review article metal species in biology: bottom-up and top...

Hindawi Publishing CorporationISRN ChromatographyVolume 2013 Article ID 801840 21 pageshttpdxdoiorg1011552013801840

Review ArticleMetal Species in Biology Bottom-Up and Top-Down LCApproaches in Applied Toxicological Research

Juumlrgen Gailer

Department of Chemistry University of Calgary 2500 University Drive NW Calgary AB Canada T2N 1N4

Correspondence should be addressed to Jurgen Gailer jgailerucalgaryca

Received 30 January 2013 Accepted 19 February 2013

Academic Editors B K Mandal A Namera Y Qiu and A I Suarez

Copyright copy 2013 Jurgen GailerThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Since the inception of liquid chromatography (LC) more than 100 years ago this separation technique has been developed into apowerful analytical tool that is frequently applied in life science research To this end unique insights into the interaction of metalspecies (throughout this manuscript ldquometal speciesrdquo refers to ldquotoxic metals metalloid compounds and metal-based drugsrdquo andldquotoxic metalsrdquo to ldquotoxic metals and metalloid compoundsrdquo) with endogenous ligands can be obtained by using LC approaches thatinvolve their hyphenation with inductively coupled plasma-based element specific detectorsThis review aims to provide a synopsisof the different LC approaches which may be employed to advance our understanding of these interactions either in a ldquobottom-uprdquoor a ldquotop-downrdquo manner In the ldquobottom-uprdquo LC-configuration endogenous ligands are introduced into a physiologically relevantmobile phase buffer and the metal species of interest is injected Subsequent ldquointerrogationrdquo of the on-column formed complex(es)by employing a suitable separation mechanism (eg size exclusion chromatography or reversed-phase LC) while changing theligand concentration(s) the column temperature or the pH can provide valuable insight into the formation of complexes undernear physiological conditionsThis approach allows to establish the relative stability and hydrophobicity of metal-ligand complexesas well as the dynamic coordination of a metal species (injected) to two ligands (dissolved in the mobile phase) Conversely theldquotop-downrdquo analysis of a biological fluid (eg blood plasma) by LC (eg using size exclusion chromatography) can be used todetermine the size distribution of endogenous metalloproteins which are collectively referred to as the ldquometalloproteomerdquo Thisapproach can provide unique insight into the metabolism and the plasma protein binding of metal species and can simultaneouslyvisualize the dose-dependent perturbation of the metalloproteome by a particular metal speciesThe concerted application of theseLC approaches is destined to provide new insight into biochemical processes which represent an important starting point to advancehuman health in the 21st century

1 Introduction

Based on his work on plant pigments MS Tswett serendip-itously invented adsorption chromatography as a universalanalytical method to separate chemical compounds in 1906[1] After an interregnumofsim40 years the discovery of liquid-liquid partition chromatography by AJP Martin and RLMSynge breathed new life into this ground-breaking separationapproach [2] and wasmdashtogether with the development of thefirst general theory of chromatographymdashawarded the NobelPrize in Chemistry in 1952 [3] Over the intervening yearsliquid chromatography (LC) has matured into one of themost enabling and widely used separation technologies inmodern analytical chemistry [4]This evolution has involvedthe development of stationary phases with progressively

smaller particle diameters [5] the miniaturization of columndimensions [6 7] and resulted in the beginning of highperformance liquid chromatography (HPLC) in the early1970s [8] The need to further increase the column efficiencyrequired the utilization of even smaller particle diameters andculminated in the introduction of ultra performance liquidchromatography (UPLC) in 2004 [9] In the context of sepa-ration science these LC techniques allowed for the first timeto analyze complicated biological fluids such as blood plasmafor proteins [10ndash12] or small molecular weight metabolites[13 14] and put analytical chemists in the unique positionto tackle biological complexity Advances in LC technologyhave therefore played a pivotal role in setting the stage formodern proteomics [15] metabonomicsmetabolomics [16ndash18] and systems biology research [19] Concomitant with

2 ISRN Chromatography

these technological developments a variety of separationmechanismswere establishedwhich allowedLC to become anindispensable tool for the purification of active pharmaceuti-cal ingredients (APIs) such as blood coagulation factors fromplasma and recombinant proteins from bacterial lysates [20]Reflecting on this brief historic trajectory of LC and consid-ering the crucial role that this versatile separation techniquehas served in the context of food analysis and safety [21 22]it is tempting to equate its significance in contemporary lifescience research with the role that the microscope plays inbiology the spectroscope has in chemistry and the Hubbletelescope holds in astronomy

Against this background it is interesting to note thatwithin the specific research area of ldquometal species in bio-logyrdquomdashloosely defined as the elucidation of health relevantinteractions between environmentally abundant toxic met-als or metal-based drugs with ligands within organismsmdashapproaches which involve the hyphenation of LC separationswith inductively coupled plasma- (ICP-) based detectors [23]have also become more frequently applied over the past10 years [24] To understand this trend one may identifythree contributing reasons The first one is the capability ofLC-based approaches to provide fundamentally new insightinto the complex interactions between metal species andendogenous ligands in a variety of ways [25ndash28] This notionconstitutes themain focus of this paper andwill be elaboratedon in detail later The second reason is the realization thatthese interactions provide an important starting point topotentially unravel the mechanisms which link the exposureto certainmetal speciesmdashunwitting in the context of environ-mentally abundant toxic metals [29ndash31] and deliberate withregard to intravenously administered metal-based drugsmdashwith adverse human health effects [32 33] Thus the applica-tion of LC-based approaches has potential to provide insightinto the etiology of hitherto elusive metal species relateddisease processes [34 35] The third reason is the inherentability that LC-based approaches offer in terms of developingmore effective antidotes against specific metal species toultimately advance human health [36 37] As a result theapplication of LC-based approaches will likely continue toplay an important role in ldquometal species in biologyrdquo researchin the years to come Given that several excellent reviewshave detailed either their historic development [38] or variousrecent methodological advances [24 27 28 39ndash41] the focusof the present one is to illustrate the different ways in whichLC has been employed as a tool to gain insight into thecomplex interactions that unfold after metal species entermammalian organism Furthermore particular emphasis willbe given to studies that have attempted to specifically probecertain aspects of these interactions under near physiologicalconditions It is hoped that the presented synopsis maycontribute to foster progress toward tackling the by farbiggest adversary in the context of revealing the bioinorganicprocesses which link the exposure of mammals to certainmetal species with disease processes biological complexity[19]

Within the scope of elucidating the role that metalsplay in health and disease one may differentiate betweenthree distinguished categories essential metalsmetalloids

toxic metalsmetalloids and pharmacologically active metalcompoundsdrugs Owing to the central role that essentialmetals play in health [42] extensive research has been andcontinues to be directed toward the elucidation of theexceedingly complex homeostatic regulation mechanismswhich maintain their tissue concentrations within a narrowrange throughout life [43ndash49] Comparatively less howeveris known about the role that environmentally abundant toxicmetals may play in the etiology of human disease processes[34 35 50 51] and progress towards the development ofmoreeffective chelating agents to efflux these from intoxicatedorganisms (without harming them) is slow [52] In a similarvein the biomolecular basis of the side effects that areassociated with clinically used metal-based drugs (and oftenlimit the treatment of patients) remains fragmentary [53ndash55]and surprisingly little is known about the biochemicalmecha-nism(s) by which so-called ldquoameliorating agentsrdquomitigate theside-effects of metal-based drugs in patients [37 56 57]

From a conceptual point of view the effect that any metalspecies exerts in an organism above a minimum dose andlength of exposuremdashthis includes acute toxicity and chronictoxicitymdashmay be investigated by two principle approachesThe traditional ldquobottom-uprdquo or ldquoreductionisticrdquo approach isbased on the isolation of the toxicological problem fromthe organism followed by in depth studies of the presumedunderlying biochemical mechanism(s) using appropriatetechniques For example to explain why the ingestion ofan improperly prepared puffer fish is fatal in sim60 of thereported poisoning cases (lethal dose in adults 10120583gkg bodywt) one may want to uncover the molecular mechanismof action of the responsible toxin tetrodotoxin To do sohowever one needs to identify first a putative biological targetmolecule Since early studies indicated that tetrodotoxinmay adversely affect voltage-sensitive Na+ channels [58]definitive proof of this hypothesis required detailed studiesinto the binding of tetrodotoxin to this putative Na+ channelprotein target [59] Importantly this approach can onlybe employed if some a priori information about a likelymolecular mechanism of action (eg inhibition of voltage-sensitive Na+ channels) is available In contrast the ldquotop-downrdquo or ldquosystems biologyrdquo approach attempts to directlyanalyze complex biological samples with an appropriateanalytical technique to unravel the molecular mechanism ofaction of a particular toxin therein [19 60] This alternativeapproach hinges on identifying an analytical technique thatis capable of detecting a toxin-induced change in a likelybiological compartment (eg blood plasma [26 61]) or to inferthat a relevant biotransformation of the toxin of interest hasoccurred in a particular organ (eg the liver) by analyzing anappropriate effluent stream (eg bile [62ndash65]) Importantlythe ldquotop-downrdquo approach offers the advantage that no apriori information about a likely mechanism of toxicity isrequired and that fundamentally new insights into hithertounknown mechanisms of action may be obtained Eventhough the ldquobottom-uprdquo approach has been in use longer thanthe ldquotop-downrdquo approach the latter is receiving progressivelymore attention lately This may be attributed to the fact thatorganisms are comprised of diverse anatomical structureswhich maintain intricately intertwined metabolic networks

ISRN Chromatography 3

+H3N

O

OH

HN

N

SH

CO2minus

CO2minus

(a)

+H3N

SH

CO2minus

(b)

O

HN

SH

CO2minus

(c)

+H3N

+H3N

OO

O

O

HN

HN

HN

As

S

SSeminus

CO2minusCO2

minus

CO2minus

CO2minus

NH

(d)

+H3N

+H3N

O

O

OO

HN

HN

HN

S

S

CO2minusCO2

minus

CO2minus

CO2minus

NH

(e)

CO2minus

CO2minus

SH

SH

(f)

CO2minus

CO2minus

CO2minusNN

N

minusO2C

minusO2C

(g)

SH

SH

SO3minus

(h)

CO2minusAs+

H3CH3C

CH3

(i)

H3N

H 3NPt Cl

Cl

(j)

OO

OO

H3N

H3NPt

C

C

(k)

O

OO

O

Pt

NH2

NH2

(l)

O O

Sminus

S Ominus

(m)

Figure 1 Structures of (a) L-glutathione (b) L-cysteine (c) N-acetyl-L-cysteine (d) the seleno-bis(S-glutathionyl) arsinium ion (e) oxidizedL-glutathione (f) meso-23-dimercaptosuccinic acid (g) diethylenetriaminepentaacetic acid (h) 23-dimercapto-1-propanesulfonic acid (i)arsenobetaine (j) cis-platin (k) carboplatin (l) oxaliplatin and (m) sodium thiosulfate

(ldquothe sum is more than its partsrdquo) [66] whose perturbationby a toxin requires appropriate visualization tools [67 68]Despite this trend it needs to be stressed that it does notmatter in principle whether one starts ldquobottom-uprdquo or ldquotop-downrdquo to solve a particular toxicological problem at handsince the overall goal is to obtain new insight by whatevermeans [69] Ultimately the information that is obtainedby either approach should corroborate that of the other tovalidate a novel toxicological mechanism of action

Based on the overall focus of this paper to stress theunique niche that LC-based methodologies occupy in ldquometalspecies in biologyrdquo research it is divided into four partsThe first one provides an overview of ldquobottom-uprdquo LCapproaches which have provided insight into the reactionof toxic metals with relevant endogenous ligands Part twosummarizes the results of ldquobottom-uprdquo LC approaches whichhave contributed to better understand the interaction ofmetal-based drugs with individual ligands Part three reviewsldquotop-downrdquo LC approaches which have been applied to studythe interaction of toxic metals with blood plasma and theirabstraction from proteins by chelating agents The last part

aims to highlight investigations that have employed ldquotop-downrdquo LC approaches to investigate themetabolism ofmetal-based drugs in blood plasma andor cells Owing to thisrather broad overall scope a few select examples whichillustrate in what way each LC approach has allowed insightwill be presented To further stress the role that LC playedin these studies the element-specific detector(s) that wereemployed will only be identified if it was deemed to be usefulfor the reader

2 Interaction of AbsorbedInjected MetalSpecies with Endogenous Ligands

In principle the toxicity of any metal species may beattributed to interactions with endogenous target ligandsthat occur either in the bloodstream [70] in case of agastrointestinally absorbed metal one must also considermetal-amino acid complexes since these may be the speciesthat are absorbed into the bloodstream [71] with regardto the metal-based drugs cis-platin (Figure 1(j)) or carbo-platin (Figure 1(k)) only their hydrolysis product(s) may


appreciably interact with endogenous ligands [53] andorwithin organs [72 73] It is also important to note that thebiological response of an organism to any givenmetal speciesis dose-dependent (ldquothe dose makes the poisonrdquo) and that adifferent set of endogenous ligands appear to be targeted inacute [74] as opposed to chronic AsIII toxicity [64] Bloodplasma alone may contain up to 10000 potential protein lig-ands [75] Given the large number of possible metal species-ligand interactions and considering that these occur in water[76]mdashwhich is considerably more complicated to predictfrom a thermodynamic point of view than interactionsthat take place in aprotic solvents [77]mdashit is unsurprisingthat they remain rather poorly understood with regard toenvironmentally abundant toxic metals [35 78] as well asmetal-based drugs [79ndash81]

In the bloodstream the ligand(s) which a particular metalspecies will bind to is determined by (a) its affinity foreach available binding site (eg on plasma proteins externalmembrane receptors andor on cell membranes [85]) and(b) the relative concentration of all binding sites [86] Thisbinding event often involves the coordination of the metalspecies to nitrogen oxygen and sulfur donor atoms onpeptides and proteins [87] In principle a metal species-ligand interaction may be either reversible (noncovalentbonding) irreversible (covalent bonding [82]) or involve anactual chemical reaction (eg cis-platin reduces S-S bonds inhuman serum albumin HSA [88]) Reversible interactionsbetween a metal species and endogenous ligands in thebloodstream play a central role in the distribution of theformer within the organism as they willmdashtogether with therelative availability of uptake mechanisms into organs [89]mdashdetermine the toxicological target organ(s) [35 90] Irre-versible interactions between a metal species and a particularligand in the bloodstreammdashwhich are the result of a chemicalreaction [91]mdashare of particular relevance [32 84 91] as theycan exert far-reaching systemic adverse health effects at theorgan level if the ligand is an essential ultratrace element suchas Se (Figure 2 red arrows) Once the metal species andorits metabolites enter(s) a particular organ cell processes (a)and (b) will unfold anew within the cytosol albeit with adifferent set of proteins and at reducing conditions sinceall mammalian cells contain between 01ndash10mM glutathione(GSH Figure 1(a)) [92] The intracellularly formed metal-ligand complex may then either disrupt vital biochemicalprocesses in the cytosol [93 94] or enter subcellular compart-ments (eg mitochondria lysosomes and nuclei [95]) where(a) and (b) will unfold and may result in cell death [96ndash98](Figure 2 dashed red arrows)

Although interactions of metal species with endogenousligands that occur in the bloodstream are in principle as tox-icologically relevant as those that unfold within organs [32]the former may be more important than we currently thinkThis is because irreversible interactions in this central biologi-cal compartmentmay result in systemic toxicity (Figure 2 redarrows) whereas reversible interactions will be involved indetermining the target organ (Figure 2 dashed red arrows)In human blood plasma the 20 most abundant plasmaproteinsmdashthey comprise sim97 of the total protein [99] and

Organism

Blood

Cellsorgans

Subcellularmechanisms

Pathways

Proteins

Genes

Toxic metal species

Higher-levelcontrols of

geneexpression

Proteinmachinery

readsgenes

Higher-leveltriggers of

cellsignalling

Figure 2 Conceptual depiction of the reductionist causal chain toconstruct an organism (black arrows) as well as the systems biologyview of downward causation (blue arrows) adapted from [69]Thereare twoprincipleways bywhich a toxicmetal species that has enteredthe bloodstream may exert adverse health effects Red arrows ametal species interacts with an endogenous target ligand in thebloodstream (eg formation of compounds with As-Se and Hg-Sebonds [32 82] binding of cis-platin derived hydrolysis productswithHSA block the Zn2+ binding site [83] or the displacement of Znfrom the extracellular glycoprotein Cu Zn superoxide dismutase[26]) which may affect cell signalling gene expression andorthe distribution of an essential ultratrace element to organs andtherefore affect the whole organism [84] Dashed red arrows ametalspecies enters a specific cell type where it interacts with intracellulartarget ligands (eg binding of cis-platin derived hydrolysis productsto DNA [55]) to induce apoptosis Note that the red arrows pertainto all organs within an organism (systemic toxicity) whereas thedashed red arrows correspond to the target organ only (selectivetoxicity) In the context of developing more targeted metal-basedanticancer drugs it is therefore critical not only to increase theirselective toxicity (ie their preferential delivery toward cancer cells)but also to minimizeavoid systemic toxicity caused by unintendedinteractions with endogenous ligands in the bloodstream [83]

almost half of them are Cu Fe and Zn-metalloproteins [100]mdashhave so far received most attention as target ligands formetal species By far the most abundant plasma proteinhuman serum albumin (HSA) for example has binding sitesfor Cd2+ [101] cis-platin derived hydrolysis products [80]and numerous other metal species [102] Despite the factthat extensive research has been directed to investigate theinteraction of individualmetal specieswith individual plasmaproteins (ldquobottom-uprdquo approach) comparatively few studieshave attempted to probe the interaction of metal specieswith more than one ligand (which is the norm in biologicalsystems) with whole plasmablood in vitro let alone in vivo(ldquotop-downrdquo approach) A comprehensive understanding ofall interactions that unfold in vivo is particularly challengingsince the biotransformationmetabolism of a metal specieseither in the bloodstream [32 34 35 53 61 84 91] or in


the liver (from which metabolites may be expelled backinto the bloodstream for urinary excretion) may result inspecies that could also bind to particular plasma proteinsover time Considering the logistical challenges that aregenerally associated with the execution of in vivo studies itis not surprising that our understanding of the metabolismof metal species is incomplete Our lack of understandingthe biomolecular basis of the side-effects of intravenouslyadministered metal-based drugs may be similarly attributedto the comparatively few studies that have been conductedto probe their interactions and that of their hydrolysis prod-ucts with extracellular (plasma proteins) and intracellular(cytosolic) proteins [103 104] Ultimately the outcome of anymetal species that has entered a mammalian organism andinteracted with a variety of endogenous ligands will be thatit is either deposited as an insoluble and possibly nontoxiccomplex (eg in case of mercury or arsenic [105ndash107]) orthat the parent metal species together with its metabolites isexcreted in urine as has been reported for Pt-based anticancerdrugs [108] or in feces as is the case for Hg2+ CH

3Hg+ Cd2+

AsIII and Pb2+ [109]

3 Why Use LC to Probe Interactions ofMetal Species with Endogenous Ligands

A large variety of instrumental techniques have been appliedto study interactions between individual metal species andendogenous ligands including heteronuclear NMR spec-troscopy [112] electrospray-ionization mass spectrometry(ESI-MS) [113] extended X-ray absorption fine structurespectroscopy (EXAFS) [113ndash115] and electron paramagneticresonance spectroscopy (EPR) [116] Since none of the afore-mentioned techniques can probe the platinum speciationat micromolar concentrations inside cells [117] howeverthere is a need for methods that are capable of monitoringthe reaction of Pt compounds in biological fluids [118] Inthis context one may ask what specific information LC canprovide in terms of probing the interaction of a metal specieswith endogenous ligandsThis question will be answered anddiscussed later (see Sections 4ndash8) In addition what practicaladvantages are there in using an LC approachOne advantageof LC approaches is the fact that the required equipmentsuch as HPLC pumps as well as the ICP-based detectors(needed to monitor metal species in the column effluent) areavailable in many analytical laboratories In addition nearphysiological conditions during the LC separation can bemaintained by using phosphate-buffered saline (PBS)-buffer(note that certain seemingly useful biological buffers mustbe strictly avoided to preclude the generation of artifactswhen blood plasma is analyzed [119]) and thermostating theLC column With regard to ldquotop-downrdquo LC approachesmdashwhich generally involve the analysis of a complex biologicalsample (eg blood plasma) with an LC separation systemthat is coupled online to a ICP-detectormdashthe analyticalresult will be inherently less complex than that of con-ventional proteomic techniques [120] This in turn greatlyfacilitates the interpretation of the obtained results [100]Another advantage of ldquotop-downrdquo LC approaches is that they

allow one to rapidly analyze a biological fluid The plasmametalloproteome which is defined as all Cu Fe and Znmetalloproteins that are present in plasma for example canbe obtained in lt25 min [100] Importantly this capabilityopens the door to probe dynamic processes that involvemetalspecies in biological fluids in vitro in almost real time Giventhat the analysis of any biological fluid by ldquotop downrdquo LCapproaches essentially results in the detection of multiplemetal peaks it is important to point out that the analysisof collected metal-containing fractions with some of theaforementioned techniques (eg ESI-MS [119] EXAFS [121]or immunoassays [100]) can provide vital information tostructurally characterize a detected novel metal species inorder to gain insight into a bioinorganicmechanismof action

4 lsquolsquoBottom-Uprsquorsquo LC Approaches toProbe the Interaction of Toxic Metals withEndogenous Ligands

The interaction of immobilized metal ions for protein lig-ands is specifically exploited in the LC-based methodologyreferred to as immobilized metal affinity chromatography(IMAC) [122] Since IMAC has been successfully applied togain insight into the binding of Co2+ Ni2+ Cu2+ and Zn2+ toserum albumins from different mammalian organisms [123]one may anticipate that IMAC has also been applied to studythe interaction of toxic metals with biological ligands Fewstudies have been applied to probe the interaction of toxicmetals (eg Hg2+) with endogenous ligands by this approach[124] LC-based approaches however have been appliedto investigate the interaction of toxic metals with relevantendogenous ligands although in a different manner (seebelow) In terms of choosing relevant endogenous ligandsL-glutathione (GSH) must be considered as this tripeptideis found in a large variety of mammalian cells (includingerythrocytes) at intracellular concentrations up to 10mM[125] The vital role that GSH plays in critical biochemicaldetoxification reactions (eg the degradation of H

2O2[126])

and the affinity that it exhibits for several toxic metals basedon Pearsonrsquos HSAB principle [127] makes it a toxicologicallyhighly relevant target GSHhas in fact been referred to as thefirst line of defense against the acute toxicity of Cd2+ andHg2+[128 129] and its reaction with numerous toxic metals hastherefore been extensively investigated [130] The interactionof GSH with toxic metalsmetalloid compounds however isnot only relevant with regard to their acute toxicity but alsotheir chronic toxicity since the formation of complexes canresult in the activation of the metalmetalloid [131] Since L-cysteine (Cys Figure 1(b)) is present in hepatocyte cytosolat 02ndash05mM [126] and at 10 120583M in blood plasma [132]it represents another toxicologically relevant endogenousligand Because of its structural analogy to Cys N-acetyl-L-cysteine (NAC Figure 1(c)) is another potentially interestingligand

41 Interaction of a Toxic Metal with a Single Ligand Theinteraction of a single toxic metal with GSH has been studiedusing size-exclusion chromatography (SEC) employing theldquoretention analysis methodrdquo (Figure 3(a)) The latter was


Information obtained

Toxic metalmetalloid

compound

Ligand 1+ Ligand 2 in MP(eg GSH and Cys

or DMPS)

Micelles (eg positive surface charge)in MP or MP only

∙ SEC effect of 119879 pH and ligand concentrationon formation of metal-ligand complex

∙ SEC comparative stability of differentspecies of same metalloid with ligand

∙ RP-HPLC comparative hydrophobicity ofmetal-ligand complexes [CH3HgSG versusCH3HgCys] [143]

∙ SEC competitive binding of As(OH)3 toGSH and DMPS [135]∙ RP-HPLC coordination behavior of different species

∙ RP-HPLC discovery of novel metal-ligandcomplexes (GS-Hg-Cys) [121]

∙ SEC confirmation of spectroscopically

∙ SECMP only isolation of metal speciesfrom reaction mixture [(HgSe)100(GS)5] [61]

(a)

(b)

(c)

Ligand 1 in MP(eg GSH or Cys)

∙ SECMP only stability of toxicologically

∙ SECMP only identification of thiol ligandof (GS)2 AsSe in bile [63]

derived solution structure [(GS)2AsSeminus] [155]

[(GS)3As] [135]

of same metal (Hg2+ versus CH3Hg+) toward mixturesof (GSHCys) [121] and CysNAC [145]

[(GS)3As (GS)2AsMe GSAsMe2] [136]

relevant species [(GS)2AsSemiddot middot middotHgCH3] [161]

Figure 3 ldquoBottom-uprdquo LC approaches which have been employed to probe interactions between toxic metals and endogenous ligands (a)The retention time of the toxic metal is measured as a function of the ligand concentration in the mobile phase the pH or the columntemperature (b) The retention time of the toxic metalmetalloid compound is measured as a function of an increasing concentration ofLigand 2 while leaving the concentration of Ligand 1 in the mobile phase constant (c) The retention time of the metal-containing entity ismeasured as a function of an increased concentration of micelles in the mobile phase Abbreviations size exclusion chromatography (SEC)reversed-phase high performance liquid chromatography (RP-HPLC) mobile phase buffer (MP) L-glutathione (GSH) L-cysteine (Cys) and23-dimercapto-1-propanesulfonic acid (DMPS)

originally conceived to study the binding of L-tryptophanto HSA [133 134] which involved the measurement of theretention volume of the former as a function of the HSAconcentration in the mobile phase A progressive decreasein the retention time of L-tryptophan with an increase inthe HSA concentration indicated a binding event since theon-column formed L-tryptophan-HSA complex was excludedfrom the pores of the stationary phase Hence this LCapproach was deemed to be potentially useful to investigatethe interaction between a toxic metal with an endogenousligand at near physiological conditions Conceptually thedynamic equilibrium between the metal-species (injected)and the endogenous ligand (in the mobile phase) is keptconstant throughout the entire chromatographic process(Figure 3(a)) which is closely related to what happens whena toxic metal enters a ligand-laden biological compartmentsuch as blood plasma or a cell (Figure 4)

Using the aforementioned approach and employing aSephadex G-10 SEC column and PBS-buffer (pH 74) as themobile phase the reaction between arsenite [As(OH)

3] and

GSH (1) was investigated [135]

As(OH)3+ 119909GSH

997888rarr (GS)119909As(OH)

3minus119909+ 119909H2O 119909 = 1ndash3

(1)

The injection of 30 120583g As in form of As(OH)3onto

this system resulted in the elution of an As peak afterthe inclusion volume which was indicative of an unknowninteraction with the stationary phase backboneThe additionof 05mM GSH to the mobile phase decreased the reten-tion time of the injected As(OH)

3and was rationalized in

terms of the formation of (GS)119909As complexes (119909 = 1ndash3)

Applying this LC approach the effects of pH (range 2ndash10) column temperature (4 25 and 37∘C) and the GSHconcentration (05ndash75mM) on the formation of (GS)

119909As

complexes were investigated It was found that their for-mation was facilitated by low temperatures (4∘C) pH 60ndash80 and high GSH concentrations (75mM) Simulatingthe physicochemical conditions of erythrocyte cytosol (sim30mMGSH) and hepatocytes (sim75mMGSH) by adding thecorresponding GSH concentrations to the mobile phase (pH74) and maintaining the column to 37∘C provided evidencefor the formation of (GS)

119909As complexes To ascertain that

the GSH-induced retention shift of the injected As(OH)3to

shorter retention times was truly caused by the formationof (GS)

119909As complexes the chelating agent 23-dimercapto-

1-propanesulfonate (DMPS Figure 1(h)) was added (10 and20mM) to a 75mM GSH containing mobile phase Asexpected the injection of As(OH)

3shifted the elution of


Hepatocyte Nucleus

Biologicalcomplexity

Blood plasma

Erythrocyte

Tum

or

P = protein (cytosolicplasma)Metabolism of metal speciesModulation of metabolism of metal species

STS

STS

Top-down

Bottom-up

a

bc

d

ef

Hg2+

Hg2+

AsIII

AsIII

SeIV MeHg+

(GS)3As GSHGSH

GSH

GSH

GSH

GSH GSH

CA

CA

ZnCACACd PtSTS

CAAs

Urine

Bile

Zn2+ ZnP

CdP

Cd2+

Cd2+

P

P

CdP

CdP

[(SeHg)100(GS)5]SeP darr

PtH2O+ PtP

(NAC)2Hg

GSHgCys (Cys)2Hg

Cys Cys NAC

(GS)2Hg

(GS)2AsMe(GS)2AsSeminus

GSHgMeMeHg+Se-II

SeIV

MeAsIII

Me2AsIII(GS)2AsSeHgMe

GSAsMe2

cisPt

cisPt

(SeHg)100(GS)5

Figure 4 Unraveling the metabolism of metal species is a prerequisite to gain deeper insight into the molecular basis of their adverse healtheffects inmammalian organisms Toxicologically relevant interactions betweenmetal species and endogenous ligands unfold in blood plasmaerythrocytes and hepatocytes and can be investigated by applying ldquobottom-uprdquo or ldquotop-downrdquo techniques Interactions between toxic metals(Hg2+ AsIII SeIV CH

3Hg+) and small molecular weight ligands (GSH Cys NAC) that unfold within erythrocytes andor hepatocyte cytosol

and have been studied with ldquobottom uprdquo LC approaches (Figure 3) Interactions betweenmetal species (Cd2+ cisPt) and cytosolic and plasmaproteins have been studied using ldquotop-downrdquo LC-based approaches (a) Detection of Cd-metallothionein complexes in liver cytosol [110] (b)Displacement of Zn2+ from a plasmametalloprotein by Cd2+ [26] (c) Binding of Cd2+ to plasma proteins [26 36 111] (d) Abstraction of Cd2+from plasma proteins by chelating agents (CA) [36] (e) Comparative binding of cis-platincarboplatin to plasma proteins [53] (f)Modulationof the interaction between cis-platin and plasma proteins by sodium thiosulfate (STS) [37] Note that the combined application of ldquobottom-uprdquo and ldquotop-downrdquo approaches will be required to better understand the interplay between metal species small molecular weight ligandsand proteins to ultimately unravel their adverse health effects and to develop better mitigation strategies by modulating their metabolism bythe administration of chelating agentsameliorating agents

the As peak toward the inclusion volume which was rational-ized in terms of the stronger binding ofAs(OH)

3to the vicinal

dithiol groups of DMPS (chelate effect) as compared to GSH(monothiol Figure 1(a)) Thus this principle LC approachallowed insight into the dynamic on-column formation of(GS)119909As species under near physiological conditions To

obtain the same information with other techniques suchas 1H-NMR can be challenging because of line-broadeningphenomena at higher temperatures and a comparative lackof sensitivity [135] In addition the chosen LC approachcan be ideally employed to study competitive interactionsbetween a toxic metalmetalloid compound and two ligandsby dissolving them in themobile phase (ie GSHandDMPS)The disadvantages of this approach are that no complex

formation constant can be extracted and that the structuralcharacterization of an on-column formed metal-peptide-complex by ESI-MS is next to impossible due to the utilizationof PBS-buffer and the excess GSH therein In addition noinformation about the specific binding site on the employedligand can be obtained (this information however can beeasily obtained by 1H-NMR spectroscopy) Overall thisLC approach nevertheless provided unique insight into thephysicochemical conditions under which the highly toxicmetalloid species As(OH)

3will react with GSH to form labile

(GS)119909As complexes

Encouraged by this outcome this LC approach was alsoapplied to study the interaction of other AsIII compounds


namely methylarsonous acid [MeAs(OH)2] and dimethy-

larsinous acid [Me2AsOH] with GSH [136] Both of these

methylated AsIII compounds are of toxicological significancebecause their GS-complexes methylarsonous diglutathione[(GS)2AsMe] and dimethylarsinous glutathione [GSAsMe

2]

are believed to play a pivotal role during the methylationof As(OH)

3in the mammalian liver [137ndash139] To probe

these interactions a Sephadex G-15 SEC column was usedIn order to mimic the physico-chemical conditions thatare prevalent in protein-free hepatocyte cytosol [136] themobile phase was comprised of 100mM GSH in PBS-buffer Varying the column temperature between 4 25 and37∘C 10 120583g of As in form of As(OH)

3 methylarsonous

acid [MeAs(OH)2] dimethylarsinous acid [Me

2AsOH] or

arsenobetaine (AsB Figure 1(i)) were chromatographed sep-arately AsB eluted in the inclusion volume irrespective ofthe column temperature which indicated that it did notform a complex with GSH At 4∘C the injection of As(OH)

3

resulted in the elution of (GS)3As which was inferred from

its elution before a simMW600 GSSG standard Interestinglyan increase of the column temperature from 4∘C to 37∘Cprogressively shifted the elution of the injected As(OH)

3

toward the inclusion volume whereas the retention times ofthe injectedMeAs(OH)

2andMe

2AsOH remained essentially

unchanged [136] These findings implied that the As-S bondsin MeAs(GS)

2and Me

2AsGS are comparatively more stable

than those in (GS)3AsThis is toxicologically relevant because

all of these GS-complexes are substrates for ATP-drivenhepatocyte export pumps which are located at the apicaland the basolateral side of hepatocytes [138ndash140] The effluxof GS-conjugates from hepatocytes to the bloodstream forexample can be mediated by multidrug resistant protein 1(MRP1) [140 141] It is therefore conceivable that the morestable species MeAs(GS)

2and Me

2AsGS are more efficiently

expelled from hepatocytes to the bloodstream via a MRPthan the comparatively unstable (GS)

3As (Figure 5) If this

more efficient efflux of MeAs(GS)2and Me

2AsGS compared

to (GS)3As by a MRP is experimentally confirmed it would

provide a potential explanation for the evolution of hepaticenzymes that methylate As(OH)

3 This is because organisms

that had developed the capability to methylate As(OH)3

to MeAs(OH)2and Me

2AsOH in the liver would more

efficiently effluxAsIII via the kidneys [136] and therefore havean evolutionary advantage

More recently this LC approach was applied to system-atically develop a RP-HPLC separation of mercuric mercury(Hg2+) and methylmercury (CH

3Hg+) based on using aque-

ousmobile phases andGSH or Cys asmobile phasemodifiers(Figure 3(a)) Optimal separation was achieved with 10mMCys in 50mM phosphate-buffer (pH 75) and using a GeminiRP-HPLC column [143] During the optimization processthe pH dependent retention behavior of Hg2+ and CH

3Hg+

was investigated by separately injecting these mercurialsusing a GSH or Cys-containing mobile phase Between pH5 and 8 the formation of complexes between CH

3Hg+ and

each thiol was essentially independent of the mobile phasewhereas the formation of complexes between Hg2+ andGSH was unaffected in the pH range 60ndash80 but strikingly

As(OH)3

As(OH)3 + 3GSH As(GS)3

CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-

Transport to kidneys andexcretion of methylated

AsV species in urine

lowast

lowast

CH3As(GS)2(CH3)2AsGS

(CH3)2AsGS

Figure 5 Proposed model which can provide a feasible explanationfor the evolution of enzymes which methylate AsIII in hepato-cytes As(OH)

3is enzymatically converted (lowast) to the trivalent As

compounds MeAs(OH)2and Me

2AsOH which readily react with

cytosolic glutathione (GSH) to MeAs(GS)2and Me

2AsGS The

increased stability of As-S bonds in MeAs(GS)2and Me

2AsGS

compared to the much less stable (GS)3As will facilitate the efflux of

the former species to the bloodstream by MRP export pumps [136]The translocation of these species to the kidneys and subsequentoxidation will result in the excretion of methylated AsV species inurine [142]

different at pH 50 and 55 The stronger retention ofthe complexes that were formed between each mercurialand GSH (GS-Hg-SG and GS-Hg-CH

3) compared to the

structurally related Cys (Cys-Hg-Cys and Cys-Hg-CH3) was

attributed to the comparatively greater hydrophobicity ofGSHThe observed differences in the hydrophobicity of thesemetal complexes may be of toxicological significance as thisphysicochemical characteristic is likely to play an importantrole during the intracellular disposition of Hg2+ and CH

3Hg+

[144]

42 Interaction of a ToxicMetal with TwoEndogenous LigandsMost recently the aforementioned RP-HPLC separation ofHg2+ and CH

3Hg+ was improved because the on-column

formed Hg2+ complex essentially eluted in the void volumeThis improvement was achieved by partially replacing Cysof the mobile phase by the comparatively more hydrophobicNAC (Figures 1(c) and 3(b) respectively) A baseline sepa-ration of Hg2+ and CH

3Hg+ with the former species being

retained was achieved with 75mM NAC and 25mM Cys in50mM phosphate buffer (pH 74) [145] The separation wasattributed to the formation of comparatively more hydropho-bic mercurial-complexes with NAC compared to Cys Theobserved formation of hydrophobicHg2+-NACcomplexes (atphysiological pH) is of potential relevance to explain whythe administration of rats with NAC effectively protectedthem against the nephrotoxicity of HgCl

2[146] In addition

the formation of a CH3Hg-NAC complex at pH 74 may

be involved in the profound NAC-induced mobilization of


4

3

2090807060504030201

0 1 2 3 4 5 6 7 8 9 10Concentration of Cys (mM) in 5 mM GSH mobile phase

119896998400

CH3Hg+

Hg2+

Cd2+

Figure 6 Retention behavior of Hg2+ CH3Hg+ and Cd2+ on a

Gemini C18 RP-HPLC column (150 times 46mm ID) at 37∘C Mobilephase 50mMGSH with 0ndash10mMCys in PBS buffer (pH 74) Flowrate 10mLmin Detector ICP-AES (Hg at 194227 nm or Cd at226502 nm) Injection volume 20 120583L (5120583g Hg or Cd) Detachedsymbols on right side correspond to the 1198961015840 of each metal speciesusing PBS buffer with 50mM Cys as the mobile phase Errors in119896

1015840 are within the area of plot markers unless otherwise indicated(reproduced with permission from [121] and Elsevier)

CH3Hg+ from tissue compartments (including the brain)

to urine which was observed in mice [147] and possiblyinvolves the renal organic anion transporter-1 (OAT-1) [148]Considering that the clearance of Hg2+ from organs was notaccelerated by NAC in mice [147] and taking into accountthat NAC did not alter the plasma concentration and theurinary excretion of the essential metals Ca Mg Fe Zn andCu in humans after the administration of an oral dose of600mgday for 2 weeks [149] this thiol ligand appears to berather selective for the mobilization of CH

3Hg+ Since NAC

is inexpensive well tolerated by patients (it is used to treatacetaminophen toxicity) and was orally effective to removenearly 90 of a CH

3Hg+ dose in mice within 2 days further

investigations should explore the utility ofNACas an antidoteagainst CH

3Hg+ in clinical settings [147 150]

The aforementioned results indicated that this LCapproach is amenable to study the interaction between atoxic metal with two ligands Although this very idea mayat first seem irrelevant considering the underlying biologicalcomplexity one must not forget that the individual concen-trations of GSH and Cys in hepatocyte cytosol may greatlyexceed those of every single cytosolic protein [121] Henceinteractions of a toxic metal with these thiols may play animportant role in their intracellular distribution [72 73]Such competitive interactions can be investigated with LC byleaving the concentration of ligand A in the mobile phaseconstant and observing the retention behavior of the metalspecies of interest as a function of increasing concentrationsof ligand B in the mobile phase (Figure 3(b)) [121] Using aGemini RP-HPLC column (37∘C) and a mobile phase thatwas comprised of 50mMGSH in PBS-buffer (pH 74) 5120583g ofHg2+ CH

3Hg+ or Cd2+ were separately injected (Figure 6)

A single Cd-species eluted essentially in the void volume

(1198961015840 = 01) which was structurally characterized as tetrahe-dral (GS)

4Cd by X-ray absorption spectroscopy (XAS) In

contrast a Hg2+ speciesmdashpresumably the linear GS-Hg-SGmdashwas somewhat retained on this column (1198961015840 = 09) whereasa CH

3Hg+ species was retained considerably (1198961015840 = 38)

(Figure 6)WhenCys was incrementally added to the 50mMGSH containing mobile phase (05mM10mM incrementsup to 10mM) and each metal species was injected theretention time of Cd2+ remained unchanged The retentiontime of the injected CH

3Hg+ continuously decreased and

was rationalized in terms of the rapid displacement of GS-moieties fromCH

3Hg+ bymore hydrophilic Cys-moieties In

contrast the retention behavior of Hg2+ displayed a charac-teristic stepwise decrease of its retention time with graduallyincreasing Cys concentrations This behavior was explainedin terms of the consecutive displacement of two GS-moietiesthat were coordinated to Hg2+ by Cys-moieties These resultsrevealed fundamental differences with regard to the Hg-Sbonding in these mercury complexes and provided directexperimental evidence for the formation of the proposedspecies GS-Hg-Cys [151] under near physiological conditions

43 Interaction of Two or Three Toxic Metals with OneEndogenous Ligand To elucidate in what way the interplay oftoxic metals with endogenous ligands may contribute to theetiology of disease processes [34] one must be aware of thefact that in reality human populations are rarely exposed to asingle toxic metal [152] Furthermore toxic metals are alwaysingested together with essential elements and themetabolismof certain combinations of toxicmetals and essential elementsmay therefore be biochemically intertwined and of toxicolog-ical significance Direct experimental evidence in support ofthis possibility was reported in 1941 when the simultaneousadministration of ratswithAs(OH)

3and selenite (HSeO

3

minus)mdashan essential ultratrace elementmdashresulted in themutual detox-ification of these individually highly toxic metalloid species[153] Considering that free selenite has been detected inhuman blood plasma [154] this intriguing trace elementantagonism was hypothesized to be implicated in the chronictoxicity of As(OH)

3 Investigations aimed at uncovering its

biomolecular basis revealed that it appeared to be based onthe rapid in vivo assembly of the seleno-bis(S-glutathionyl)arsinium ion (GS)

2AsSeminus (Figure 1(d)) in the bloodstream

(in erythrocytes [91]) followed by its translocation to theplasma the liver and its eventual excretion in bile [62 64]Since the structure of this species was entirely derived fromEXAFS and Raman spectroscopy its unequivocal confirma-tion required an alternative technique Surprisingly this atfirst difficult task could be accomplished using an LC-basedapproach To synthesize (GS)

2AsSeminus equimolar As(OH)

3

and HSeO3

minus are reacted at physiological pH with ge8 mole-equivalents GSH as follows

As(OH)3+HSeO

3

minus

+ 8GSH

997888rarr (GS)2AsSeminus + 3GSSG + 6H

2O

(2)

Given that the obtained reaction mixture was supposedto contain (GS)

2AsSeminus and 3 mole equivalents of GSSG


(Figure 1(e)) the only notable difference between thesespecies is their net charge minus3 on (GS)

2AsSeminus and minus2 on

GSSG (see Figures 1(d) and 1(e))Thus a LC-based separationapproach that could exploit this charge difference wouldindirectly confirm the spectroscopically derived structure Itwas surmised that the separation of these species might bepossible by employing a micellar chromatography approachbased on SEC (Figure 3(c)) and the detection of the cor-responding species by ICP-AES [155] A Sephadex G-25SEC column served as the stationary phase and 01M Tris-buffer with 50mM hexadecyltrimethylammonium bromide(HDTAB critical micellar concentration 092mM pH 80)served as the mobile phase The analysis of the reactionmixture revealed the elution of a species that containedAs Seand S which was reasonably well separated from GSSG Theintegration of the obtained S-peaks confirmed that indeedtwo S-atoms (derived from two GS-entities) coeluted withan As and Se-containing species Remarkably the elutionof (GS)

2AsSeminus followed by GSSG implied additional neg-

ative charge on the former species and thus corroboratedthe spectroscopically derived solution structure [155] Togain insight into the biochemical mechanism of forma-tion of (GS)

2AsSeminus equimolar bis-glutathionylarsenous acid

[(GS)2AsOH] was reacted with HSeminus in aqueous solution

Analysis of the reaction mixture on a Sephadex G-10 SECcolumn using 01M Tris-buffer (pH 80) as the mobile phaseclearly demonstrated that (GS)

2AsSeminus had formed (this was

confirmed by XAS) Thus the investigated reaction mecha-nism can explain how (GS)

2AsSeminus might be formed in vivo

[131]A related trace element antagonism was discovered in

1967 when the coadministration of rats with HgCl2and

HSeO3

minus resulted in negligible toxicity whereas essentiallyall animals in the HgCl

2group died [156] Although this

antagonism was later demonstrated to involve the formationof a nontoxic HgSe species in the bloodstream [157] thestructural characterization of this species remained elusiveTo synthesize a model compound that was intended touncover the molecular basis of this HgCl

2HSeO

3

minus antag-onism equimolar HgCl

2HSeO

3

minus was reacted with 5 moleequivalents of GSH in PBS-buffer [61] The analysis of theobtained black solution by 199Hg-NMR spectroscopy 77Se-NMR spectroscopy and a variety of mass spectroscopic tech-niques (eg FAB-MS and ESI-MS) however did not provideany useful information toward the structural characterizationof the contained black solution species It was eventuallydecided to directly analyze this mixture on a Sephadex G-25 SEC column and detecting Hg Se and S by means ofan ICP-AES The analysis of the black solution revealed asingle Hg Se and S-containing entity which eluted in thevoid volume followed by GSSG (this indicated that a redoxreaction had occurred) Detailed analysis of the obtainedS-specific chromatogram implied that 5 GS-moieties wereassociated with the detected Hg-Se species and EXAFSanalysis of the isolated HgSeS-species finally revealed thisspecies as (Hg-Se)

100(GS)5 a mercuric selenide core with

ligation to 5 GS-moieties via S-Se bonds The analysis ofblood plasma obtained from New Zealand white rabbits

25min after they had been injected with HgCl2and HSeO

3

minus

revealed that the Se and Hg near edge XAS spectra mostclosely resembled those of the synthetic (Hg-Se)

100(GS)5

Thus conventional SEC (without adding any ligand to themobile phase buffer) coupled with an ICP-AES played apivotal role in the elucidation of the structural basis of thismineral antagonism

Evidence in favor of the existence of an even more fas-cinating ldquothree-wayrdquo antagonism between AsIII SeIV andCH3Hg+mdashthree individually highly toxic metalmetalloid

speciesmdashwas first reported based on feeding experimentswith Japanese quail in 1982 [158] In particular the survivalrates of quail which were fed CH

3Hg+ (10mgHg kgminus1)

increased from 5 to 75 (after 16 weeks) when selenite(6mg Se kgminus1) was included into the CH

3Hg+ laced diet

Quite counterintuitively the survival rate increased to 100when AsIII (15mg As kgminus1) was fed together with SeIVand CH

3Hg+ Taking into consideration that (GS)

2AsSeminus is

assembled in erythrocyte lysate [91] and that CH3Hg+ is

known to cross the erythrocyte membrane [159] and giventhe high affinity of CH

3Hg+ for selenide [160] it was deemed

possible that this antagonism might involve the reaction of(GS)2AsSeminus with CH

3Hg+ within erythrocytes to yield the

complex [(GS)2AsSeHgCH

3] The addition of CH

3HgOH to

(GS)2AsSeminus and the analysis of the obtained aqueous solution

by EXAFS revealed that the proposed complex had indeedformed [161] To elucidate if this reaction might also unfoldin erythrocytes rabbit erythrocyte lysate was supplementedwith synthetic (GS)

2AsSeminus and equimolar CH

3HgOH XAS

analysis revealed that 90 [(GS)2AsSeHgCH

3] was present

To investigate the stability of this toxicologically relevantspecies [(GS)

2AsSeHgCH

3] was chromatographed on a

Sephadex G-15 SEC column using PBS-buffer as the mobilephase and an ICP-AES as the simultaneous As Se S and Hg-specific detector (Figure 3(c)) The coelution of As Se andS was indicative of (GS)

2AsSeminus and was followed by a Hg-

species These findings indicated that [(GS)2AsSeHgCH

3]

had dissociated during the chromatographic process and thatthe Se-Hg bond was labile when PBS-buffer was used as themobile phase (this however does not necessarily imply thatthis complex is unstable in vivo) Although the biochemicalfate of [(GS)

2AsSeHgCH

3] is currently unknown these

results firmly established the plausibility of its formationunder near physiological conditions to explain the remark-able ldquothree-wayrdquo antagonism

5 lsquolsquoBottom-Uprsquorsquo LC Approaches toProbe the Interaction of Metal-Based Drugswith Endogenous Ligands

Although metal-based drugs constitute a minority amongthe plethora of medicinal drugs that are in use today [162]their fraction appears to be slowly increasing [163 164]The serendipitously discovered cis-platin for example isstill one of the worldrsquos best selling anticancer drug [165]which is extraordinarily effective to treat certain types ofcancers [166 167] Since only two other Pt-based anticancerdrugs carboplatin (Figure 1(k)) and oxaliplatin (Figure 1(l))


have received worldwide approval for their treatment ofhuman cancer patients [168] only these three Pt-based drugswill be discussed For a historical perspective of cis-platinand its proposed mode of action the reader is referred toa recent review [165] In general comparatively few studieshave been reported in which LC has been employed to studythe interaction of these Pt-based drugs with either individualplasma proteins small molecular peptides (GSH) andoramino acids (Cys) [169 170] This must be attributed tothe fact that this interaction is complicated by the fact thatthese Pt-drugs produce multiple Pt-containing hydrolysisproducts [171] which will then bind to plasma proteins [53]thiol-containing biomolecules [170] andor the pharmaco-logical target DNA [172 173] Thus the few studies thathave employed LC to gain insight into the interaction ofPt-based anticancer drugs with plasma proteins effectivelystudied primarily the protein binding of the in situ generatedhydrolysis products These studies involved the incubationof individual Pt-based drugs with individual plasma proteins(eg HSA) at physiologically relevant conditions (01MNaClin pH 74 buffer) followed by the subsequent LC-analysisof the reaction mixture at different time intervals In thismanner the binding of cis-platin to the key plasma proteinHSA was investigated [174] Subsequent studies revealed thatboth isomers induced the formation of the albumin dimerwith the transisomer exhibiting a much greater tendencyto induce protein dimerization [175] Furthermore trans-platin cleaved the SndashS bonds in HSA more readily thancis-platin [175 176] More recent studies investigated thecomparative binding of cis-platin to transferrin (Tf) or HSAAfter the incubation of 1120583M cis-platin with either apo-transferrin (Tf 3mgmL) or HSA (30mgmL) in 09 NaCland 10mM Tris-acetic acid (pH 74) subsequent analysisby anion exchange HPLC-ICP-MS (Mono Q) revealed com-plete binding of cis-platin derived species to HSA in 24 h(PtHSA ratio = 4 1) but only 50 in case of apo-Tf after96 h (Ptapo-Tf = 1 1) [177] The fact that the binding ofcis-platin derived hydrolysis products to Tf does not involvethe displacement of Fe [178] is of pharmacological relevancesince only the fully Fe3+-loaded Tf is capable of bindingto the Tf receptor on the surface of cells These findingsstrongly suggest that cis-platin derived species may entercancer cells via receptor-mediated endocytosis of Tf [179]In addition the comparative binding of cis-platin and aruthenium(II)arene complex (RAPTA-T) to HSA and Tf(both holo-Tf and apo-Tf) at different molar ratios has beeninvestigated by SEC-ICP-MS [180] Importantly these inves-tigations revealed that cis-platin bound to HSA and Tf to thesame extent whereas RAPTA-T showed a marked preferenceto bind to Tf In addition RAPTA-T showed a markedlyhigher preference for holo-Tf than apo-Tf Thus ldquobottom uprdquoLC approaches allow to assess the comparative binding ofmetal-based drugs andor their hydrolysis products to keyplasma proteins under near physiological conditions Theresults that are derived from such studies allow to predictwhether it is likely that an investigated metal-based drugand or its hydrolysis products may enter cells via establishedpathways

6 lsquolsquoTop-Downrsquorsquo LC Approaches toProbe the Interaction of Toxic Metals withEndogenous Ligands

Blood represents the principal medium for the transportof gastrointestinally absorbed nutrients including essentialmetals as well as dietary toxic metals to organs Interactionsbetween toxic metals and constituents of the bloodstreammay adversely affect the transport of sufficient amounts ofessential elements to their intended destinations within anorganism [26 181] To this end the interactions of As(OH)

3

and Hg2+ with the essential ultratrace element Se representthe first examples for this toxicological mechanism of action(Figure 2 red arrows) [32 84]Thus the bloodstream in itselfrepresents a biological compartment where relevant mech-anisms of action may unfold Accordingly the detection ofinorganic pollutant-induced biochemical changes in plasmaor erythrocyte lysate represents a feasible approach to possi-bly gain insight into the etiology of pollutant induced adversehealth effects in mammals In this context it is importantto stress that everyone who embarks on the quest to probethese interactions should be aware of the difficulty that isassociated with sampling a living organism without changingthe composition of any given sample before or during theanalysis [26 182] In principle one has to distinguish betweenperturbations that may be detected within minutes after anappropriate dose of a particular toxin has been added toblood plasma in vitro [26] or administered to animals [183]and perturbations that are only detectable in blood plasmaor erythrocyte lysate after a given model organism has beenexposed to a particular toxin for weeks or months [184 185]With regard to the nature of these perturbations one mustdistinguish between those that are displayed at the mRNA-level [186] the small molecule level [13 17] the protein level[12] and those at the metalloprotein level [26 75]

At present numerous proteomic approaches are avail-able to visualize changes of any given proteome over timesuch as increased or decreased concentrations of specificproteins (eg in erythrocyte lysate [75]) but many of thesetechniques are time consuming expensive and incapableof detecting changes at the metalloprotein level Converselythere are comparatively few LC-based approaches that can beemployed to determine the ldquometalloproteomerdquo of any biolog-ical fluid Fortuitously however all of these can accomplishthis task in a comparatively rapid manner

The first notable LC-based approaches that were appliedto determine the metalloproteome of biological fluids in atruly ldquotop-downrdquo manner are the pioneering studies of KTSuzuki who analyzed rat liver cytosol (SEC-AAS) [110] andblood plasma (SEC-ICP-AES) [111] After the injection ofrats with Cd2+ (04mgCdkg bodywt) blood plasma wasanalyzed (after 1 2 3 5 10 20 and 30min) using a systemthat was comprised of an Asahipak GS-520 SEC column10mM Tris-HCl buffer in 09 NaCl (pH 74) as the mobilephase and an ICP-AES to monitor the emission lines of SCd Zn Cu Fe and P [111] At the 3min time point thecoelution of a single Cd-peak with Zn and S implied thebinding of Cd2+ to the most abundant plasma protein rat


serum albumin (RSA) In addition these authors detecteda comparatively broad Zn-peak (which eluted before Zn-RSA) along with a single Cu-peak (ceruloplasmin) and threeunidentified and nonbaseline separated Fe-peaks as well astwo unidentified P-entities (a small one in the highmolecularweight and an intense one in the lt60 kDa elution range)Interestingly the analysis of plasma 20min after the injectionrevealed that the Cd-peak that was detected after 3minwas bearly detectable which implied rapid translocation ofCd2+ from the bloodstream to organs Concomitantly theintensity of the broad Zn-peak had increased in intensitycompared to that at the 3min time point Collectively theseresults demonstrated that the analysis of blood plasma bySEC-ICP-AES can provide valuable insight not only into thedirect interaction of a toxic metal ion with biological ligandsin blood plasma (ie the rapid binding of Cd2+ to RSAand the subsequent translocation of Cd2+ to organs) butalso into changes of the metalloproteome (eg the observedchanges of the Zn-metalloproteome in response to Cd2+)More recently the application of the same analytical approach(stationary phase Superdex 200 SEC column mobile phasePBS-buffer [100]) was applied to analyze rabbit plasma towhich increasing doses of Cd2+ (05 2 and 200 120583g Cd2+mL)had been added in vitro [26] The results revealed at leasttwo Cd2+ binding proteins after the addition of the lowCd2+ doses At the high Cd2+ dose however an altered Zn-metalloproteome pattern was detected which was attributedto aCd2+-induced displacement of Zn2+ ions from its bindingsites on aZn-metalloproteinmdashpresumablyCu Zn superoxidedismutasemdashwhich has been previously observed [187]

LC-based approaches may not only be applied to detectmetalloproteins but can also be employed to detect toxi-cologically relevant species in complex biological fluids Tothis end it has been reported that (GS)

2AsSeminus is excreted in

rabbit bile after their injection with As(OH)3and HSeO

3

minus

[62] Even though the presence of this species was inferredfrom the analysis of bile byXAS this technique can inherentlynot conclusively identify the sulfur donor in this speciesThe analysis of bile from similarly treated rabbits by SEC-ICP-AES revealed a single As Se and S-containing entityand S-containing bile components The addition of synthetic(GS)2AsSeminus to this bile and reanalysis revealed an increased

intensity of the As and the Se peak and therefore unequivo-cally identified GS-moieties as the ligands in the species thatis excreted from the liver [63]

In the context of toxic metals interacting with endoge-nous ligands within organisms however their abstractionfrom proteins by a chelating agent is also eminently healthrelevant since a better understanding of these events willdirectly contribute to the development of safer chelatingagents for their effective tissuemobilization Since themanip-ulation of the distribution of metal ions in biological organ-isms is a very complicated process (ie not very amenable totheoretical modeling [188]) one relies on empirical evidencefrom biological experiments to gain insight This notion isexemplified by the recent application of SEC to systematicallyassess the efficiency of three chelating agents to abstract Cd2+from rabbit plasma proteins in vitro [36] Using a Superdex

200 SEC column PBS-buffer as the mobile phase and anICP-AES the analysis of rabbit plasma that had been spikedwith 20 120583g Cd2+mL revealed two distinct Cd-peaks whichwere tentatively identified as 120572

2-macroglobulin and rabbit

serum albumin The dose-dependent addition of meso-23-dimercaptosuccinic acid (DMSA Figure 1(f)) diethylen-etriamine pentaacetic acid (DTPA Figure 1(g)) or 23-dimercapto-1-propanesulfonic acid (DMPS) to the Cd-spikedplasma (033ndash099mM chelating agent) revealed DTPA tobe most effective in abstracting Cd2+ from plasma proteins(100 removal at 033mM) to a Cd2+-chelate complex whicheluted in the 5 kDa elution rangeThe simultaneous detectionof the emission lines of Ca Cu Fe and Zn howeverprovided arguably the most valuable insight into unintendedconsequences of the investigated chelating agents Whereasnone of the chelating agents adversely effected the plasmadistribution of Fe at all investigated doses all of themmobilized Zn2+ from plasma proteins to sim5 kDa Zn-species(at 033mM DTPA removed 80 DMPS 63 and DMSA29) Furthermore DTPA resulted in a dose-dependentelution of a [Ca-DTPA]3minus complex which is undesirable froma clinical point of view since a decrease in the concentrationof free plasma Ca2+ is associated with twitching and titanicconvulsions in humans [189] These unintended chelatingagent-induced perturbations of the plasma distribution ofCa2+ andZn2+ could therefore result in the dyshomeostasis ofthese essential elements in vivo andmdashif they were repeatedlyadministered intravenously to patientsmdashpotentially resultin pathologies [48] The obtained results were thereforeassessed in terms of finding a chelating agent and dose whichoffer a compromise between maximizing the abstraction ofCd2+ from plasma proteins and minimizing that of Zn2+and ideally avoiding the complexation of free Ca2+ Thiscompromise was a dose of 033mM DMSA Based on thesein vitro results the employed LC-approach is destined to playan increasingly important role in the context of developingsafer chelating agents for the treatment of patients that sufferfrom metal intoxications [190ndash192]

7 lsquolsquoTop-Downrsquorsquo LC Approaches toProbe the Interaction of Metal-Based Drugswith Endogenous Ligands

In the context of the significant recent decline in the numberof therapeutic medicinal drugs that are being approved [54193] metal-based drugs offer several conceptual advantagescompared to molecularly targeted ones that are constructedfrom organic fragments [54 162] but their side-effectsremain a major problem [166] Owing to the rising costs thatare required to bring a new drug to the market [194] theresulting greater scrutiny to the return on RampD investment[195] translates to an increased need to be able to predictthe efficacy and potential side-effects of promising drugcandidates before expensive clinical studies are initiated[196] Therefore a better understanding of the interactionof promising metal-based drug candidates with endogenousligands is becoming more important and in vitro stud-ies can offer valuable insight in this regard In terms of


developing better metal-based anticancer drugs for exampletheir efficacy can be assessed in cell culture experiments usingappropriate cancer cell lines (Figure 2 dashed red arrows)but one needs to be aware that this may not be sufficient topredict in vivo activity [197] The prediction of their systemicside-effects in model organisms or patients (Figure 2 redarrows) however remains challenging [83] Conceptuallythe side effects that intravenously administered metal-basedanticancer drugs such as cis-platin exert in patients mustbe attributed to their limited selectivity toward the tumortissue compared to healthy tissues (they are therefore alsoreferred to as shotgun cytotoxics [162]) and the complexinteractions that unfold between the parent drug and itshydrolysis products with plasma proteins [198] erythrocytes[199] or cytosolic ligands [200] Since interactions betweenclinically used metal-based drugs and proteins are not wellunderstood [201] in vitro studies with blood plasma mayprovide insight into the biomolecular basis of their sideeffects To this end comparatively few studies have beenconducted which employed ldquotop-downrdquo LC-approaches toprobe such interactions [202] One of the first studies whichutilized SEC-ICP-MS to analyze blood serumwhich had beenspikedwith cis-platin over a 24 h period used a Supelco ProgelTSK SEC column and 30mM Tris-buffer (pH 72) [198] Analternative approach for the analysis of rabbit blood plasmawas developed by our group using a Superdex 200 SEC col-umn andPBS-buffer as themobile phase [100]Thedevelopedmethod was first applied to study the binding of the surrogatemetal-based drug arsenobetaine (Figure 1(i)) to human andrabbit blood plasma to which it had been added in vitro [203]The results revealed that arsenobetainemdashwhich is present inmany marine organisms that are frequently used for humanconsumptionmdashdid not bind to plasma proteins gt300Da overa 6 h period which provided a feasible explanation for therapid excretion of this compound in urineThe first clinicallyusedmetal-based drugs that were investigated employing thisldquotop downrdquo LC approach were the anticancer drugs cis-platinand carboplatin [53] The addition of pharmacologicallyrelevant doses of each Pt-drug to human plasma in vitro andthe subsequent analysis of plasma by SEC-ICP-AES over a24 h period revealed a considerably faster hydrolysis of cis-platin than carboplatin In addition this study revealed thatthe hydrolysis products of both Pt-compounds appeared tobind to the same plasma proteins Even though results that areobtained with blood plasma in vitro are in itself insufficientto predict if the same dynamic changes will occur in vivo[204] the fact that cis-platin and carboplatin are known toexhibit entirely different side effects in patients [53] stronglysuggests that their interaction with plasma constituents couldbe involved in mediating their side effects Another studyapplied RP-HPLC using a ODS-3 column to analyze ratblood to which oxaliplatin had been added [70] The resultsrevealed that 4 h after incubation 53 of oxaliplatin wasassociated with erythrocytes and that 58 of that was boundto cytosolic proteins (the rest was bound to membrane orin the ultrafiltrate) Interestingly the analysis of erythrocytelysate revealed several chemically unreactive Pt-containingspecies (oxaliplatinmetaboliteswhich containedGSHorCys)and the parent oxaliplatin The analysis of plasma revealed

that the major biotransformation products were the mainbreakdown product of oxaliplatin Pt(dach)Cl

2 In addition

Cys and methionine adducts of Pt(dach)Cl2were detected

In view of the severe side effects of Pt-based anticancerdrugs the intravenous administration of model organismswith so-called ameliorating agents (either shortly before orafter the metal-based drug) has been demonstrated to beeffective [37] Sodium thiosulfate (STS) for example hasbeen demonstrated to effectively ameliorate the side-effectsof cis-platin In order to gain insight into the biomolecularmechanism by which STS may exert this desirable effect apharmacologically relevant dose of STS was added to humanplasma in vitro before or after a pharmacologically relevantdose of cis-platin The subsequent determination of the Pt-distribution in plasma by SEC-ICP-AES revealed that a Pt-STS complex was formed Although the structure of thiscomplex has not been elucidated its formation in bloodplasma provides a feasible explanation for the STS-mediatedamelioration of the side-effects of cis-platin in mammalianmodel organisms Importantly these results represent a start-ing point for the systematic exploration of other amelioratingagents in in vitro studies

8 Outlook

The increased anthropogenic emission of toxic metals intothe environment [34] will be inevitably associated with anincreased influx of inorganic pollutants into the humanbloodstream of various populations including children Animproved understanding of the biochemistry of toxic metalstherein as well as in target organs is therefore urgentlyneeded not only to better understand the biomolecularmechanisms of their chronic toxicity but also to developinexpensive efficient and safe therapies to mitigate toxicmetal-induced adverse health effects in affected populationsTo date the application of ldquobottom-uprdquo LC approaches hasprovided valuable insight into the formation of toxic metal-ligand complexes at near physiological conditions (GS)

3As

[135] their stability (GS)3As MeAs(GS)

2and Me

2AsGS

[136] and (GS)2AsSeHgCH

3[161] their hydrophobicity GS-

Hg-SG versus Cys-Hg-Cys and GS-Hg-CH3versus Cys-

Hg-CH3[143] Cys-Hg-Cys versus NAC-Hg-NAC Cys-Hg-

CH3versus NAC-Hg-CH

3[145] provided evidence for

the formation of novel species Cys-Hg-SG [121] andplayed an important role in the structural confirmationof a spectroscopically-derived structure (GS)

2AsSeminus [155]

Perhaps most importantly LC has been shown to be capableof probing the dynamic and competitive binding of a toxicmetal to two ligands As(OH)

3to GSHDMPS [135] Hg2+

to CysGSH CH3Hg+ to CysGSH [121] In contrast ldquotop-

downrdquo LC approaches have allowed to investigate the bindingof the toxic metal Cd2+ to proteins in plasma in vitro[36] and to visualize a toxic metal induced perturbation(Cd2+ displaced Zn2+ from a plasma metalloprotein [26])which constitutes a relevant mechanism of toxicity [205]Furthermore the analysis of bile by SEC-ICP-AES allowed toidentify the thiol ligands of the detected As-Se species as GSwhich established (GS)

2AsSeminus as the molecular species that

is excreted from the liver [63] Last but not least ldquotop-downrdquo


LC approaches can be employed to study the dose-dependentabstraction of Cd2+ from plasma proteins by chelating agents[36]Thus the application of ldquobottom-uprdquo andor ldquotop-downrdquoLC approaches each offers unique albeit different insightsinto themetabolism of toxicmetals (Figure 4)The combinedapplication of these techniques in conjunction with othertechniques can contribute to better understand the inter-play of toxic metals with endogenous ligands in mammalsConsidering that the exposure of various populations totoxic metals is of increasing societal concern [206ndash214] LCapproaches can be applied to develop practical solutions toaddress toxic metal-related problems in the near future

Another societal issue that is indirectly related tometals isthe significant decline in the number of therapeuticmedicinaldrugs that are being approved This undesirable trend maybe reversed if metal-based drugs can be developed that offerimproved efficacy and reduced side effects compared to thosethat are in use today To this end ldquobottom-uprdquo LC approachescan provide insight into the binding of a metal-based drugandor its hydrolysis products to mixtures of key plasmaproteins (eg HSA Tf) Results from such studies are usefulto predict the absorption of a metal-based drug into thetarget organ cells via established uptake mechanisms Onthe other hand the addition of a metal-based drug to bloodplasma followed by the LC analysis of the obtained mixtureallows to simultaneously observe the hydrolysis of the parentdrug and the plasma protein binding of the in situ gen-erated hydrolysis products This approach has considerablepotential to advance our understanding of the biochemicalfate of established or synthetic metal-based drug candidatesin plasma which will contribute to potentially unravel themolecular basis of their side effects In addition ldquotop-downrdquoLC approaches are also ideally suited to gain fundamentallynew insight into mechanism of action by which amelioratingagents (eg NAC) modulate the metabolism of metal-baseddrugs (eg cis-platin) in plasma LC can therefore play animportant role with regard to the development of novelstrategies to decrease the adverse health effects ofmetal-baseddrugs while leaving their antitumor effect largely intact [37]

9 Conclusion

In terms of defining the bioinorganic mechanisms by whichmetal species exert toxicity which may eventually resultin pathologies at the organ level we are confronted withconsiderable knowledge gapsThe biomolecular mechanismswhich determine the distribution of particular toxic metalsto target organs for example are largely unknown eventhough their binding sites on key plasma proteins have beenstructurally characterized [181] Similarly the biomolecularbasis of the severe side-effects that clinically usedmetal-baseddrugs exert in patients (eg the intravenously administeredcis-platin) remains elusive and the mechanisms by whichameliorating agents modulate the metabolism of cis-platin tomitigate its side effects essentially remain a terra incognitaCollectively this undesirable situation must be attributed tothe lack of a comprehensive understanding of the reversibleand irreversible interactions that unfold between metalspecies (and their metabolites) with endogenous ligands in

various biological compartments in vivo Since it is preciselythese interactions which definemdashin their entirety mdashthetoxicity that any given metal species (or mixtures thereof)will exert at the organ level (Figure 2) the application ofldquobottom-uprdquo and ldquotop-downrdquo LC-based approaches has thepotential to provide much needed insight into toxicologicallyrelevant aspects of metal-ligand interactions under nearphysiological conditions The concerted application of LC-based methodologies in conjunction with other advancedspectroscopic techniques will play an important role in thequest to better understand the disposition the metabolismthe detoxification the toxicology the abstraction of toxicmetalsmetal-based drugs from proteins and themodulationof the metabolism of metal-based drugs by amelioratingagents Importantly the information that can be obtainedfrom such studies must be integrated into the wider bio-chemical context to establish progressively more refinedmodels of the metabolism of the metal species of interestOverall the application of LC to ldquometal species in biologyrdquorelated problems has the potential to establish maximumtolerable doses of a given metal species that may be ingestedor injected before a particular biological networksystemis perturbed which would facilitate the interpretation ofbiomonitoring studies [75] ldquoTop-downrdquo LC approaches areparticularly suited to play a leading role in the context ofdeveloping better chelating agents to mobilize toxic metalsfrom organisms to possibly gain insight into the side effectsof metal-based drugs and to identify ldquoameliorating agentsrdquowhich may be coadministered along with metal-based drugsto mitigate their often severe side-effects in patients Theinterdisciplinary nature of this research endeavormdashat theinterface of physiology biology inorganic chemistry bio-chemistry analytical chemistry toxicology and medicinemdashis very much in the spirit of the father of LC In fact theinscription on MS Tswettrsquos gravestone reads ldquoHe inventedchromatography separating molecules but uniting peoplesrdquoMay this notion continue to serve as a guiding principle asLC-based approaches will continue to serve humanity wellinto the 21st century

References

[1] M Tswett ldquoPhysical chemical studies on chlorophyll adsorp-tionsrdquo Berichte der Deutschen botanischen Gesellschaft vol 24pp 316ndash326 1906

[2] A J PMartin andR LM Synge ldquoAnew formof chromatogramemploying two liquid phasesrdquo Biochemical Journal vol 35 pp1358ndash1368 1941

[3] L S Ettre and K I Sakodynskii ldquoMS Tswett and the dis-covery of chromatography I early work (1899ndash1903)rdquo Chro-matographia vol 35 no 3-4 pp 223ndash231 1993

[4] A Zotou ldquoAn overview of recent advances inHPLC instrumen-tationrdquo Central European Journal of Chemistry vol 10 no 3 pp554ndash569 2012

[5] K K Unger and A I Liapis ldquoAdsorbents and columns in ana-lytical high-performance liquid chromatography a perspectivewith regard to development and understandingrdquo Journal ofSeparation Science vol 35 no 10-11 pp 1201ndash1212 2012


[6] D Ishii K Asai K Hibi T Jonokuchi andM Nagaya ldquoA studyof micro-high-performance liquid chromatography I Devel-opment of technique for miniaturization of high-performanceliquid chromatographyrdquo Journal of Chromatography vol 144no 2 pp 157ndash168 1977

[7] C G Horvath B A Preiss and S R Lipsky ldquoFast liquidchromatography an investigation of operating parameters andthe separation of nucleotides on pellicular ion exchangersrdquoAnalytical Chemistry vol 39 no 12 pp 1422ndash1428 1967

[8] P D McDonald and D Patrick ldquoFifty years of innovation inanalysis and purificationrdquo Chemical Heritage vol 26 no 2 pp32ndash37 2008

[9] M E Swartz ldquoUPLC an introduction and reviewrdquo Journal ofLiquid Chromatography amp Related Technologies vol 28 no 7-8pp 1253ndash1263 2005

[10] L Anderson and C L Hunter ldquoQuantitative mass spectro-metric multiple reaction monitoring assays for major plasmaproteinsrdquoMolecularampCellular Proteomics vol 5 no 4 pp 573ndash588 2006

[11] D J States G S Omenn T W Blackwell et al ldquoChallengesin deriving high-confidence protein identifications from datagathered by a HUPO plasma proteome collaborative studyrdquoNature Biotechnology vol 24 no 3 pp 333ndash338 2006

[12] S P Gygi B Rist T J Griffin et al ldquoProteome analysis of low-abundance proteins using multidimensional chromatographyand isotope-coded affinity tagsrdquo Journal of Proteome Researchvol 1 no 1 pp 47ndash54 2002

[13] D S Wishart ldquoAdvances in metabolite identificationrdquo Bioanal-ysis vol 3 no 15 pp 1769ndash1782 2011

[14] K A Francesconi and F Pannier ldquoSelenium metabolites inurine a critical overview of past work and current statusrdquoClinical Chemistry vol 50 no 12 pp 2240ndash2253 2004

[15] R F Service ldquoProteomics ponders prime timerdquo Science vol321 no 5897 pp 1758ndash1761 2008

[16] J K Nicholson and J C Lindon ldquoSystems biology metabo-nomicsrdquo Nature vol 455 no 7216 pp 1054ndash1056 2008

[17] S C Booth M L Workentine A M Weljie and R J TurnerldquoMetabolomics and its application to studying metal toxicityrdquoMetallomics vol 3 pp 1142ndash1152 2011

[18] J van der Greef P Stroobant and R van der Heijden ldquoTherole of analytical sciences in medical systems biologyrdquo CurrentOpinion in Chemical Biology vol 8 no 5 pp 559ndash565 2004

[19] L Hood ldquoA systems approach to medicine will transformhealthcarerdquo in Physical Biology from Atoms to Medicine A HZewail Ed pp 337ndash366 Imperial College Press London UK2008

[20] G Xindu and W Lili ldquoLiquid chromatography of recombinantproteins and protein drugsrdquo Journal of Chromatography B vol866 no 1-2 pp 133ndash153 2008

[21] R J McGorrin ldquoOne hundred years of progress in foodanalysisrdquo Journal of Agricultural and Food Chemistry vol 57 no18 pp 8076ndash8088 2009

[22] L S Jackson ldquoChemical food safety issues in the United Statespast present and futurerdquo Journal of Agricultural and FoodChemistry vol 57 no 18 pp 8161ndash8170 2009

[23] S J Hill A Fisher and M Foulkes ldquoBasic concepts and instru-mentation for plasma spectrometryrdquo in Inductively CoupledPlasma Spectrometry and Its Applications S J Hill Ed pp 61ndash97 Blackwell Publishing Ames Iowa USA 2007

[24] F van Heuveln H Meijering and J Wieling ldquoInductivelycoupled plasma-MS in drug development bioanalytical aspectsand applicationsrdquo Bioanalysis vol 4 no 15 pp 1933ndash1965 2012

[25] S Mounicou J Szpunar and R Lobinski ldquoMetallomics theconcept and methodologyrdquo Chemical Society Reviews vol 38no 4 pp 1119ndash1138 2009

[26] J L Gomez-Ariza E Z Jahromi M Gonzalez-FernandezT Garcıa-Barrera and J Gailer ldquoLiquid chromatography-inductively coupled plasma-based metallomic approaches toprobe health-relevant interactions between xenobiotics andmammalian organismsrdquoMetallomics vol 3 no 6 pp 566ndash5772011

[27] J P Barnett D J Scanlan and C A Blindauer ldquoProtein frac-tionation and detection for metalloproteomics challenges andapproachesrdquo Analytical and Bioanalytical Chemistry vol 402no 10 pp 3311ndash3322 2012

[28] J Szpunar ldquoAdvances in analytical methodology for bioinor-ganic speciation analysis metallomics metalloproteomics andheteroatom-tagged proteomics and metabolomicsrdquo Analystvol 130 no 4 pp 442ndash465 2005

[29] R Dufault B LeBlanc R Schnoll et al ldquoMercury from chlor-alkali plants measured concentrations in food product sugarrdquoEnvironmental Health vol 8 article 2 2009

[30] D Gilbert-Diamond K L Cottingham J F Gruber et al ldquoRiceconsumption contributes to arsenic exposure in US womenrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 51 pp 20656ndash20660 2011

[31] H H Harris I J Pickering and G N George ldquoThe chemicalform ofmercury in fishrdquo Science vol 301 no 5637 p 1203 2003

[32] J Gailer ldquoArsenic-selenium and mercury-selenium bonds inbiologyrdquo Coordination Chemistry Reviews vol 251 no 1-2 pp234ndash254 2007

[33] A Bhatnagar ldquoEnvironmental cardiology studying mecha-nistic links between pollution and heart diseaserdquo CirculationResearch vol 99 no 7 pp 692ndash705 2006

[34] J Gailer ldquoProbing the bioinorganic chemistry of toxic metals inthemammalian bloodstream to advance humanhealthrdquo Journalof Inorganic Biochemistry vol 108 pp 128ndash132 2012

[35] E Z Jahromi and J Gailer ldquoProbing bioinorganic chemistryprocesses in the bloodstream to gain new insights into the originof human diseasesrdquoDalton Transactions vol 39 no 2 pp 329ndash336 2010

[36] E Z Jahromi and J Gailer ldquoIn vitro assessment of chelatingagents with regard to their abstraction efficiency of Cd2+ boundto plasma proteinsrdquo Metallomics vol 4 no 9 pp 995ndash10032012

[37] M Sooriyaarachchi A Narendran and J Gailer ldquoThe effect ofsodium thiosulfate on the metabolism of cis-platin in humanplasma in vitrordquoMetallomics vol 4 no 9 pp 960ndash967 2012

[38] M Montes-Bayon K DeNicola and J A Caruso ldquoLiquidchromatography-inductively coupled plasma mass spectrome-tryrdquo Journal of Chromatography A vol 1000 no 1-2 pp 457ndash476 2003

[39] J Lopez-Barea and J L Gomez-Ariza ldquoEnvironmental pro-teomics and metallomicsrdquo Proteomics vol 6 supplement 1 ppS51ndashS62 2006

[40] M A O da Silva A Sussulini and M A Z Arruda ldquoMetallo-proteomics as an interdisciplinary area involving proteins andmetalsrdquo Expert Review of Proteomics vol 7 no 3 pp 387ndash4002010

[41] W Shi and M R Chance ldquoMetalloproteomics forward andreverse approaches in metalloprotein structural and functionalcharacterizationrdquo Current Opinion in Chemical Biology vol 15no 1 pp 144ndash148 2011


[42] W Mertz ldquoThe essential trace elementsrdquo Science vol 213 no4514 pp 1332ndash1338 1981

[43] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReviews of Physiology vol 69 pp 69ndash85 2007

[44] WMaret andY Li ldquoCoordination dynamics of zinc in proteinsrdquoChemical Reviews vol 109 no 10 pp 4682ndash4707 2009

[45] J T Rubino and K J Franz ldquoCoordination chemistry ofcopper proteins how nature handles a toxic cargo for essentialfunctionrdquo Journal of Inorganic Biochemistry vol 107 no 1 pp129ndash143 2012

[46] S M Yannone S Hartung A L Menon M W W Adamsand J A Tainer ldquoMetals in biology definingmetalloproteomesrdquoCurrent Opinion in Biotechnology vol 23 no 1 pp 89ndash95 2012

[47] S L Sensi P Paoletti A I Bush and I Sekler ldquoZinc inthe physiology and pathology of the CNSrdquo Nature ReviewsNeuroscience vol 10 no 11 pp 780ndash791 2009

[48] K J Waldron J C Rutherford D Ford and N J RobinsonldquoMetalloproteins and metal sensingrdquo Nature vol 460 no 7257pp 823ndash830 2009

[49] W Maret ldquoMetalloproteomics metalloproteomes and theannotation of metalloproteinsrdquo Metallomics vol 2 no 2 pp117ndash125 2010

[50] J K Nicholson and I D Wilson ldquoUnderstanding lsquoglobalrsquo sys-tems biology metabonomics and the continuum of metabol-ismrdquo Nature Reviews Drug Discovery vol 2 no 8 pp 668ndash6762003

[51] J L Peters T S Perlstein M J Perry E McNeely and JWeuveldquoCadmium exposure in association with history of stroke andheart failurerdquo Environmental Research vol 110 no 2 pp 199ndash206 2010

[52] O Andersen ldquoChemical and biological considerations in thetreatment of metal intoxications by chelating agentsrdquo Mini-Reviews in Medicinal Chemistry vol 4 no 1 pp 11ndash21 2004

[53] M Sooriyaarachchi A Narendran and J Gailer ldquoComparativehydrolysis and plasma protein binding of cis-platin and carbo-platin in human plasma in vitrordquo Metallomics vol 3 no 1 pp49ndash55 2011

[54] T W Hambley ldquoDeveloping new metal-based therapeuticschallenges and opportunitiesrdquo Dalton Transactions no 43 pp4929ndash4937 2007

[55] J Reedijk ldquoWhy does cisplatin reach guanine-N7 with compet-ing S-donor ligands available in the cellrdquoChemical Reviews vol99 no 9 pp 2499ndash2510 1999

[56] B H Ali and M S Al Moundhri ldquoAgents ameliorating oraugmenting the nephrotoxicity of cisplatin and other platinumcompounds a review of some recent researchrdquo Food andChemical Toxicology vol 44 no 8 pp 1173ndash1183 2006

[57] K C M Campbell R P Meech J J Klemens et al ldquoPreventionof noise- and drug-induced hearing loss with d-methioninerdquoHearing Research vol 226 no 1-2 pp 92ndash103 2007

[58] G D Zeevalk and W J Nicklas ldquoMechanisms underlying ini-tiation of excitotoxicity associated with metabolic inhibitionrdquoJournal of Pharmacology and Experimental Therapeutics vol257 no 2 pp 870ndash878 1991

[59] S Cestele and W A Catterall ldquoMolecular mechanisms of neu-rotoxin action on voltage-gated sodium channelsrdquo Biochimievol 82 no 9-10 pp 883ndash892 2000

[60] K Cottingham ldquoSystems biology a boon for analytical chem-istsrdquo Analytical Chemistry vol 77 no 9 pp 197Andash200A 2005

[61] J Gailer G N George I J Pickering et al ldquoStructural basisof the antagonism between inorganic mercury and selenium in

mammalsrdquo Chemical Research in Toxicology vol 13 no 11 pp1135ndash1142 2000

[62] J Gailer G N George I J Pickering et al ldquoA metabolic linkbetween arsenite and selenite the seleno-bis(S- glutathionyl)arsinium ionrdquo Journal of the American Chemical Society vol122 no 19 pp 4637ndash4639 2000

[63] J Gailer S Madden G A Buttigieg M B Denton and H SYounis ldquoIdentification of [(GS)

2AsSe]minus in rabbit bile by size-

exclusion chromatography and simultaneous multielement-specific detection by inductively coupled plasma atomic emis-sion spectroscopyrdquo Applied Organometallic Chemistry vol 16no 2 pp 72ndash75 2002

[64] J Gailer L Ruprecht P Reitmeir B Benker and P SchramelldquoMobilization of exogenous and endogenous selenium to bileafter the intravenous administration of environmentally rel-evant doses of arsenite to rabbitsrdquo Applied OrganometallicChemistry vol 18 no 12 pp 670ndash675 2004

[65] J Gailer G N George I J Pickering R C Prince H SYounis and J J Winzerling ldquoBiliary excretion of [(GS)

2AsSe]minus

after intravenous injection of rabbits with arsenite and selenaterdquoChemical Research in Toxicology vol 15 no 11 pp 1466ndash14712002

[66] P Hunter P Robbins and D Noble ldquoThe IUPS humanphysiome projectrdquo Pflugers Archiv vol 445 no 1 pp 1ndash9 2002

[67] L Hood J R Heath M E Phelps and B Lin ldquoSystems biol-ogy and new technologies enable predictive and preventativemedicinerdquo Science vol 306 no 5696 pp 640ndash643 2004

[68] L M Gierasch and A Gershenson ldquoPost-reductionist proteinscience or putting Humpty Dumpty back together againrdquoNature Chemical Biology vol 5 pp 774ndash777 2009

[69] D Noble The Music of Life Biology Beyond Genes OxfordUniversity Press New York NY USA 2006

[70] F R Luo S D Wyrick and S G Chaney ldquoBiotransformationsof oxaliplatin in rat blood in vitrordquo Journal of Biochemical andMolecular Toxicology vol 13 no 3-4 pp 159ndash169 1999

[71] C Jumarie C Fortin M Houde P G C Campbell and FDenizeau ldquoCadmium uptake by Caco-2 cells effects of Cdcomplexation by chloride glutathione and phytochelatinsrdquoToxicology and Applied Pharmacology vol 170 no 1 pp 29ndash382001

[72] R K Zalups ldquoMolecular interactions with mercury in thekidneyrdquo Pharmacological Reviews vol 52 no 1 pp 113ndash1432000

[73] R K Zalups and S Ahmad ldquoMolecular handling of cadmium intransporting epitheliardquo Toxicology and Applied Pharmacologyvol 186 no 3 pp 163ndash188 2003

[74] J L Webb ldquoArsenicalsrdquo in Enzyme and Metabolic Inhibitors JL Webb Ed vol 3 pp 595ndash793 Academic Press London UK1966

[75] S A Manley and J Gailer ldquoAnalysis of the plasma met-alloproteome by SEC-ICP-AES bridging proteomics andmetabolomicsrdquo Expert Review of Proteomics vol 6 no 3 pp251ndash265 2009

[76] GChillemi GMancini N Sanna et al ldquoEvidence for sevenfoldcoordination in the first solvation shell of Hg(II) aqua ionrdquoJournal of the American Chemical Society vol 129 no 17 pp5430ndash5436 2007

[77] G M Whitesides P W Snyder D T Moustakas and KA Mirica ldquoDesigning ligands to bind tightly to proteinsrdquo inPhysical Biology from Atoms to Medicine A H Zewail Ed pp189ndash215 Imperial College Press London UK 2008


[78] J P K Rooney ldquoThe role of thiols dithiols nutritional factorsand interacting ligands in the toxicology of mercuryrdquo Toxicol-ogy vol 234 no 3 pp 145ndash156 2007

[79] A Casini and J Reedijk ldquoInteractions of anticancer Pt com-pounds with proteins an overlooked topic in medicinal inor-ganic chemistryrdquoChemical Science vol 3 no 11 pp 3135ndash31442012

[80] A R Timerbaev C G Hartinger S S Aleksenko and B KKeppler ldquoInteractions of antitumor metallodrugs with serumproteins advances in characterization using modern analyticalmethodologyrdquoChemical Reviews vol 106 no 6 pp 2224ndash22482006

[81] MKnipp ldquoMetallothioneins and platinum(II) anti-tumor com-poundsrdquo Current Medicinal Chemistry vol 16 pp 522ndash5372009

[82] J Gailer ldquoReactive selenium metabolites as targets of toxicmetalsmetalloids in mammals a molecular toxicological per-spectiverdquo Applied Organometallic Chemistry vol 16 no 12 pp701ndash707 2002

[83] W Hu Q Luo K Wu et al ldquoThe anticancer drug cisplatincan cross-link the interdomain zinc site on human albuminrdquoChemical Communications vol 47 no 21 pp 6006ndash6008 2011

[84] J Gailer ldquoChronic toxicity of AsIII in mammals the role of(GS)2AsSeminusrdquo Biochimie vol 91 no 10 pp 1268ndash1272 2009

[85] M T Le J Gailer and E J Prenner ldquoHg2+ and Cd2+ interactdifferently with biomimetic erythrocyte membranesrdquo BioMet-als vol 22 no 2 pp 261ndash274 2009

[86] N Ballatori ldquoTransport of toxic metals by molecular mimicryrdquoEnvironmental Health Perspectives vol 110 no 5 pp 689ndash6942002

[87] R H Holm P Kennepohl and E I Solomon ldquoStructural andfunctional aspects of metal sites in biologyrdquo Chemical Reviewsvol 96 no 7 pp 2239ndash2314 1996

[88] B P Esposito and R Najjar ldquoInteractions of antitumoralplatinum-group metallodrugs with albuminrdquo CoordinationChemistry Reviews vol 232 no 1-2 pp 137ndash149 2002

[89] N Ballatori and A T Truong ldquoMechanisms of hepaticmethylmercury uptakerdquo Journal of Toxicology and Environmen-tal Health vol 46 no 3 pp 343ndash353 1995

[90] N Shimojo Y Kumagai and J Nagafune ldquoDifference betweenkidney and liver in decreased manganese superoxide dismutaseactivity caused by exposure of mice to mercuric chloriderdquoArchives of Toxicology vol 76 no 7 pp 383ndash387 2002

[91] S A Manley G N George I J Pickering et al ldquoThe selenobis(S-glutathionyl) arsinium ion is assembled in erythrocytelysaterdquo Chemical Research in Toxicology vol 19 no 4 pp 601ndash607 2006

[92] A Meister ldquoGlutathione metabolism and its selective modifica-tionrdquo Journal of Biological Chemistry vol 263 no 33 pp 17205ndash17208 1988

[93] J M Mates J A Segura and F J Alonso ldquoRoles of dioxins andheavy metals in cancer and neurological diseases using ROS-mediated mechanismsrdquo Free Radical Biology and Medicine vol49 no 9 pp 1328ndash1341 2010

[94] T J Hagele J N Mazerik A Gregory et al ldquoMercury activatesvascular endothelial cell phospholipase D through thiols andoxidative stressrdquo International Journal of Toxicology vol 26 no1 pp 57ndash69 2007

[95] S Bhattacharya S Bose B Mukhopadhyay et al ldquoSpecificbinding of inorganic mercury to Na- K-ATPase in rat liverplasma membrane and signal transductionrdquo BioMetals vol 10no 3 pp 157ndash162 1997

[96] D R Green and G Kroemer ldquoThe pathophysiology of mito-chondrial cell deathrdquo Science vol 305 no 5684 pp 626ndash6292004

[97] SOrrenius andB Zhivotovsky ldquoThe future of toxicologymdashdoesitmatter how cells dierdquoChemical Research in Toxicology vol 19pp 729ndash733 2006

[98] J D Robertson and S Orrenius ldquoMolecular mechanisms ofapoptosis Induced by cytotoxic chemicalsrdquo Critical Reviews inToxicology vol 30 no 5 pp 609ndash627 2000

[99] U Landegren J Vanelid M Hammond et al ldquoOpportunitiesfor sensitive plasma proteome analysisrdquo Analytical Chemistryvol 84 no 4 pp 1824ndash1830 2012

[100] S A Manley S Byrns A W Lyon P Brown and J GailerldquoSimultaneous Cu- Fe- and Zn-specific detection of met-alloproteins contained in rabbit plasma by size-exclusionchromatography-inductively coupled plasma atomic emissionspectroscopyrdquo JBIC Journal of Biological Inorganic Chemistryvol 14 no 1 pp 61ndash74 2009

[101] P J Sadler and J H Viles ldquo1H and 113Cd NMR investigationsof Cd2+ and Zn2+ binding sites on serum albumin competitionwith Ca2+ Ni2+ Cu2+ and Zn2+rdquo Inorganic Chemistry vol 35no 15 pp 4490ndash4496 1996

[102] D C Carter and J X Ho ldquoStructure of serum albuminrdquoAdvances in Protein Chemistry vol 45 pp 153ndash203 1994

[103] V Sahni D Choudhury and Z Ahmed ldquoChemotherapy-associated renal dysfunctionrdquo Nature Reviews Nephrology vol5 pp 450ndash462 2009

[104] D Wang and S J Lippard ldquoCellular processing of platinumanticancer drugsrdquo Nature Reviews Drug Discovery vol 4 pp307ndash320 2005

[105] F Caurant M Navarro and J C Amiard ldquoMercury in pilotwhales possible limits to the detoxification processrdquo Science ofthe Total Environment vol 186 no 1-2 pp 95ndash104 1996

[106] M Korbas J L O rsquoDonoghue G E Watson et al ldquoThe chemi-cal nature of mercury in human brain following poisoning orenvironmental exposurerdquo ACS Chemical Neuroscience vol 1no 12 pp 810ndash818 2010

[107] J P Berry and P Galle ldquoSelenium-arsenic interaction in renalcells role of lysosomes Electron microprobe studyrdquo Journal ofSubmicroscopic Cytology and Pathology vol 26 no 2 pp 203ndash210 1994

[108] S Hann G Koellensperger Z Stefanka et al ldquoApplication ofHPLC-ICP-MS to speciation of cisplatin and its degradationproducts in water containing different chloride concentrationsand in human urinerdquo Journal of Analytical Atomic Spectrometryvol 18 pp 1391ndash1395 2003

[109] Z Gregus and C D Klaassen ldquoDisposition of metals in rats acomparative study of fecal urinary and biliary excretion andtissue distribution of eighteen metalsrdquo Toxicology and AppliedPharmacology vol 85 no 1 pp 24ndash38 1986

[110] K T Suzuki ldquoDirect connection of high-speed liquid chro-matograph (equipped with gel permeation column) to atomicabsorption spectrophotometer for metalloprotein analysismetallothioneinrdquoAnalytical Biochemistry vol 102 no 1 pp 31ndash34 1980

[111] K T Suzuki H Sunaga E Kobayashi and N ShimojoldquoMercaptalbumin as a selective cadmium-binding protein in ratserumrdquo Toxicology and Applied Pharmacology vol 86 no 3 pp466ndash473 1986

[112] S J Berners-Price and P J Sadler ldquoCoordination chemistryof metallodrugs insights into biological speciation from NMR


spectroscopyrdquo Coordination Chemistry Reviews vol 151 pp 1ndash40 1996

[113] M Groessl and P J Dyson ldquoBioanalytical and biophysicaltechniques for the elucidation of the mode of action of metal-based drugsrdquo Current Topics in Medicinal Chemistry vol 11 no21 pp 2632ndash2646 2011

[114] V Mah and F Jalilehvand ldquoCadmium(II) complex formationwith glutathionerdquo JBIC Journal of Biological Inorganic Chem-istry vol 15 no 3 pp 441ndash458 2010

[115] R Ortega A Carmona I Llorens and P L Solari ldquoX-rayabsorption spectroscopy of biological samples A tutorialrdquo Jour-nal of Analytical Atomic Spectrometry vol 27 pp 2054ndash20652012

[116] N Cetinbas M I Webb J A Dubland and C J WalsbyldquoSerum-protein interactions with anticancer Ru(III) complexesKP1019 and KP418 characterized by EPRrdquo JBIC Journal ofBiological Inorganic Chemistry vol 15 no 2 pp 131ndash145 2010

[117] Y Kasherman S Sturup and D Gibson ldquoIs glutathione themajor cellular target of cisplatin A study of the interactionsof cisplatin with cancer cell extractsrdquo Journal of MedicinalChemistry vol 52 no 14 pp 4319ndash4328 2009

[118] D Gibson ldquoThe mechanism of action of platinum anticanceragentsmdashwhat do we really know about itrdquoDalton Transactionsno 48 pp 10681ndash10689 2009

[119] E Z Jahromi W White Q Wu R Yamdagni and J GailerldquoRemarkable effect of mobile phase buffer on the SEC-ICP-AESderived Cu Fe and Zn-metalloproteome pattern of rabbit bloodplasmardquoMetallomics vol 2 no 7 pp 460ndash468 2010

[120] C C Wu P H Peng Y T Chang et al ldquoIdentification ofpotential serum markers for nasopharyngeal carcinoma from axenograftedmousemodel usingCy-dye labeling combinedwiththree-dimensional fractionationrdquo Proteomics vol 8 no 17 pp3605ndash3620 2008

[121] K L Pei M Sooriyaarachchi D A Sherrell G N George andJ Gailer ldquoProbing the coordination behavior of Hg2+ CH

3Hg+

and Cd2+ towards mixtures of two biological thiols by HPLC-ICP-AESrdquo Journal of Inorganic Biochemistry vol 105 no 3 pp375ndash381 2011

[122] E K M Ueda P W Gout and L Morganti ldquoCurrent andprospective applications of metal ion-protein bindingrdquo Journalof Chromatography A vol 988 no 1 pp 1ndash23 2003

[123] L Andersson E Sulkowski and J Porath ldquoImmobilized metalion affinity chromatography of serumalbuminsrdquoBioseparationvol 2 no 1 pp 15ndash22 1991

[124] L Gelunaite V Luksa O Sudziuviene V Bumelis and HPesliakas ldquoChelated mercury as a ligand in immobilized metalion affinity chromatography of proteinsrdquo Journal of Chromatog-raphy A vol 904 no 2 pp 131ndash143 2000

[125] A Meister and M E Anderson ldquoGlutathionerdquo Annual Reviewof Biochemistry vol 52 pp 711ndash760 1983

[126] N Kaplowitz T Y Aw and M Ookhtens ldquoThe regulationof hepatic glutathionerdquo Annual Review of Pharmacology andToxicology vol 25 pp 715ndash744 1985

[127] K L Haas and K F Franz ldquoApplication of metal coordinationchemistry to explore and manipulate cell biologyrdquo ChemicalReviews vol 109 no 10 pp 4921ndash4960

[128] R K Singhal M E Anderson and A Meister ldquoGlutathione afirst line of defense against cadmium toxicityrdquo FASEB Journalvol 1 no 3 pp 220ndash223 1987

[129] A Naganuma M E Anderson and A Meister ldquoCellularglutathione as a determinant of sensitivity to mercuric chlo-ride toxicity prevention of toxicity by giving glutathione

monoesterrdquo Biochemical Pharmacology vol 40 no 4 pp 693ndash697 1990

[130] D L Rabenstein ldquoMetal complexes of glutathione and theirbiological significancerdquo in Glutathione Chemical BiochemicalandMedical Aspects DDolphinOAvramovic andR PoulsonEds pp 147ndash186 JohnWiley amp Sons NewYork NY USA 1989

[131] J Gailer G N George I J Pickering G A Buttigieg M BDenton and R S Glass ldquoSynthesis X-ray absorption spec-troscopy and purification of the seleno-bis (S-glutathionyl)arsinium anion from selenide arsenite and glutathionerdquo Journalof Organometallic Chemistry vol 650 no 1-2 pp 108ndash113 2002

[132] L H Lash and D P Jones ldquoDistribution of oxidized andreduced forms of glutathione and cysteine in rat plasmardquoArchives of Biochemistry and Biophysics vol 240 no 2 pp 583ndash592 1985

[133] B Sebille N Thuaud and J P Tillement ldquoRetention datamethods for the determination of drug-protein binding param-eters by high-performance liquid chromatographyrdquo Journal ofChromatography vol 204 pp 285ndash291 1981

[134] B Sebille and N Thuaud ldquoDetermination of tryptophan-human serum albumin binding from retention data and sep-aration of tryptophan enantiomer by high performance liquidchromatographyrdquo Journal of Liquid Chromatography vol 3 no2 pp 299ndash308 1980

[135] J Gailer and W Lindner ldquoOn-column formation of arsenic-glutathione species detected by size-exclusion chromatographyin conjunction with arsenic-specific detectorsrdquo Journal of Chro-matography B vol 716 pp 83ndash93 1998

[136] A J Percy and J Gailer ldquoMethylated trivalent arsenic-glutathione complexes are more stable than their arseniteanalogrdquo Bioinorganic Chemistry and Applications vol 2008Article ID 539082 8 pages 2008

[137] K Rehman and H Naranmandura ldquoArsenic metabolism andthioarsenicalsrdquoMetallomics vol 4 no 9 pp 881ndash892 2012

[138] SVKalaMWNeelyGKala et al ldquoTheMRP2cMOAT trans-porter and arsenic-glutathione complex formation are requiredfor biliary excretion of arsenicrdquo Journal of Biological Chemistryvol 275 no 43 pp 33404ndash33408 2000

[139] E M Leslie ldquoArsenic-glutathione conjugate transport by thehuman multidrug resistance proteins (MRPsABCCs)rdquo Journalof Inorganic Biochemistry vol 108 pp 141ndash149 2012

[140] E M Leslie R G Deely and S P C Cole ldquoToxicological rele-vance of themultidrug resistance protein 1 MRP1 (ABCC1) andrelated transportersrdquo Toxicology vol 167 no 1 pp 3ndash23 2001

[141] N Ballatori S M Krance R Marchan and C L HammondldquoPlasma membrane glutathione transporters and their rolesin cell physiology and pathophysiologyrdquo Molecular Aspects ofMedicine vol 30 no 1-2 pp 13ndash28 2009

[142] R S Braman and C C Foreback ldquoMethylated forms of arsenicin the environmentrdquo Science vol 182 no 4118 pp 1247ndash12491973

[143] A J Percy M Korbas G N George and J Gailer ldquoReversed-phase high-performance liquid chromatographic separationof inorganic mercury and methylmercury driven by theirdifferent coordination chemistry towards thiolsrdquo Journal ofChromatography A vol 1156 no 1-2 pp 331ndash339 2007

[144] S Balaz M Wiese and J K Seydel ldquoA time hierarchy-basedmodel for kinetics of drug disposition and its use in quantita-tive structure-activity relationshipsrdquo Journal of PharmaceuticalSciences vol 81 no 9 pp 849ndash857 1992


[145] J D Meers E Z Jahromi B Heyne and J Gailer ldquoImprovedRP-HPLC separation of Hg2+ and CH

3Hg+ using a mixture of

thiol-based mobile phase additivesrdquo Journal of EnvironmentalScience and Health A vol 47 no 1 pp 149ndash154 2012

[146] GGirardi andMM Elias ldquoEffectiveness ofN-acetylcysteine inprotecting against mercuric chloride-induced nephrotoxicityrdquoToxicology vol 67 no 2 pp 155ndash164 1991

[147] N Ballatori MW Lieberman andWWang ldquoN-acetylcysteineas an antidote in methylmercury poisoningrdquo EnvironmentalHealth Perspectives vol 106 no 5 pp 267ndash271 1998

[148] A S Koh T A Simmons-Willis J B Pritchard S M Grassland N Ballatori ldquoIdentification of a mechanism by whichthe methylmercury antidotes N-acetylcysteine and dimercap-topropanesulfonate enhance urinary metal excretion transportby the renal organic anion transporter-1rdquoMolecular Pharmacol-ogy vol 62 no 4 pp 921ndash926 2002

[149] E Hjortsoslash J S Fomsgaard and N Fogh-Andersen ldquoDoes N-acetylcysteine increase the excretion of trace metals (calciummagnesium iron zinc and copper) when given orallyrdquo Euro-pean Journal of Clinical Pharmacology vol 39 no 1 pp 29ndash311990

[150] L Chapman and H M Chan ldquoThe influence of nutrition onmethyl mercury intoxicationrdquo Environmental Health Perspec-tives vol 108 no 1 pp 29ndash56 2000

[151] R K Zalups and L H Lash ldquoInteractions between glutathioneand mercury in the kidney liver and bloodrdquo in Toxicology ofMetals L W Chang Ed pp 145ndash163 CRC Lewis PublishersBoca Raton Fla USA 1996

[152] EMitchell S Frisbie and B Sarkar ldquoExposure tomultiple met-als from groundwater-a global crisis geology climate changehealth effects testing and mitigationrdquo Metallomics vol 3 no9 pp 874ndash908 2011

[153] K P DuBois A L Moxon and O E Olson ldquoFurther studies onthe effective ness of arsenic in preventing selenium poisoningrdquoJournal of Nutrition vol 19 pp 477ndash482 1940

[154] B Michalke ldquoSelenspeziation mit SAX-ICP-MS und RPLC-ICP-MSrdquo in Moderne Techniken Der Ionenanalyse K Fischerand D Jensen Eds pp 50ndash60 ECOMED VerlagsgesellschaftLandsberg Germany 2002

[155] J Gailer S Madden M F Burke M B Denton and H VAposhian ldquoSimultaneous multielement-specific detection of anovel glutathione-arsenic-selenium ion [(GS)

2AsSe]minus by ICP

AES after micellar size- exclusion chromatographyrdquo AppliedOrganometallic Chemistry vol 14 no 7 pp 355ndash363 2000

[156] J Parizek and I Ostadalova ldquoThe protective effect of smallamounts of selenite in sublimate intoxicationrdquo Experientia vol23 no 2 pp 142ndash143 1967

[157] S Yoneda and K T Suzuki ldquoEquimolar Hg-Se complex bindsto selenoprotein Prdquo Biochemical and Biophysical Research Com-munications vol 231 no 1 pp 7ndash11 1997

[158] M M El-Begearmi H E Ganther and M L Sunde ldquoDietaryinteraction between methylmercury selenium arsenic andsulfur amino acids in Japanese quailrdquo Poultry Science vol 61no 2 pp 272ndash279 1982

[159] A Naganuma and N Imura ldquoMethylmercury binds to a lowmolecular weight substance in rabbit and human erythrocytesrdquoToxicology andApplied Pharmacology vol 47 no 3 pp 613ndash6161979

[160] Y Sugiura Y Tamai and H Tanaka ldquoSelenium protectionagainst mercury toxicity high binding affinity of methylmer-cury by selenium-containing ligands in comparisonwith sulfur-containing ligandsrdquo Bioinorganic Chemistry vol 9 no 2 pp167ndash180 1978

[161] M Korbas A J Percy J Gailer and G N George ldquoA possiblemolecular link between the toxicological effects of arsenicselenium and methylmercury methylmercury(II) seleno bis(S-glutathionyl) arsenic(III)rdquo JBIC Journal of Biological InorganicChemistry vol 13 no 3 pp 461ndash470 2008

[162] T W Hambley ldquoChemistry metal-based therapeuticsrdquo Sciencevol 318 no 5855 pp 1392ndash1393 2007

[163] K HThompson and C Orvig ldquoBoon and bane of metal ions inmedicinerdquo Science vol 300 no 5621 pp 936ndash939 2003

[164] M J Abrams and B A Murrer ldquoMetal compounds in therapyand diagnosisrdquo Science vol 261 no 5122 pp 725ndash730 1993

[165] A M Pizarro and P J Sadler ldquoUnusual DNA binding modesfor metal anticancer complexesrdquo Biochimie vol 91 no 10 pp1198ndash1211 2009

[166] SVanZutphen and J Reedijk ldquoTargeting platinumanti-tumourdrugs overview of strategies employed to reduce systemictoxicityrdquo Coordination Chemistry Reviews vol 249 no 24 pp2845ndash2853 2005

[167] T W Hambley ldquoThe influence of structure on the activityand toxicity of Pt anti-cancer drugsrdquo Coordination ChemistryReviews vol 166 pp 181ndash223 1997

[168] B W Harper A M Krause-Heuer M P Grant M ManoharK B Garbutcheon-Singh and J R Aldrich-Wright ldquoAdvancesin platinum chemotherapeuticsrdquo Chemistry vol 16 no 24 pp7064ndash7077 2010

[169] J C Dabrowiak J Goodisman and A K Souid ldquoKinetic studyof the reaction of cisplatin with thiolsrdquo Drug Metabolism andDisposition vol 30 no 12 pp 1378ndash1384 2002

[170] T Ishikawa and F Ali-Osman ldquoGlutathione-associatedcis-diamminedichloroplatinum(II) metabolism andATP-dependent efflux from leukemia cells Molecularcharacterization of glutathione-platinum complex and itsbiological significancerdquo Journal of Biological Chemistry vol268 no 27 pp 20116ndash20125 1993

[171] K W Lee and D S J Martin ldquoCis-dichlorodiamminepl-atinum(II) Aquation equilibria and isotopic exchange of chlo-ride ligands with free chloride and tetrachloroplatinate(II)rdquoInorganica Chimica Acta vol 17 pp 105ndash110 1976

[172] A V Klein and T W Hambley ldquoPlatinum drug distribution incancer cells and tumorsrdquo Chemical Reviews vol 109 no 10 pp4911ndash4920 2009

[173] X Wang and Z Guo ldquoThe role of sulfur in platinum anticancerchemotherapyrdquoAnti-Cancer Agents inMedicinal Chemistry vol7 no 1 pp 19ndash34 2007

[174] A I I Ivanov J Christodoulou J A Parkinson et al ldquoCisplatinbinding sites on human albuminrdquo The Journal of BiologicalChemistry vol 273 no 24 pp 14721ndash14730 1998

[175] L Trynda-Lemiesz H Kozlowski and B K Keppler ldquoEffect ofcis- trans-diamminedichloroplatinum(II) and DBP on humanserum albuminrdquo Journal of Inorganic Biochemistry vol 77 no3-4 pp 141ndash146 1999

[176] N Ohta D Chen S Ito T Futo T Yotsuyanagi andK Ikeda ldquoEffect of trans-diamminedichloroplatinum(II) onhuman serum albumin conformational changes through par-tial disulfide bond cleavagerdquo International Journal of Pharma-ceutics vol 118 no 1 pp 85ndash93 1995


[177] D Esteban-Fernandez M Montes-Bayon E B Gonzalez MM Gomez-GomezM A Palacios and A Sanz-Medel ldquoAtomic(HPLC-ICP-MS) and molecular mass spectrometry (ESI-Q-TOF) to study cis-platin interactions with serum proteinsrdquoJournal of Analytical Atomic Spectrometry vol 23 pp 378ndash3842008

[178] H Sun H Li and P J Sadler ldquoTransferrin as a metal ionmediatorrdquo Chemical Reviews vol 99 no 9 pp 2817ndash2842 1999

[179] J M El Hage Chahine M Hemadi and N T Ha-DuongldquoUptake and release of metal ions by transferrin and interactionwith receptorrdquoBiochimica et Biophysica Acta vol 1820 no 3 pp334ndash347 2012

[180] M Groessl M Terenghi A Casini L Elviri R Lobinski andP J Dyson ldquoReactivity of anticancer metallodrugs with serumproteins new insights from size exclusion chromatography-ICP-MS and ESI-MSrdquo Journal of Analytical Atomic Spectrom-etry vol 25 no 3 pp 305ndash313 2010

[181] A J Stewart C A Blindauer S Berezenko D Sleep and P JSadler ldquoInterdomain zinc site on human albuminrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 100 no 7 pp 3701ndash3706 2003

[182] C B Kissinger and P T Kissinger ldquoYour bioanalytical data areonly as good as your samplesrdquoBioanalysis vol 4 no 12 pp 1411ndash1415 2012

[183] S Doker S Mounicou M Dogan and R Lobinski ldquoProbingthe metal-homeostatis effects of the administration of chrom-ium(vi) tomice by ICPMS and size-exclusion chromatography-ICP MSrdquoMetallomics vol 2 no 8 pp 549ndash555 2010

[184] M Gonzalez-Fernandez M A Garcıa-Sevilliano R Jara-Biedma et al ldquoSize characterization of metal species in liverand brain from free-living (Mus spretus) and laboratory (MusMusculus) mice by SEC-ICP-MS application to environmentalcontamination assessmentrdquo Journal of Analytical Atomic Spec-trometry vol 26 no 1 pp 141ndash149 2011

[185] RMontes-Nieto C A Fuentes-Almagro D Bonilla-Valverde etal ldquoProteomics in free-livingMus spretus tomonitor terrestrialecosystemsrdquo Proteomics vol 7 no 23 pp 4376ndash4387 2007

[186] J Riuz-Laguna N Abril T Garcıa-Barrera J L Gomez-ArizaJ Lopez-Barea and C Pueyo ldquoAbsolute transcript expressionsignatures of Cyp and Gst genes in Mus spretus to detectenvironmental contaminationrdquo Environmental Science amp Tech-nology vol 40 no 11 pp 3646ndash3652 2006

[187] Y H Huang C M Shih C J Huang et al ldquoEffects of cadmiumon structure and enzymatic activity of Cu Zn-SOD and oxida-tive status in neural cellsrdquo Journal of Cellular Biochemistry vol98 no 3 pp 577ndash589 2006

[188] L E Scott and C Orvig ldquoMedicinal inorganic chemistryapproaches to passivation and removal of aberrant metal ionsin diseaserdquo Chemical Reviews vol 109 no 10 pp 4885ndash49102009

[189] W B Cannon ldquoOrganization for physiological homeostasisrdquoPhysiological Reviews vol 9 no 3 pp 399ndash431 1929

[190] W J Crinnion and J Q Tran ldquoOrganic foods contain higherlevels of certain nutrients lower levels of pesticides and mayprovide health benefits for the consumerrdquo Alternative MedicineReview vol 15 pp 303ndash310 2010

[191] L Charlet Y Chapron P Faller R Kirsch A T Stone andP C Baveye ldquoNeurodegenerative diseases and exposure to theenvironmentalmetalsMn Pb andHgrdquoCoordinationChemistryReviews vol 256 no 19-20 pp 2147ndash2163 2012

[192] G N George R C Prince J Gailer et al ldquoMercury binding tothe chelation therapy agents DMSA and DMPS and the rational

design of custom chelators for mercuryrdquo Chemical Research inToxicology vol 17 no 8 pp 999ndash1006 2004

[193] J W Scannell A Blanckley H Boldon and B WarringtonldquoDiagnosing the decline in pharmaceutical RampD efficiencyrdquoNature Reviews Drug Discovery vol 11 pp 191ndash200 2012

[194] D Malakoff ldquoCan treatment costs be tamedrdquo Science vol 331no 6024 pp 1545ndash1547 2011

[195] S Castellino ldquoMALDI imagingMS analysis of drug distributionin tissue the right time()rdquo Bioanalysis vol 4 no 21 pp 2549ndash2551 2012

[196] S Tuncel F Dumoulin J Gailer et al ldquoA set of highly water-soluble tetraethyleneglycol-substituted Zn(II) phthalocyaninessynthesis photochemical and photophysical properties inter-action with plasma proteins and in vitro phototoxicityrdquo DaltonTransactions vol 40 no 16 pp 4067ndash4079 2011

[197] J Moretto B Chauffert F Ghiringhelli J R Aldrich-Wrightand F Bouyer ldquoDiscrepancy between in vitro and in vivoantitumor effect of a new platinum(II) metallointercalatorrdquoInvestigational New Drugs vol 29 no 6 pp 1164ndash1176 2011

[198] J Szpunar A Makarov T Pieper B K Keppler and R Lobin-ski ldquoInvestigation of metallodrug-protein interactions by size-exclusion chromatography coupled with inductively coupledplasma mass spectrometry (ICP-MS)rdquo Analytica Chimica Actavol 387 no 2 pp 135ndash144 1999

[199] R Mandal R Kalke and X F Li ldquoInteraction of oxaliplatincisplatin and carboplatin with hemoglobin and the resultingrelease of a heme grouprdquo Chemical Research in Toxicology vol17 no 10 pp 1391ndash1397 2004

[200] R Baliga Z Zhang M Baliga N Ueda and S V Shah ldquoInvitro and in vivo evidence suggesting a role for iron in cisplatin-induced nephrotoxicityrdquo Kidney International vol 53 no 2 pp394ndash401 1998

[201] V Calderone A Casini SMangani LMessori and P L OriolildquoStructural investigation of cisplatin-protein interactions selec-tive platination of His19 in a cuprozinc superoxide dismutaserdquoAngewandte Chemie vol 45 no 8 pp 1267ndash1269 2006

[202] X Sun C N Tsang and H Sun ldquoIdentification and charac-terization of metallodrug binding proteins by (metallo) pro-teomicsrdquoMetallomics vol 1 no 1 pp 25ndash31 2009

[203] K L Pei and J Gailer ldquoProbing the interaction of arsenobetainewith blood plasma constituents in vitro an SEC-ICP-AESstudyrdquoMetallomics vol 1 no 5 pp 403ndash408 2009

[204] DA Smith L Di and EHKerns ldquoThe effect of plasma proteinbinding on in vivo efficacy misconceptions in drug discoveryrdquoNature Reviews Drug Discovery vol 9 pp 929ndash939 2010

[205] M Kirberger and J J Yang ldquoStructural differences betweenPb2+- and Ca2+-binding sites in proteins implications withrespect to toxicityrdquo Journal of Inorganic Biochemistry vol 102no 10 pp 1901ndash1909 2008

[206] J M Swanson S Entringer C Buss and P DWadhwa ldquoDevel-opmental origins of health and disease environmental expo-suresrdquo Seminars in ReproductiveMedicine vol 27 no 5 pp 391ndash402 2009

[207] L Trasande P J Landrigan and C Schechter ldquoPublic healthand economic consequences of methyl mercury toxicity to thedeveloping brainrdquo Environmental Health Perspectives vol 113no 5 pp 590ndash596 2005

[208] L D Hylander and M E Goodsite ldquoEnvironmental costs ofmercury pollutionrdquo Science of the Total Environment vol 368no 1 pp 352ndash370 2006


[209] J J Wirth and R S Mijal ldquoAdverse effects of low level heavymetal exposure onmale reproductive functionrdquo Systems Biologyin Reproductive Medicine vol 56 no 2 pp 147ndash167 2010

[210] L Jarup ldquoHazards of heavy metal contaminationrdquo BritishMedical Bulletin vol 68 no 1 pp 167ndash182 2003

[211] B H Robinson ldquoE-waste an assessment of global productionand environmental impactsrdquo Science of the Total Environmentvol 408 no 2 pp 183ndash191 2009

[212] G Zheng X Xu K Wu T A Yekeen and X Huo ldquoAssociationbetween lung function in school children and exposure to threetransition metals from an e-waste recycling areardquo Journal ofExposure Science and Environmental Epidemiology vol 23 pp67ndash72 2013

[213] S-R Lim D Kang O A Ogunseitan and J M SchoenungldquoPotential environmental impacts from the metals in incandes-cent compact fluorescent lamp (CFL) and light-emitting diode(LED) bulbsrdquo Environmental Science and Technology vol 47 no2 pp 1040ndash1047 2013

[214] Y Liu C Wen and X Liu ldquoChinarsquos food security soiled bycontaminationrdquo Science vol 339 pp 1382ndash1383 2013

Submit your manuscripts athttpwwwhindawicom

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Inorganic ChemistryInternational Journal of

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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these technological developments a variety of separationmechanismswere establishedwhich allowedLC to become anindispensable tool for the purification of active pharmaceuti-cal ingredients (APIs) such as blood coagulation factors fromplasma and recombinant proteins from bacterial lysates [20]Reflecting on this brief historic trajectory of LC and consid-ering the crucial role that this versatile separation techniquehas served in the context of food analysis and safety [21 22]it is tempting to equate its significance in contemporary lifescience research with the role that the microscope plays inbiology the spectroscope has in chemistry and the Hubbletelescope holds in astronomy

Against this background it is interesting to note thatwithin the specific research area of ldquometal species in bio-logyrdquomdashloosely defined as the elucidation of health relevantinteractions between environmentally abundant toxic met-als or metal-based drugs with ligands within organismsmdashapproaches which involve the hyphenation of LC separationswith inductively coupled plasma- (ICP-) based detectors [23]have also become more frequently applied over the past10 years [24] To understand this trend one may identifythree contributing reasons The first one is the capability ofLC-based approaches to provide fundamentally new insightinto the complex interactions between metal species andendogenous ligands in a variety of ways [25ndash28] This notionconstitutes themain focus of this paper andwill be elaboratedon in detail later The second reason is the realization thatthese interactions provide an important starting point topotentially unravel the mechanisms which link the exposureto certainmetal speciesmdashunwitting in the context of environ-mentally abundant toxic metals [29ndash31] and deliberate withregard to intravenously administered metal-based drugsmdashwith adverse human health effects [32 33] Thus the applica-tion of LC-based approaches has potential to provide insightinto the etiology of hitherto elusive metal species relateddisease processes [34 35] The third reason is the inherentability that LC-based approaches offer in terms of developingmore effective antidotes against specific metal species toultimately advance human health [36 37] As a result theapplication of LC-based approaches will likely continue toplay an important role in ldquometal species in biologyrdquo researchin the years to come Given that several excellent reviewshave detailed either their historic development [38] or variousrecent methodological advances [24 27 28 39ndash41] the focusof the present one is to illustrate the different ways in whichLC has been employed as a tool to gain insight into thecomplex interactions that unfold after metal species entermammalian organism Furthermore particular emphasis willbe given to studies that have attempted to specifically probecertain aspects of these interactions under near physiologicalconditions It is hoped that the presented synopsis maycontribute to foster progress toward tackling the by farbiggest adversary in the context of revealing the bioinorganicprocesses which link the exposure of mammals to certainmetal species with disease processes biological complexity[19]

Within the scope of elucidating the role that metalsplay in health and disease one may differentiate betweenthree distinguished categories essential metalsmetalloids

toxic metalsmetalloids and pharmacologically active metalcompoundsdrugs Owing to the central role that essentialmetals play in health [42] extensive research has been andcontinues to be directed toward the elucidation of theexceedingly complex homeostatic regulation mechanismswhich maintain their tissue concentrations within a narrowrange throughout life [43ndash49] Comparatively less howeveris known about the role that environmentally abundant toxicmetals may play in the etiology of human disease processes[34 35 50 51] and progress towards the development ofmoreeffective chelating agents to efflux these from intoxicatedorganisms (without harming them) is slow [52] In a similarvein the biomolecular basis of the side effects that areassociated with clinically used metal-based drugs (and oftenlimit the treatment of patients) remains fragmentary [53ndash55]and surprisingly little is known about the biochemicalmecha-nism(s) by which so-called ldquoameliorating agentsrdquomitigate theside-effects of metal-based drugs in patients [37 56 57]

From a conceptual point of view the effect that any metalspecies exerts in an organism above a minimum dose andlength of exposuremdashthis includes acute toxicity and chronictoxicitymdashmay be investigated by two principle approachesThe traditional ldquobottom-uprdquo or ldquoreductionisticrdquo approach isbased on the isolation of the toxicological problem fromthe organism followed by in depth studies of the presumedunderlying biochemical mechanism(s) using appropriatetechniques For example to explain why the ingestion ofan improperly prepared puffer fish is fatal in sim60 of thereported poisoning cases (lethal dose in adults 10120583gkg bodywt) one may want to uncover the molecular mechanismof action of the responsible toxin tetrodotoxin To do sohowever one needs to identify first a putative biological targetmolecule Since early studies indicated that tetrodotoxinmay adversely affect voltage-sensitive Na+ channels [58]definitive proof of this hypothesis required detailed studiesinto the binding of tetrodotoxin to this putative Na+ channelprotein target [59] Importantly this approach can onlybe employed if some a priori information about a likelymolecular mechanism of action (eg inhibition of voltage-sensitive Na+ channels) is available In contrast the ldquotop-downrdquo or ldquosystems biologyrdquo approach attempts to directlyanalyze complex biological samples with an appropriateanalytical technique to unravel the molecular mechanism ofaction of a particular toxin therein [19 60] This alternativeapproach hinges on identifying an analytical technique thatis capable of detecting a toxin-induced change in a likelybiological compartment (eg blood plasma [26 61]) or to inferthat a relevant biotransformation of the toxin of interest hasoccurred in a particular organ (eg the liver) by analyzing anappropriate effluent stream (eg bile [62ndash65]) Importantlythe ldquotop-downrdquo approach offers the advantage that no apriori information about a likely mechanism of toxicity isrequired and that fundamentally new insights into hithertounknown mechanisms of action may be obtained Eventhough the ldquobottom-uprdquo approach has been in use longer thanthe ldquotop-downrdquo approach the latter is receiving progressivelymore attention lately This may be attributed to the fact thatorganisms are comprised of diverse anatomical structureswhich maintain intricately intertwined metabolic networks


+H3N

O

OH

HN

N

SH

CO2minus

CO2minus

(a)

+H3N

SH

CO2minus

(b)

O

HN

SH

CO2minus

(c)

+H3N

+H3N

OO

O

O

HN

HN

HN

As

S

SSeminus

CO2minusCO2

minus

CO2minus

CO2minus

NH

(d)

+H3N

+H3N

O

O

OO

HN

HN

HN

S

S

CO2minusCO2

minus

CO2minus

CO2minus

NH

(e)

CO2minus

CO2minus

SH

SH

(f)

CO2minus

CO2minus

CO2minusNN

N

minusO2C

minusO2C

(g)

SH

SH

SO3minus

(h)

CO2minusAs+

H3CH3C

CH3

(i)

H3N

H 3NPt Cl

Cl

(j)

OO

OO

H3N

H3NPt

C

C

(k)

O

OO

O

Pt

NH2

NH2

(l)

O O

Sminus

S Ominus

(m)











Organism

Blood

Cellsorgans


Pathways

Proteins

Genes

Toxic metal species


geneexpression

Proteinmachinery

readsgenes


cellsignalling





3Hg+ Cd2+







2O2[126])






compound


or DMPS)









(a)

(b)

(c)





[(GS)3As] [135]







3] and


As(OH)3+ 119909GSH

997888rarr (GS)119909As(OH)

3minus119909+ 119909H2O 119909 = 1ndash3

(1)






119909As









Hepatocyte Nucleus


Blood plasma

Erythrocyte

Tum

or


STS

STS

Top-down

Bottom-up

a

bc

d

ef

Hg2+

Hg2+

AsIII

AsIII

SeIV MeHg+

(GS)3As GSHGSH

GSH

GSH

GSH

GSH GSH

CA

CA

ZnCACACd PtSTS

CAAs

Urine

Bile

Zn2+ ZnP

CdP

Cd2+

Cd2+

P

P

CdP

CdP


PtH2O+ PtP

(NAC)2Hg

GSHgCys (Cys)2Hg

Cys Cys NAC

(GS)2Hg


GSHgMeMeHg+Se-II

SeIV

MeAsIII


GSAsMe2

cisPt

cisPt

(SeHg)100(GS)5





3to the vicinal











2]




3 methylarsonous


2AsOH] or


3



3


2andMe



2and Me




2and Me





2AsGS compared





to MeAs(OH)2and Me






3Hg+


3Hg+ and


As(OH)3


CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-



lowast

lowast


(CH3)2AsGS






2AsGS The


2AsGS




3) compared to the



3Hg+

[144]






2[146] In addition




4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


+H3N

O

OH

HN

N

SH

CO2minus

CO2minus

(a)

+H3N

SH

CO2minus

(b)

O

HN

SH

CO2minus

(c)

+H3N

+H3N

OO

O

O

HN

HN

HN

As

S

SSeminus

CO2minusCO2

minus

CO2minus

CO2minus

NH

(d)

+H3N

+H3N

O

O

OO

HN

HN

HN

S

S

CO2minusCO2

minus

CO2minus

CO2minus

NH

(e)

CO2minus

CO2minus

SH

SH

(f)

CO2minus

CO2minus

CO2minusNN

N

minusO2C

minusO2C

(g)

SH

SH

SO3minus

(h)

CO2minusAs+

H3CH3C

CH3

(i)

H3N

H 3NPt Cl

Cl

(j)

OO

OO

H3N

H3NPt

C

C

(k)

O

OO

O

Pt

NH2

NH2

(l)

O O

Sminus

S Ominus

(m)











Organism

Blood

Cellsorgans


Pathways

Proteins

Genes

Toxic metal species


geneexpression

Proteinmachinery

readsgenes


cellsignalling





3Hg+ Cd2+







2O2[126])






compound


or DMPS)









(a)

(b)

(c)





[(GS)3As] [135]







3] and


As(OH)3+ 119909GSH

997888rarr (GS)119909As(OH)

3minus119909+ 119909H2O 119909 = 1ndash3

(1)






119909As









Hepatocyte Nucleus


Blood plasma

Erythrocyte

Tum

or


STS

STS

Top-down

Bottom-up

a

bc

d

ef

Hg2+

Hg2+

AsIII

AsIII

SeIV MeHg+

(GS)3As GSHGSH

GSH

GSH

GSH

GSH GSH

CA

CA

ZnCACACd PtSTS

CAAs

Urine

Bile

Zn2+ ZnP

CdP

Cd2+

Cd2+

P

P

CdP

CdP


PtH2O+ PtP

(NAC)2Hg

GSHgCys (Cys)2Hg

Cys Cys NAC

(GS)2Hg


GSHgMeMeHg+Se-II

SeIV

MeAsIII


GSAsMe2

cisPt

cisPt

(SeHg)100(GS)5





3to the vicinal











2]




3 methylarsonous


2AsOH] or


3



3


2andMe



2and Me




2and Me





2AsGS compared





to MeAs(OH)2and Me






3Hg+


3Hg+ and


As(OH)3


CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-



lowast

lowast


(CH3)2AsGS






2AsGS The


2AsGS




3) compared to the



3Hg+

[144]






2[146] In addition




4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





Organism

Blood

Cellsorgans


Pathways

Proteins

Genes

Toxic metal species


geneexpression

Proteinmachinery

readsgenes


cellsignalling





3Hg+ Cd2+







2O2[126])






compound


or DMPS)









(a)

(b)

(c)





[(GS)3As] [135]







3] and


As(OH)3+ 119909GSH

997888rarr (GS)119909As(OH)

3minus119909+ 119909H2O 119909 = 1ndash3

(1)






119909As









Hepatocyte Nucleus


Blood plasma

Erythrocyte

Tum

or


STS

STS

Top-down

Bottom-up

a

bc

d

ef

Hg2+

Hg2+

AsIII

AsIII

SeIV MeHg+

(GS)3As GSHGSH

GSH

GSH

GSH

GSH GSH

CA

CA

ZnCACACd PtSTS

CAAs

Urine

Bile

Zn2+ ZnP

CdP

Cd2+

Cd2+

P

P

CdP

CdP


PtH2O+ PtP

(NAC)2Hg

GSHgCys (Cys)2Hg

Cys Cys NAC

(GS)2Hg


GSHgMeMeHg+Se-II

SeIV

MeAsIII


GSAsMe2

cisPt

cisPt

(SeHg)100(GS)5





3to the vicinal











2]




3 methylarsonous


2AsOH] or


3



3


2andMe



2and Me




2and Me





2AsGS compared





to MeAs(OH)2and Me






3Hg+


3Hg+ and


As(OH)3


CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-



lowast

lowast


(CH3)2AsGS






2AsGS The


2AsGS




3) compared to the



3Hg+

[144]






2[146] In addition




4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



3Hg+ Cd2+







2O2[126])






compound


or DMPS)









(a)

(b)

(c)





[(GS)3As] [135]







3] and


As(OH)3+ 119909GSH

997888rarr (GS)119909As(OH)

3minus119909+ 119909H2O 119909 = 1ndash3

(1)






119909As









Hepatocyte Nucleus


Blood plasma

Erythrocyte

Tum

or


STS

STS

Top-down

Bottom-up

a

bc

d

ef

Hg2+

Hg2+

AsIII

AsIII

SeIV MeHg+

(GS)3As GSHGSH

GSH

GSH

GSH

GSH GSH

CA

CA

ZnCACACd PtSTS

CAAs

Urine

Bile

Zn2+ ZnP

CdP

Cd2+

Cd2+

P

P

CdP

CdP


PtH2O+ PtP

(NAC)2Hg

GSHgCys (Cys)2Hg

Cys Cys NAC

(GS)2Hg


GSHgMeMeHg+Se-II

SeIV

MeAsIII


GSAsMe2

cisPt

cisPt

(SeHg)100(GS)5





3to the vicinal











2]




3 methylarsonous


2AsOH] or


3



3


2andMe



2and Me




2and Me





2AsGS compared





to MeAs(OH)2and Me






3Hg+


3Hg+ and


As(OH)3


CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-



lowast

lowast


(CH3)2AsGS






2AsGS The


2AsGS




3) compared to the



3Hg+

[144]






2[146] In addition




4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




compound


or DMPS)









(a)

(b)

(c)





[(GS)3As] [135]







3] and


As(OH)3+ 119909GSH

997888rarr (GS)119909As(OH)

3minus119909+ 119909H2O 119909 = 1ndash3

(1)






119909As









Hepatocyte Nucleus


Blood plasma

Erythrocyte

Tum

or


STS

STS

Top-down

Bottom-up

a

bc

d

ef

Hg2+

Hg2+

AsIII

AsIII

SeIV MeHg+

(GS)3As GSHGSH

GSH

GSH

GSH

GSH GSH

CA

CA

ZnCACACd PtSTS

CAAs

Urine

Bile

Zn2+ ZnP

CdP

Cd2+

Cd2+

P

P

CdP

CdP


PtH2O+ PtP

(NAC)2Hg

GSHgCys (Cys)2Hg

Cys Cys NAC

(GS)2Hg


GSHgMeMeHg+Se-II

SeIV

MeAsIII


GSAsMe2

cisPt

cisPt

(SeHg)100(GS)5





3to the vicinal











2]




3 methylarsonous


2AsOH] or


3



3


2andMe



2and Me




2and Me





2AsGS compared





to MeAs(OH)2and Me






3Hg+


3Hg+ and


As(OH)3


CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-



lowast

lowast


(CH3)2AsGS






2AsGS The


2AsGS




3) compared to the



3Hg+

[144]






2[146] In addition




4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Hepatocyte Nucleus


Blood plasma

Erythrocyte

Tum

or


STS

STS

Top-down

Bottom-up

a

bc

d

ef

Hg2+

Hg2+

AsIII

AsIII

SeIV MeHg+

(GS)3As GSHGSH

GSH

GSH

GSH

GSH GSH

CA

CA

ZnCACACd PtSTS

CAAs

Urine

Bile

Zn2+ ZnP

CdP

Cd2+

Cd2+

P

P

CdP

CdP


PtH2O+ PtP

(NAC)2Hg

GSHgCys (Cys)2Hg

Cys Cys NAC

(GS)2Hg


GSHgMeMeHg+Se-II

SeIV

MeAsIII


GSAsMe2

cisPt

cisPt

(SeHg)100(GS)5





3to the vicinal











2]




3 methylarsonous


2AsOH] or


3



3


2andMe



2and Me




2and Me





2AsGS compared





to MeAs(OH)2and Me






3Hg+


3Hg+ and


As(OH)3


CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-



lowast

lowast


(CH3)2AsGS






2AsGS The


2AsGS




3) compared to the



3Hg+

[144]






2[146] In addition




4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





2]




3 methylarsonous


2AsOH] or


3



3


2andMe



2and Me




2and Me





2AsGS compared





to MeAs(OH)2and Me






3Hg+


3Hg+ and


As(OH)3


CH3As(GS)2

Hepatocyte

Bloodstream

GS-Xconjugate

exportpumpMRP-



lowast

lowast


(CH3)2AsGS






2AsGS The


2AsGS




3) compared to the



3Hg+

[144]






2[146] In addition




4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


4

3

2090807060504030201


119896998400

CH3Hg+

Hg2+

Cd2+






3Hg+ Since NAC


















3and selenite (HSeO

3







3

and HSeO3


As(OH)3+HSeO

3

minus

+ 8GSH


2O

(2)

















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















HSeO3




2HSeO

3


2HSeO

3





3

minus


100(GS)5






3Hg+ laced diet



2AsSeminus is








3HgOH to




3HgOH XAS


3] was present


2AsSeHgCH





3]


2AsSeHgCH









3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





3









3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






3

minus












2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




2 In addition



8 Outlook


3As


2and Me

2AsGS





CH3versus NAC-Hg-CH



2AsSeminus [155]










9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




9 Conclusion



References







































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


































































2AsSe]minus





























































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















































3Hg+








































2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















2AsSe]minus by ICP









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

review article metal species in biology: bottom-up and top...

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