is the regulation of the transsulfuration pathway , due to the fact that cadmium exposure caused a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3     .
these results were corroborated by additional studies which demonstrated that pcs-1 mutants were an order of magnitude more sensitive to cadmium than were the metallothionein mutants .
furthermore , the mt - pcs-1 triple mutant was found to display an additive sensitivity toward cadmium     .
metallothioneins ( mts ) belong to a family of cysteine - rich low - molecular - weight metal - binding proteins ( mw 3,50014,000 da ) involved in metal detoxification and homeostasis .
mts bind both metals of physiological importance such as copper and zinc , as well as xenobiotics including arsenic , cadmium , mercury , and silver .
the binding of these metals occurs via the interaction of the cysteine residues with thiol groups .
binding of metals by mts may be transient , as mts are capable of rapidly releasing metal ions .
the protective roles of mts can be ascribed to their three primary functions : ( 1 ) metal homeostasis , ( 2 ) heavy metal detoxification , and ( 3 ) protection from oxidative stress . additionally , these proteins have been identified as contributors to the hormetic response     [ 39 , 61 ] .  
mammals express four known metallothionein isoforms ( mt - i , mt - ii , mt - iii , mt - iv )     .
mt - i and mt - ii are expressed in almost all tissues and have been best characterized with regard to their protection of the brain     .
mt - iii is especially enriched in the central nervous system , although its role has not yet been clearly defined     .
mt - iv is most abundantly expressed in the stratified squamous epithelia     [ 6466 ] .
mt expression has been shown to be induced under stressful cellular conditions such as exposure to cytokines , glucocorticoids , reactive oxygen species ( ros ) , and metal ions .
mts can bind directly and sequester the toxicant ; they also can provide protection by acting as antioxidants   ( figure 1 ) .
further , mts can limit apoptosis and promote the survival of mitochondrial dysfunctional cells by serving as highly efficient reducing elements against reactive oxygen species ( ros ) . c. elegans contain two distinct isoforms of mts , known as mtl-1 and mtl-2 , which can be induced in response to exposure to various metals     .   
jiang and colleagues examined the effects of mts on depleted uranium ( du ) in c. elegans .
results from their study showed that mtl-1 was an important factor in uranium accumulation in c. elegans as knockouts displayed increased cellular accumulation .   in a study investigating lead and methylmercury toxicity
, ye and colleagues   demonstrated the involvement of mts in affording a protective cross - adaptation response to neurobehavioral toxicity .
this endpoint was assessed by observing behavioral alterations ( head thrashing and body bending ) in worms that were exposed during the l2 phase to either pb or mehg .
the study was conducted in conjunction with mild heat shock , wherein pretreatment of the larva with heat shock prevented the neurobehavioral deficits and the stress response at lower concentrations ( 50100  m ) but not at higher concentrations ( 200  m ) .
additionally , mild heat shock coupled with exposure to a low concentration of either metal was found to induce mtl-1 and mtl-2 promoter activity and gfp gene expression , results that were not observed in either the metal - exposed or heat - shocked cohort alone .
finally , the overexpression of mtl-1 or mtl-2 at the l2 stage was shown to significantly repress neurobehavioral toxicity , suggesting that the accumulation of mt protein is necessary to confer the protective response to the toxicant .  
similarly , helmcke and aschner   reported that mtl knockouts displayed increased lethality upon exposure to mehg .
this group also demonstrated increases in mtl-1::gfp fluorescence in response to acute mehg exposure at the l1 stage ; however , chronic mehg exposure produced no change in florescence .
their results indicate that mtl-1 is important in mediating mehg toxicity and that the effects occur in a concentration- and time - dependent manner .
meyer and colleagues   examined the aggregation of silver nanoparticles ( agnps ) in wild type and mtl-2   c. elegans .
results from these studies showed that the mtl-2 strain displayed greater agnp sensitivity than did the wild - type controls .
these data indicate that there may be a differential preference for mtl-1 over mtl-2 depending on the particular metal to which an organism is exposed .   in a 2004 study by swain and colleagues ,
using gfp - expressing transgenes , mt - null alleles , and the rnai knockdown of mts , this group demonstrated that cadmium but not copper or zinc was able to influence a concentration - dependent , temporal transcription response .
cadmium exposure caused a reduction in body size , generation time , brood size , and lifespan , effects that were magnified in the mt knockdown worms .   
hughes and colleagues      studied metabolic profiles using proton nmr spectroscopy and uplc - ms following cadmium exposure in single and double mtl knockouts .
results showed that the metallothionein status did not influence the metabolic profile in cadmium - exposed or unexposed worms .
the primary response to low levels of cadmium was the regulation of the transsulfuration pathway , due to the fact that cadmium exposure resulted in a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3     .
these results were corroborated by data showing that pcs-1 mutants ( phytochelation synthase-1 ) were an order of magnitude more sensitive to cadmium than were mt mutants .
further , an additive sensitivity toward cadmium was observed in the mt - pcs-1 triple mutant     .  
a study by bofill and colleagues   examined zinc and cadmium toxicity ; results indicated differential metal binding behavior for mt-1 as compared to mt-2 .
specifically , the mt-1 isoform showed optimal behavior when binding zn , and mt-2 showed optimal behavior when binding cd .
accordingly , it was hypothesized that , due to its induction following cd exposure , mt2 is primarily responsible for detoxification , whereas mt1 possesses some degree of constitutive expression and is , therefore , primarily involved in physiological metal metabolism ( e.g. zinc ) .
these findings were corroborated by additional studies which showed that mt - knockout worms exhibited significantly decreased levels of overall fitness after the knockout of mt1 than after mt2 knockout .
further , both mt isoforms displayed a clear preference for divalent metal ion binding as opposed to copper coordination , likely due to the presence of histidines in the mts .   using
both in   vitro and in vivo models , zeitoun - ghandour and colleagues      examined zinc and cadmium exposures and showed different roles for mtl-1 and mtl-2 . both isoforms
their affinities and stoichiometries were measured , and both isoforms displayed equal zinc- binding ability ; however , mtl-2 had a higher affinity for cd than did mtl-1 .
these experiments were repeated in vivo in mtl-1 , mtl-2 , and double knockouts following exposure to 340  m zn or 25  m cd .
zinc levels were found to be significantly increased in all knockout strains , but mtl-1 knockout worms demonstrated the most acute level of sensitivity .
however , cadmium accumulation was found to be the highest in the mtl-2 knockout and double mutant strains .
additional studies assessed metal speciation , and results indicated that o - donating ligands play an important role in maintaining zinc levels , independent of metallothioneins status .
further , cadmium was shown to interact with thiol groups , and cd speciation was significantly different in the mtl-1 strain when compared with both the mtl-2 strain and the double knockout strain , suggesting that the two mt isoforms have distinct in vivo roles     .
the authors suggested that mts are not functioning as metal storage proteins but , rather , are mediating the accumulation and excretion of metals . a follow - up study , showed in vitro evidence for the partitioning of zinc and cadmium with different metallothionein isoforms .
employing electrospray ionization mass spectrometry ( esi - ms ) to directly observe zinc and cadmium binding preferences , more cadmium ions were found to be preferentially bound to mt-2 than to mt-1 ; however , cd was shown to be capable of inducing both isoforms .
finally , partitioning was also demonstrated to be more effective at lower cd  :  zn ratios .   
using daf-2 ( insulin receptor - like protein ) and age-1 ( phosphatidylinositol-3-oh kinase catalytic subunit ) mutants , barsyte et al .     
examined the expression of mt genes under noninducing conditions and after exposure to cadmium and copper .
they reported that mt-1 mrna levels were significantly higher in daf-2 mutants compared to both age-1 mutants and wild - type worms under basal conditions .
exposure to cadmium treatment resulted in a three - fold induction of mt-1 and a two - fold induction of mt-2 mrna in daf-2 mutants as compared to wild - type controls .
copper did not induce mt-1 or mt-2 mrna expression in any of the strains tested     .  
collectively , these studies show differential metal preferences for one mt isoform over another depending on the metal to which an organism is exposed .
most significantly , these studies indicate that the mts play crucial roles in metal detoxification ( table 2 ) .
indeed , mts have been associated with a protective effect in cells under numerous states of disease and stress .  
interestingly , serum mt levels of cancer patients are three times higher than those of control patients .
a study conducted in denmark revealed the increased expression of mt-1 and mt-2 mrna and protein in many human cancers such as breast , kidney , lung , nasopharynx , ovary , prostate , salivary gland , testes , urinary bladder , cervical endometrial skin carcinoma , melanoma , acute lymphoblastic leukemia , and pancreatic cancers .
this information is of particular import given the use of metals for the treatment of certain cancers , for example , arsenic as treatment for promyelocytic leukemia .
it is interesting to postulate that higher levels of mts may enhance the efficacy of metal therapeutic agents or , conversely , may lead to resistance to such therapies .
understanding the factors that modulate mt expression will allow for the improved understanding of metalloid toxicity and will provide more effective therapeutic approaches to metalloid - based chemotherapy .
there are a number of pumps and transporters that have been implicated in metal detoxification .
these include atp - binding cassette ( abc ) transporters , such as the multidrug resistance - associated protein ( mrp ) as well as two members of the p - glycoprotein subfamily ( pgp-1 and pgp-3 ) , which have been shown to contribute to heavy metal tolerance through the use of c. elegans deletion mutants . in c. elegans
, there are approximately 60 genes encoding abc transporters , and these genes make up the largest family of transporters     . c. elegans have four mrp homologues      and fifteen pgp homologues     .
the pgps are ubiquitously expressed and are most abundantly found in the apical membranes of the gut and in the excretory organs of the worm .
pgp-2 is expressed in the intestine and is required for the acidification of lysosomes and lipid storage ; pgp-1 and -3 contribute to heavy metal and drug resistance     .  
tseng and colleagues   investigated the arsa protein - mediated detoxification of the metalloids , as(iii ) and sb(iii ) .
bacterial arsa atpase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite and antimonite . in this study ,
wild - type and asna-1-mutant nematodes were evaluated for as and sb response and toxicity .
the asna-1 gene of c. elegans was found to be stimulated by as(iii ) ; further , sb(iii ) was determined to be crucial for establishing tolerance .
although these results occurred in response to as and sb exposure , the ubiquity of the arsa atpase - dependent pathway has not been observed in other species or in response to other metals .
the role of multidrug resistance - associated protein ( mrp ) in arsenite and cadmium toxicity was explored in a study by broeks and colleagues     .
the targeted inactivation of mrp-1 rendered the arsenite  and cd  exposed worms incapable of recovering from temporary exposure to high arsenic and cadmium , whereas the wild - type controls were able to recover .
additionally , worms were also shown to be hypersensitive to arsenite and cd exposures when both mrp-1 and pgp-1 ( p - glycoprotein-1 ) were deleted     .
lastly , no increased sensitivity in response to exposure to antimony was observed in mrp-1-deletion mutants as compared to wild - type controls     .
vatamaniuk and colleagues      characterized the half - molecule abc transporter of the heavy metal tolerance family-1 ( hmt-1 ) subfamily in response to cadmium exposure .
the suppression of hmt-1 expression by rnai was shown to produce punctuate refractive inclusions within the vicinity of the nucleus of the intestinal epithelial cells upon exposure to toxic levels of cadmium     .
similarly , schwartz and colleagues described the c. elegans hmt-1 following exposure to arsenic , copper , and cadmium .
hmt-1 conferred tolerance in response to exposure to all three metals as shown by lethality testing following the knockdown of hmt-1     .  
kurz and colleagues      demonstrated the three - fold induction of pgp-5 following cadmium exposure .
results of this study showed that strong fluorescence was induced in the intestinal cells of pgp-5::gfp worms , where the gfp - encoding gene is under the control of the upstream pgp-5 promoter     .
copper and zinc were also found to be capable of inducing pgp-5 expression in these worms .  
accordingly , it was concluded that pgp-5 is required for establishing full resistance to cadmium and copper .
in addition , the rnai knockdown of tir-1 , an upstream component of the p38 mapk pathway in the pgp-5 transgenic reporter strain , was shown to significantly reduce pgp-5 induction following exposure to cadmium .
however , the double - stranded rna knockdown of erk ( mpk-1 ) and jnk ( med-1 and kgb-1 ) did not affect the induction of pgp-5 in response to cadmium exposure     .  
au and colleagues      studied the divalent - metal transporter ( dmt1 ) following exposure to manganese .
the dmt1-like family of proteins has been shown to regulate manganese and iron in the cell .
the deletion of the three worm dmt1-like genes resulted in differential effects on manganese toxicity .
the deletion of smf-1 and smf-3 increased mn tolerance , whereas the deletion of smf-2 increased mn sensitivity     .
heat shock proteins ( hsps ) are cytosolic molecular chaperones . hsps promote the refolding and repair of denatured proteins and facilitate protein synthesis upon activation by cellular stress     [ 75 , 76 ] .
hsps , particularly those in the hsp70 family , have also been shown to participate in the hormetic response .
hsp70s are atp - binding proteins that convert atp to adp and bind to peptides , thereby , inactivating them and preventing aggregation ( figure 1 ) .
oxidative stress can cause a reduction in cellular atp levels . decreased levels of atp result in the continued prevention of the aggregation of damaged proteins .
the functions of the hsp70 products are mediated by the conserved n - terminal atpase and the c - terminal peptide - binding region     . the human and c. elegans hsp70 genes have a high degree of homology and share a conserved core 
the hsp16 family of stress proteins is produced in c. elegans only under stress conditions     [ 8082 ] . in a study examining the effects of mehg exposure , hsp-4::gfp was measured immediately following the treatment of l1 worms for 30 minutes and l4 worms for 15 hours with this toxicant . after 30 minutes of acute exposure to mehg ,
however , in l4 worms chronically exposed to mehg for 15 hours , hsp-4::gfp was induced .
at the same time point in the chronic treatment paradigm , a four - fold increase in gst-4 fluorescence was detected , but there were no changes in either mtl-1 or mtl-2::gfp expression .   
jones and candido   exposed nematodes to cadmium or mercury and measured feeding behavior . for these studies , transgenic lines containing the promoter sequence for hsp16 genes which regulate the production of e. coli-galactosidase were used . accordingly , to measure stress , levels of this protein were assessed .
results showed that cadmium inhibited feeding behavior significantly but not completely , as a minimal rate of feeding continued at high cadmium concentrations .
further , exposure to cadmium ( 1  ppm ) induced a detectable production of -galactosidase without inhibiting feeding behavior .
the stress response was induced at a concentration of cadmium that was ten times lower than the lc50 .
mercury also was shown to inhibit feeding at concentrations similar to those necessary for the induction of a stress response ; however , the difference in this instance was less than two fold .
the use of c. elegans as an experimental model has produced considerable insight and valuable information regarding the multiple and varied processes of metal detoxification .
conclusive biochemical evidence has indicated that different metals are not handled in the same capacity .
the selectivity and sensitivity of each of these proteins is highlighted in the large body of accumulated research on different metal toxicities as well as various systems of metal detoxification .
however , the overall mechanisms , temporal activation , and interplay between different cell detoxification systems remain elusive .
future studies are necessary in order to enhance our understanding of the complex interplay of multiple - cell detoxification systems in response to exposure to different metals .
the c. elegans model system will be critical for these investigations , as knockouts are easily generated and provide a wealth of information about metal detoxification in a genetically retractable , inexpensive , and in vivo model .
 metals have been definitively linked to a number of disease states . due to 
 the widespread existence of metals in our environment from both natural and anthropogenic sources , understanding the mechanisms of their cellular detoxification is of upmost importance . 
 organisms have evolved cellular detoxification systems including glutathione , metallothioneins , pumps and transporters , and heat shock proteins to regulate intracellular metal levels . 
 the model organism , caenorhabditis elegans ( c. elegans ) , contains these systems and provides several advantages for deciphering the mechanisms of metal detoxification . 
 this review provides a brief summary of contemporary literature on the various mechanisms involved in the cellular detoxification of metals , specifically , antimony , arsenic , cadmium , copper , manganese , mercury , and depleted uranium using the c. elegans model system for investigation and analysis . 
1. Introduction
2. Glutathione
3. Metallothioneins
4. Pumps and Transporters
5. Heat Shock Proteins
6. Conclusion