arsenic   is a toxic metalloid [ 1 , 2 ] 
 that exists throughout nature 
 in organic and inorganic forms .
it is 
 commonly present in soils on average at several mg / kg , as well as in marine 
 sediments , and is enriched in mineral deposits as oxides and sulfides .
the 
 basic element , arsenic , exists in either of three allotropic forms : yellow , 
 black , or grey with the stable , semimetallic form having a silver / steely - grey 
 colour as a brittle , crystalline solid .
the semimetallic form oxidises rapidly 
 in air , and at high temperatures produces a white cloud of arsenic trioxide ( as2o3 ) .
arsenic , with its variety of 
 chemical forms and oxidation states , is listed in table 1 .
the current iupac 
 nomenclature is listed 
 in   as well as the more common names of the arsenic - based compounds which 
 are mainly used throughout this review .
when absorbed at toxic 
 levels , arsenic causes severe health problems , including cancer .
arsenic - containing compounds are applied in 
 plant pesticides and insecticides and arsenic environmental contamination represents 
 a global health problem , particularly from leaching into ground water . when the 
 soluble levels exceed 50  g / l 
 in drinking water as in many regions of bangladesh , arsenic becomes a 
 particular health concern as it has recently been associated with increased 
 cancer rates appearing after consumption with a lag time up to 1020 years .
whilst the carcinogenic 
 aspects of arsenic compounds are not the focus of this review , nevertheless this 
 undesirable aspect needs to be raised .
thus , arsenic and its methylated species 
 are known carcinogens but this description is probably inaccurate as they act 
 more as cocarcinogens by facilitating / promoting the induction of tumors of the 
 skin , urinary bladder , and lung rather than directly inducing cell transformation 
 and oncogenesis [ 2 , 6 ] . as cocarcinogens , mechanisms include indirect effects 
 such as dna damaging genotoxicity by altered dna methylation as well as 
 inducing high levels of oxidative stress leading to altered cell proliferation and 
 tumor promotion ( detailed in ) . 
  in terms of chronic toxicity ,
the 50% lethal dose of arsenic as arsenic trioxide 
 in mice received by the oral route varies from 1548  mg / kg , whereas the acute 
 lethal dose in humans varies from 13  mg / kg body weight . despite its toxicity
, arsenic has been applied 
 in chinese medicines for thousands of years as refined preparations of realgar 
 ( as4s4 ) or orpiment ( as2s3 ) 
 which are two rare mineralised forms .
realgar , easily discerned by its bright 
 red and orpiment with a browny yellow appearance are ores often occurring close 
 to each other in hydrothermal veins of precious metal sulfide ores or hot 
 spring deposits as volcanic sublimate products ( crystallized from gases ) .
hence , 
 arsenic is a common waste product from the mining of metal ores and the name 
 realgar is possibly derived from arabic words for  powder of the 
 mine  ( rahj al ghar ) .
the long history of medical applications has included treating many 
 diseases such as cancers administering micromolar levels in patients and , in 
 particular , arsenic compounds have proven to be very effective against certain 
 hematological malignancies ( reviewed in [ 9 , 10 ] ) .
thus , arsenic oxides and 
 derivatives have been established as effective treatments for acute 
 promyelocytic leukemia ( apl ) , and they are being tested as therapies for a 
 range of other hematological cancers including myelodysplastic syndromes , 
 multiple myeloma , and chronic myelogenous leukemia ( cml ) .
the results of 
 clinical trails using arsenic - based drugs in cancer therapy have been 
 extensively reviewed [ 812 ] .
hence , this review mainly concerns those factors 
 that explain the antineoplastic mechanism of action of arsenic - based drugs , 
 distinguishing their selectivity for killing certain types of cancer cells from 
 their potential for toxicity to normal cells in the body .
the reason for their efficacy 
 and selectivity in treating certain hematological malignancies is covered in 
 the last section of this review .
however , the mechanisms of action of arsenic - basedcompounds 
 on cells must first be described in detail to provide sufficient understanding 
 to enable their selective targeting of specific types of cancer cells to be 
 duly evaluated .
water distributes the majority of 
 inorganic arsenic in the biosphere either as pentavalent 
 arsenate ( as , as(v ) ) or trivalent arsenic ( as , 
 as(iii ) ) . in solution ,
the ph and redox conditions affect 
 the chemical state of 
 arsenic that predominates .
thus , in highly oxidised environments , the arsenate ( as ) 
 form predominates as one of four major species of arsenic acid ; h3aso4 , h2aso4 , haso4 , and 
 aso4 . however , 
 mildly reducing conditions such as those generally present inside all mammalian 
 cells   will favour the reduction of the pentavalent arsenate ( as ) 
 to the trivalent arsenic ( as ) .
arsenous acid with the formula as(oh)3 results 
 after the slow hydrolysis of arsenic trioxide in water .
as the ph increases , 
 arsenous acid is converted to the arsenic oxide ions [ aso(oh)2 ] , 
 [ aso2(oh ) ] , and [ aso3 ] .
arsenic combines readily 
 with many elements ; and bacteria have evolved systems to detoxify inorganic 
 arsenic to form organic arsenic - containing compounds such as methylated forms .
plant 
 and insect pesticides have been made using organic arsenic derivatives ; and monomethylarsonic acid 
 ( mma(v ) ) and dimethylarsinic acid ( dma(v ) ) are used in products for weed 
 control .
also , mma(v ) and dma(v ) are metabolites of inorganic arsenic , formed 
 intracellularly in mammals , primarily in the liver .
the metabolic process of 
 inorganic arsenic conversion is known as biotransformation and appears to 
 enhance the excretion of arsenic from the body , involving formation of 
 methylated compounds of trivalent arsenic as intermediates .
the metabolism 
 involves reduction to a trivalent state and oxidative methylation to a 
 pentavalent state .
in addition , 
 reductases present in cells and other reactions facilitate the reduction of 
 arsenic acid [ as(v ) ] to the arsenous [ as(iii ) ] form including methylation of the 
 arsenous form , mainly via the liver , to produce mono- , di- , and trimethylated 
 species . the main enzyme involved in methylation of as(iii )
is arsenic 
 methyltransferase ( as3mt ) that requires glutathione ( gsh ) to promote the 
 reaction and a cycling redox system such as thioredoxin ( reviewed in ) to 
 detoxify arsenic - containing compounds .
many animal species convert inorganic arsenic - containing 
 compounds into mono- , di- and tri - methylated arsenic species which are mostly 
 then secreted in the urine .
this is because the methylated forms are not 
 as well absorbed by cells compared to the inorganic forms , although they have 
 much greater cytotoxicity if they do enter cells [ 15 , 17 ] .
the trivalent arsenic - containing compounds , including 
 the methylated organic forms have much greater potency than the pentavalent arsenic - containing 
 compounds as cytotoxics and carcinogens [ 1823 ] .
for example , in cytotoxicity 
 assays , the ic50 values for cultures of primary hepatocytes , keratinocytes , and 
 epithelial cells ranged from 3  m to well over 20  m for trihydroxidoarsenic salts whereas for monomethylarsonous 
 acid [ mma(iii ) ] , the values were consistently much lower at only several m for the equivalent normal cell types [ 2426 ] .
organic 
 arsenic - based ingredients are 
 commonly used as feed additives in poultry farming to 
 increase weight gain by preventing bacterial and parasitic infections , thereby 
 increasing feed efficiency and improving pigmentation .
the three major arsenic - containing compounds 
 used in this manner are arsenilic acid ( p - aminophenyl arsonic acid ) , roxarsone 
 ( 4-hydroxy-3-nitrophenylarsonic acid ) , and nitarsone ( 4-nitro - phenylarsonic 
 acid ) [ 22 , 23 ] .
the metabolism of these arsenic - containing 
 compounds and waste products produced by birds and mammals consuming them is 
 still uncertain at present and what the global environmental impact might be .
at the 
 biochemical level , inorganic arsenic in the pentavalent state ( as(v ) , arsenate , aso4 ) resembles a 
 phosphate ( po4 ) group in structure and it can replace phosphate in many 
 reactions . the mitochondria is a 
 major intracellular site where arsenate is metabolised , taken up as(v ) , rapidly 
 reducing it , and exporting the as(iii ) product back into the cytosol . the specific location of the site(s ) for 
 arsenic reduction in the mitochondria has not yet been defined .
however , arsenate can affect oxidative phosphorylation 
 by binding to the fo / f1 atp synthase .
arsenate can be used by the atp 
 synthase more efficiently than phosphate depending on the ca2 + levels , 
 producing adp - arsenate which unlike atp becomes rapidly hydrolysed and unable 
 to form stable high - energy compounds . it is suggested that the structure 
 and charge similarities of po4 , aso4 , and so4 result in 
 indiscriminate binding to at least two sites located in the mitochondrial 
 matrix .
at one site , occupation by any of these three anions results in 
 protection against uncoupling of the mitochondrial proton gradient by k ; 
 at the second site , in the fo / f1 atp synthase , aso4 , and so4 compete for binding against po4 , leading to the inhibition of atp production .
intriguingly , as2o3 , 
 whilst shown to have no effect on oxidative phosphorylation levels in hela [ 10  m ] and as-30d [ 100  m ] hepatoma cancer cells , significantly inhibited 
 glycolysis , particularly during the exponential growth phase of these cells 
 when they were actively respiring and producing 70% of their atp from 
 mitochondria .
several other studies have also shown that arsenic compounds 
 do not affect oxidative phosphorylation .
thus , with submitochondrial particles 
 in the presence of an atp regenerating system , 20  mm arsenate had no effect on 
 nadh formation , atp hydrolysis , and pi    h20 exchange . in a related study with 
 isolated liver mitochondria , as2o3 was again shown to 
 have no effect on oxygen consumption , or the respiratory control ratio at a concentration 
 ( 10  m ) found to be maximally effective in promoting 
 apoptosis in whole cells . however , at higher concentrations ( > 50  m ) , pyruvate / malate - supported 
 respiration ( via complex i ) became blocked , but there was no effect on either 
 complex ii or iv . the inhibitory effect on complex 

i was reversed by the addition of the reducing agent , dithiothreitol , 
 indicating that direct oxidative damage was involved .
in addition , it was shown that even with the 
 block in complex i , the cells continued to maintain cellular atp levels through 
 glycolysis , and hence , depletion of cellular atp was not the cause for the 
 cytotoxicity of as2o3 .
probably the most definitive evidence 
 for the importance of mitochondria in mediating as2o3 killing 
 of cancer cells comes from studying cells lacking mitochondrial function . 

a subclone of mitochondrial respiration deficient cells was derived from the 
 hl-60 human leukaemia 
 cell line by growth in the presence of ethidium bromide to mutate the 
 mitochondrial genome , and these cells are known as hl-60    cells . due to the lack of 
 mitochondrial respiration
, cells depend on glycolysis for 
 their energy source and , as would be expected , produced substantially less 
 superoxide radicals ( 20% of the control cells ) .
when these  cells were incubated with 10  m as2o3 , they were resistant 
 to the drug , revealing that mitochondrial respiratory function is required for 
 the cytotoxic actions of as2o3 .
the 
 inhibition of glycolysis by arsenic - based drugs appears unlikely to be a significant 
 factor involved in the drug - induced killing of cancer cells .
thus , incubating 
 cells in glucose - deficient medium to block glycolysis had no significant effect 
 on the as2o3 [ 30 m ] mediated levels of cell death in 
 the jurkat cell line . however , when glycolysis was blocked and 
 mitochondrial respiration inhibited using oligomycin a , the cells became very 
 sensitive to as2o3 mediated cell death . in this regard ,
it is also worth noting that studies 
 of individual glycolytic enzymes analysed in purified form in vitro have shown arsenite 
 and arsenate to be relatively weak inhibitors .
hence , for hexokinase ( ic50 : 
 15 mm for arsenate [ 37 , 38 ] ) , phosphofructokinase ( ic50 > 5  mm for arsenite ; ) 
 and pyruvate dehydrogenase ( pdh ic50 : 
 80120  m arsenite ; ) , relatively large concentrations 
 were required to inhibit these enzymes .
in fact , arsenate has been shown to stimulate 
 the activities of the two important glycolytic enzymes , hexokinase   and 
 gapdh , by overcoming product inhibition in these reactions . in the case of 
 gapdh , arsenate acts catalytically to promote the oxidation of phosphoglycerate 
  and the reaction involved the formation of an arsenate analogue of the 
 phosphate ester as an intermediate which rapidly hydrolysed , helping to drive 
 the reaction forward [ 33 , 43 ] .
this process has been commonly described in 
 relation to the effects of arsenate on numerous enzymatic reactions involving 
 phosphate and has been termed  arsenolysis  .
given the relative 
 insensitivity to the direct effects of arsenic compounds shown by the 
 glycolytic pathway , it follows that it is unlikely that glycolytic inhibition 
 results from direct binding and 
 modification of the enzymes in this pathway by arsenic - based drugs .
more likely , 
 the inhibition of glycolysis results from an indirect effect , caused by the 
 actions of arsenic compounds in modifying mitochondrial respiration leading to 
 production of ros which then acts to inhibit the glycolytic enzymes .
additional 
 support for the mitochondrial - mediated ros involvement in the action of arsenic 
 to inhibit glycolysis comes from studies where as2o3 was 
 found to be 38 times more potent in cells than in the pure preparation at 
 inhibiting pyruvate dehydrogenase .
also , inhibiting mitochondrial 
 respiration suppressed the resulting inhibition of pyruvate dehydrogenase activity 
 and h2o2 production by this 
 drug .
furthermore , the inhibition of pyruvate dehydrogenase by as2o3 was shown to require the fenton reaction occurring via hydroxyl radical intermediates 
 .
the mitochondrial effects of arsenic compounds are detailed later in this 
 review and to reiterate at this point , the evidence indicates that the actions 
 of arsenic compounds on glycolysis are not the main cause for the cytotoxic 
 effects of these drugs at clinically relevant concentrations ( 16  m ) required in plasma for the killing of cancer 
 cells in apl patients [ 8 , 44 ] . under conditions of 
 high mitochondrial respiration inside cells , it is possible that trivalent 
 arsenicals inducing significant production of ros as superoxide , peroxide , and 
 hydroxyl radicals can also result in oxidation to produce arsenate ( aso4 ) 
 ionic species [ 45 , 46 ] .
consequently , the 
 impact that arsenic compounds 
 will have on any given cell will most likely depend on the state of 
 cellular respiration and production of ros affecting the arsenic speciation and 
 whether the cell is dependant on glycolysis versus mitochondrial respiration 
 for its atp synthesis .
glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , the 
 glycolytic enzyme abundantly found in all cells and especially blood cells and 
 liver , is a major intracellular arsenate reductase   requiring gsh , nad , and 
 glycolytic substrate [ 48 , 49 ] . given that the levels and specific activity of 
 gapdh is much higher in malignant cells than in normal cells , this could 
 contribute to the rapid reduction of as(v ) species in their cytosol into more 
 toxic as(iii ) forms . in the gapdh reaction , 
 as(v )
reduction may take place during , or as a consequence of the arsenolytic 
 cleavage of the thioester bond formed between the enzyme 's cys149 residue and 
 the 3-phosphoglyceroyl moiety of the substrate .
hydrolysis of 
 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 
 the normal substrate 1,3-diphosphoglycerate under the same conditions . 

hence , gapdh is proposed as one of the key cellular converting enzymes for 
 reducing as(v ) to as(iii ) .
although purine nucleoside phosphorylase was 
 proposed to be an arsenate reductase , this was later refuted .
the other major class of as(v ) reductases in 
 cells are the glutathione s transferases and of these , the omega form or gsto1 
 appears to be most important .
thus , gsto1 can reduce arsenate to arsenite , 
 mma(v ) to mma(iii ) , and dma(v ) to dma(iii ) and deletion of the gsto1 gene in 
 mice reduced the extent of biotransformation by 3080% in most tissues examined 
 .
arsenic has a high affinity for sulfur 
 and hence , reactive sulfur - containing molecules such as reduced thiols with an 
 available sulfur atom have a significant propensity for binding to arsenic [ 55 , 56 ] . 

as(iii)-containing compounds exist as trigonal pyramidal structures and this is 
 also the structure formed upon binding of arsenic ions to cellular proteins in 
 vivo where the sulphur atoms of thiolate groups act as coordinating ligands .
the 
 resulting arsenic - thiol linkages are mainly responsible for the ability of 
 arsenic to modulate the function of various key molecules , enzymes , and ion 
 transporters inside cells and this intracellular action of arsenic is discussed 
 in detail in this section .
arsenic - containing compounds react with mono- and dithiols , 
 particularly the latter when two thiols are located in close proximity , acting 
 to cross - link the thiols together .
some debate exists 
 about the structures and speciation of arsenic - containing compounds ( both 
 inorganic and organic forms ) in solution . 
 in the absence of sulfide , as(iii )
hydroxide complexes are the major 
 arsenic - containing species and these structures probably adopt a trigonal 
 pyramidal structure with the arsenic atom at the apex [ 5759 ] .
this trigonal pyramidal structure provides 
 the potential for as(iii ) to coordinate linkages with several proximal thiol 
 groups .
this is believed to be the case 
 in bacterial enzyme systems such as the arsr repressor protein where it is 
 likely to bind three cys atoms .
thus , in the more toxic form as the 
 trivalent state ( as(iii ) ) inorganic and organic 
 ( methylated ) arsenic reacts with critical thiols in proteins , inhibiting their 
 function , as is the case with the bacterial arsr protein .
as(iii ) was shown to target the 
 reactive sulfhydryl group at the active site of thiolase(s ) involved in ketogenesis 
 from acetyl coenzyme a .
the 
 pentavalent species of inorganic arsenate ( aso4 ) 
 favoured to exist in oxidised environments , as well as organic forms of as(v ) 
 have a different structure with a trigonal - bipyramidal shape where the as(v ) 
 atom is located at the centre , co - ordinating to three equatorial and two polar 
 atoms [ 62 , 63 ] .
in comparing the two different structural states of as(iii ) and 
 as(v ) , it is not yet clear why the trivalent methylarsenic - containing compounds 
 show a much higher toxicity than their pentavalent analogues [ 24 , 64 ] .
however , 
 it could relate to cellular uptake as trivalent organoarsenic compounds are more membrane 
 permeable than the pentavalent species . also , trivalent arsenic bonded at 
 a phenyl ring is able to form much more stable covalent cross - links to cysteine 
 residues compared to arsenic in small molecules such as arsenious acid or arsenite 
 .
furthermore , the 
 organic trivalent arsenic - containing compound , phenylarsine oxide [ 0.10.5  m ] , is much more potent than the simple arsenite 
 [ 110  m ] in its cytotoxic activity in apl cells .
one major 
 drawback with phenylarsine oxide as a potential cancer therapy is its high 
 toxicity in vivo and its nonselectivity for cancer versus normal cells , 
 resulting in cytotoxicity in normal endothelial cells in the same concentration 
 range ( 0.2  m ) .
thus , phenylarsine oxide , is precluded from 
 application in clinical cancer therapy , without being further modified as a 
 drug .
the 
 greater reactivity of phenylarsene oxide and associated cytotoxicity is in 
 agreement with the results outlined above   where mass spectrometric 
 analysis of different complexes of peptides and proteins with arsenic - containing 
 species revealed that inorganic arsenite or arsenates did not interact well 
 with cysteine or glutathione , whereas the organic phenylarsine ( 3 + ) oxide did . 

in addition , three different phenyl arsenic acids and dimethylarsinic acid that 
 all contained as(v ) also formed complexes with glutathione .
hence , the bulky hydrophobic groups with 
 electron withdrawing  orbitals ( in the case of phenyl groups ) may 
 promote more stable bonds between the arsenic atom and sulfur groups inside 
 cells , modulating a larger range of enzymes and proteins with important 
 functions for maintaining cell viability . in 
 cells ,
the most common reactive species that are available for interaction with 
 arsenic are the abundant free thiol moieties in the tripeptide glutathione ( glu - cys - gly , 
 gsh ) and the free amino acid , cysteine .
thus , arsenic - based drugs can react by 
 coordinating binding to free ( reduced ) thiol groups such as those on cysteine , 
 particularly those of thioredoxin and glutathione as the major intracellular thiol 
 species important in cellular redox regulation .
it was observed early on that arsenite 
 and phenylarsine oxide in particular , but not arsenate , reacted with vicinal 
 thiol groups on proteins [ 69 , 70 ] . since 
 demonstrating that phenylarsine oxide was particularly effective at cross - linking 
 vicinal thiols in the active site of tyrosine phosphatases , the range of 
 proteins -containing vicinal cysteine residues with which phenylarsine oxide reacts 
 is increasing .
recent examples include the small gtp binding rho 
  protein family   and the mitochondrial 
 carnitine / acylcarnitine transporter .
phenylarsine 
 oxide , as a strong inhibitor of tyrosine phosphatases would increase tyrosine 
 phosphorylation levels of enzymes in cellular growth signalling pathways . 

whether the inhibition of tyrosine phosphatases and other enzymes contributes 
 to the toxic effects of arsenic - containing compounds in normal cells is not 
 clear , but is likely an important contributing factor to its general 
 cytotoxicity making phenylarsine oxide unsuitable for cancer therapy .
analysis 
 of the interactions of as(iii ) with glutathione or cysteine in vitro in aqueous 
 solutions by equilibrium binding and use of biophysical techniques including 
 nmr , electronic spectroscopy , and potentiometry revealed that as(iii ) binds either 
 of glutathione or cysteine with similar equilibrium constants .
however , several analytical studies by mass 
 spectroscopy have revealed that arsenate and arsenite do not complex readily 
 with glutathione or cysteine , but prefer to react with the thiols on reduced 
 thioredoxin molecules [ reviewed in ] .
this was confirmed by analysis of more 
 biologically relevant samples from the intracellular environment of hela cells 
 where cytosolic thioredoxin 1 ( trx1 ) and , in particular thioredoxin 2 ( trx2 ) in 
 the mitochondria , was shown to be highly reactive with arsenite ( 10  m ) 
 whereas little reactivity was detected with cellular gsh / gssg .
studies 
 with thioredoxin reductase ( txr ) purified from mouse liver showed that arsenic - containing 
 compounds with as(iii ) and arsinothiols ( complexes of as(iii ) with gsh or l - cysteine ) 
 were extremely potent inhibitors of this enzyme .
methylarsenic(iii)oxide 
 was most potent with a   ki100  nm , as an irreversible competitive inhibitor .
the 
 effects on purified glutathione reductase ( gr ) showed that the levels of 
 inhibition were not as marked with inorganic as(iii ) and as(v ) oxides showing 
 ic50s in the 1050  mm range , whereas for methylarsinic(iii)oxide it was 9  m . studies 
 on this enzyme in whole cells as hepatocytes showed the ic50 to be reduced to 
 3  m 
 for methylarsinic(iii ) oxide and for as2o3 > 100  m 
 . hence
, these observations strongly support a role for the components 
 thioredoxins and the thioredoxin reductase system as providing cellular targets 
 that are very sensitive to inhibition by arsenic - based drugs in the low 
 micromolar range .
it also explains the importance of studying the chemistry of arsenic - containing 
 compounds in the context of both purified enzymes as well as whole cells as biological 
 systems in order to obtain meaningful results .
thus , although the 
 gsh / glutathione transferase system undoubtedly plays an important role in 
 arsenic sensitivity of cells 
 ( see below in section 4 for details ) , it    would appear that the thioredoxin system might 
 represent the most immediate point of sensitive reactivity in relation to 
 cytotoxicity .
thioredoxin ( trx ) , nadph , 
 and thioredoxin reductase ( txr ) comprise the thioredoxin system that has multiple 
 functions in cells including in redox signalling via interactions with other 
 proteins , in transcriptional regulation , control of the reduced intracellular 
 redox environment , cell growth , defense against oxidative stress and control of 
 apoptosis ( reviewed in ) .
as outlined in the previous section , the thioredoxin 
 system is very sensitive to arsenic - based drugs and may well be the basis for 
 one of the important mechanisms for their actions in inducing cancer cell 
 death .
the trx system operates as a thiol - disulfide exchange reaction ( see 
 figure 1 ) .
trx1 and trx2 are key 
 regulatory isozymes that catalyse the reduction of protein disulfide bonds .
they 
 are cofactors of the apoptosis signal - regulating kinase 1 ( ask1 ) that mediates tnf 
 cytokine and oxidative stress - induced apoptosis via the mitochondrial dependent 
 pathway . in 
 their reduced forms , cytosolic trx1 and mitochondrial trx2
each contain two 
 vicinal thiol groups in their active site sequence as c  g  p 
c. trx 1 
 in the cytosol and trx2 in mitochondria bind to cys 250 and cys 30 , 
 respectively , in the regulatory n - terminal domain of ask1 and can cooperatively 
 maintain the enzyme in the inhibited state .
however , activation by tnf 
 resulting in increased production of ros and leads to the oxidation of the trx dithiol 
 group to a disulfide . under these conditions ,
the thioredoxins no longer bind 
 to ask1 and loss of trx2 
 binding to mitochondrial - located ask1 can lead to apoptosis in a 
 jnk - independent manner , whereas cytosolic ask1 upon loss of trx1 binding then 
 becomes activated as a mapkkk resulting in jnk activation , bid cleavage and bax 
 translocation to the mitochondria . since it is known that the thioredoxins 
 are major targets of arsenic - containing compounds ( see above and ) , it can 
 be predicted that arsenite - mediated oxidative binding to thioredoxins will 
 induce a similar outcome as tnf signalling , leading to the release and 
 activation of ask1 and induction of apoptosis .
another 
 thioredoxin - associated protein of importance in thiol - mediated redox regulation 
 in mitochondria is thioredoxin peroxidase ii ( tpx - ii , also known as 
 peroxiredoxin iii , prx - iii ) , an enzyme abundantly expressed in the mitochondria 
 of cancer cells that protects the cells from oxidative stress [ 79 , 80 ] .
prxiii 
 is an important antioxidant that acts in conjunction with trx2/txr ( figure 1 ) 
 in the mitochondria to remove peroxides such as h2o2 and 
 offset the apoptosis inducing effects of increased levels of h2o2 . 

however , prxiii contains three cys residues , two of which are involved as 
 redox - active sites in the formation of a stable intersubunit disulfide - bonded 
 dimer , which is then reduced by thioredoxin to the monomer .
prxiii was a more abundantly expressed 
 arsenic - binding protein when comparing arsenic resistant cells to normal cells 
 by phenylarsine oxide affinity chromatography .
hence , prxiii is very 
 likely to be another protein whose function is inhibited by arsenic - containing 
 compounds leading to the promotion of apoptosis .
increasingly , it is becoming apparent that dithiols - containing 
 redox proteins , particularly those present in the mitochondria , act as 
 controlling sensors during responses to changes in cellular redox .
many thiol 
 redox proteins contain a vicinal pair or more of reactive thiol groups [ 82 , 83 ] capable 
 of binding with arsenic - containing compounds in a similar manner to those in 
 the thioredoxin system .
the most important of these are located in the 
 mitochondria , a point of extreme sensitivity to arsenic - containing compounds 
 whose actions culminate in triggering the apoptotic pathways via the induction 
 of reactive oxygen species , leading to the killing of the cancer cells ( figures 
 1 , 2 ) .
for example , two members of the glutaredoxin ( grx ) family , including 
 grx2 located primarily in the mitochondria , catalyse gsh - dependent trx - disulfide 
 redox and protein thiol - disulfide redox reactions , particularly reversible 
 glutathionylation of protein sulfhydryl groups .
y  c and c  s  y  c active sites ; have three and two additional structural 
 cys residues , respectively ; and are therefore likely to react with arsenic - containing 
 compounds , although no reports of this could be found . another mitochondrial protein 
 targeted by arsenic - containing compounds with a vicinal dithiol group
is regulatory 
 protein  factor b.  addition of recombinant factor b back to bovine 
 submitochondrial particles depleted of this protein restored energy coupling 
 activity .
thus , reverse electron transfer from succinate to complex i enabling nad+ 
 reduction , electron transport chain function and oxidative phosphorylation / pi - atp 
 exchange of the atp synthetase complex were reactivated   as was increased 
 exchange activity of complex v .
thus , the f0-f1 atpase activity requires 
 factor b coupled to it for full activation .
however , factor b contains six 
 thiols and cys 92 and cys 94 in the bovine form were shown to bind phenylarsine 
 oxide   and phenylarsine oxide or arsenite inhibit factor b coupling 
 activity . from all of these 
 studies , it is becoming clear that redox changes to vicinal thiols affect the 
 regulation of mitochondrial function and that these thiols are also major 
 targets for inhibition by the arsenic - containing compounds ( figures 1 , 2 ) .
a channel formed by the association of two 
 proteins , the voltage dependent anion channel ( vdac ) in the outer mitochondrial 
 membrane and the adenine nucleotide transporter ( ant ) in the inner mitochondrial 
 membrane ( figure 1 ) , is a complex involved in the induction of apoptosis 
 activated via the mitochondrial pathway ( see , figure 3 ) .
the two 
 components of this complex form part of the mitochondrial permeability 
 transition pore ( mptp ) , a megachannel mediating release of molecules from the 
 mitochondria activating apoptosis .
a rapidly increased permeability of the inner 
 mitochondrial membrane ( mitochondrial permeability transition ) leads to apoptosis 
 that is mediated by the mptp .
arsenite induces apoptosis by a 
 direct effect on the mptp [ 90 , 91 ] and vdac has been shown to play an essential 
 role in opening of the permeability pore and cytochrome c release induced by 
 arsenic trioxide , which also caused vdac to homodimerise .
in addition , the 
 thiol - reactive compound 4,4-diisothiocyanostilbene-2,2-disulfonate ( dids ) has 
 been shown to block the vdac channel   and inhibit ros - mediated cytochrome c 
 release by vdac .
hence , the data indicates that vdac contains critical cys 
 residues that can undergo intermolecular cross - links mediated by reaction with 
 arsenic .
analysis of ion channel activity of 
 purified ant - containing lipid bilayers also revealed that as2o3 treatment [ 30  m ] altered the ant channel electrophysiological properties 
 .
interestingly , glutathione depletion leading to increased ros may play an 
 important role in the action of arsenic trioxide . whereas in both normal 
 and cancer cells , glutathione s transferase ( gst )
is found to interact with ant , 
 during the induction phase of apoptosis , gst dissociates from ant suggesting 
 that gst / gsh may act as a repressor of mptp and ant pore opening [ 91 , 95 ] .
this 
 is supported by the observation that increasing the expression of the enzyme 
 gstp1 in jurkat and raji leukemic cells renders them more resistant to arsenic 
 trioxide - induced apoptosis at clinically relevant levels [ 1 - 2  m ] .
gstp1 
 expression in these cells is also accompanied by accumulation of lower levels 
 of h2o2 production .
both of the 
 mammalian proteins , vdac and ant , contain two or more cys residues in their 
 structure thereby providing reactive thiol groups whose modification affects their function .
it has been 
 established that the redox state of thiol reactive groups are important for 
 activation of the mitochondrial permeability transition 
 [ 94 , 96 , 97 ] .
consequently , 
 thiol cross - linkers such as dids , diamide , and phenylarsine oxide [ 20100  m ] affect vdac and ant 
 channel function and activation of mitochondrial permeability transition [ 93 , 94 , 9698 ] . 

many published results , such as cross - linking experiments , protein / inhibitor 
 stoichiometry , chimeric dimers , analytical ultracentrifugation , or neutron 
 scattering , indicate that the ant carrier acts as a dimer and this or higher 
 oligomers are involved in membrane permeability transition [ 99 , 100 ] . facing out into the 
 mitochondrial matrix , ant has three exposed loop regions containing a conserved 
 repeat structure with one cys residue in each loop .
these cys residues are 
 important to the process of ant dimerisation , but it is not clear how this 
 operates and whether the cys residues form intermolecular disulfide bonds or 
 not .
nevertheless , copper - o - phenanthroline is able to dimerise ant by 
 intermolecular cross - linking of cys 56 ( in the first matrix loop ) . in 
 addition , phenylarsine oxide , eosin 5-maleimide , and diamide form 
 intramolecular cross - links between cys 160 and cys 257 on the other two matrix 
 loops , restricting ant in the open conformation , promoting mitochondrial 
 permeability transition .
arsenic trioxide is much weaker than 
 phenylarsine oxide at binding to the ant cys residues [ 96 , 97 ] and this may 
 explain the greater sensitivity exhibited by apl cells to phenylarsine oxide 
 [ ic50 : 0.1  m ] than to as2o3 [ ic50 : 4  m ] .
single thiol interacting compounds such 
 as n - ethylmaleimide ( nem ) can inhibit the mitochondrial permeability transition 
 and this could be either the result of direct interaction with the key cys 
 residues on the matrix loops of ant or indirectly via reaction with gsh and 
 thereby preventing gsh from being oxidized and catalysing disulfide bridging 
 between the adjacent thiol groups in the ant loops .
nem or monobromobimane , 
 in the 2550  m range , preferentially 
 react with gsh , leading to its modification in mitochondria and thereby 
 prevents gsh from being oxidised . as a result
, nem inhibits mitochondrial 
 permeability transition activation by the thiol reactive compounds , diamide or 
 t - butylhydroperoxide , implying a role for gssg in the action of these agents on 
 the permeability transition 
 [ 96 , 97 ] .
arsenites , albeit that 
 much higher 
 concentrations would be required given their lower affinity for glutathione 
 interaction , could have a similar action .
thus , high levels of arsenites could 
 modify and inhibit glutathione redox control such that glutathione - based 
 enzymes are unable to function , as well as directly mediating disulfide 
 cross - linking of ant , leading to increases in cellular ros production , mptp , 
 and apoptosis .
however , given their low reactivity with glutathione systems , 
 this appears to be unlikely as opposed to the indirect action via the 
 mitochondrial effects leading to increased ros production which then reduces 
 cellular gsh levels .
the multidrug resistance ( mdr ) protein 
 mrp1/abcc1 has been shown to transport asiii out of cells as a tri - gsh 
 conjugate ( as-(gsh)3 ) , and glutathione s - transferase ( gst ) probably facilitates 
 the process .
this is likely to be part of the normal cell and cancer cell 
 resistance mechanisms against the cytotoxic effects of arsenic - based compounds . 

gsh - depleted cells are more sensitive to killing by arsenic - containing compounds 
  and transfection of cells to express glutathione s transferase protects 
 them from arsenite inducing death by promoting arsenite transport from the 
 cells and decreasing ros levels [ 104 , 105 ] . 
 in support of this proposal , the long - term exposure of cells to arsenic - containing 
 compounds induced increased expression of glutathione s transferase and mrps . 

arsenic levels do not attain very high 
 levels in blood plasma of patients , rapidly becoming eliminated [ 8 , 44 , 107 ] . 

this removal probably results from efficient uptake of as-(gsh)3 via mrp2 in the 
 proximal tubules of the kidneys as part of the detoxification process during 
 the excretion of arsenic - based drugs predominantly into the urine [ 108 , 109 ] .
the 
 remainder is mostly removed via uptake in the liver and secretion as bile [ 110112 ] . 

as-(gsh)3 may be an important part of 
 the metabolic process for converting inorganic as(iii ) to methylated species 
 during detoxification in the liver by asmt1/cyt19 .
arsenic - containing 
 compounds substituted with organic groups such as modified phenylarsine oxides have 
 been synthesized and examined for their cytotoxic effect on human leukemic 
 cells and breast cancer cells in culture .
some of these compounds were found to 
 exhibit potent cytotoxic anticancer activity , particularly against human breast 
 cancer and leukemic cell lines , including primary leukemia cells , at micromolar 
 concentrations .
one of these 
 compounds , the novel glutathionyl peptide trivalent arsenic - containing compound 
 para 4-[n-(s - glutathionylacetyl)amino]phenylarsenoxide ( p - gsao ) shows promise 
 as a novel antineoplastic drug and is now in clinical trials .
p - gsao , like 
 phenylarsine oxide , inactivates ant - mediated atp / adp transport and triggers ca - dependent 
 mptp opening by cross - linking the critical cys residues of ant .
this leads to increased production of cellular 
 ros , atp depletion , mitochondrial depolarization , and apoptosis of angiogenic endothelial 
 cells and inhibition of tumor growth in mice with no apparent toxicity . 
 however , the action of p - gsao was indirect , and did not appear to be as a 
 result of selective tumor cell toxicity .
rather , p - gsao inhibited the 
 proliferating , but not growth - quiescent endothelial cells in vitro and angiogenesis 
 in vivo and thus acted to eliminate tumors by blocking their blood supply [ 114 , 115 ] . 

the trivalent arsenic - containing moiety of p - gsao was shown to cross - link the matrix 
 facing cys160 and cys257 thiols of ant   and effectively locks ant into the 
 open configuration .
inactivation of ant by p - gsao causes an increase in 
 superoxide levels , proliferation arrest , atp depletion , mitochondrial 
 depolarization , and apoptosis in the dividing endothelial cells .
it is likely that the arsenic - containing moiety of p - gsao reacts 
 similarly as does arsenite ( see above ) with one or two molecules of glutathione 
 before it is removed from the cell by mrps .
tumor cells export p - gsao much 
 more efficiently than endothelial cells because they have higher mrp1 or mrp2 
 activity and cellular glutathione levels   and this may explain why p - gsao 
 is not highly effective at inhibiting tumor cell growth in vivo .
in addition , 
 the greater water soluble properties of p - gsao than other arsenic - containing compounds , 
 particularly organic species should help to retain p - gsao in the intravascular 
 system where it is more likely to affect endothelial cells and inhibit tumor 
 angiogenesis .
interestingly , although the para form of 
 gsao revealed no apparent toxicity in treated animals and inhibited tumor 
 growth leading to phase i clinical trials as an anticancer agent , the 
 ortho form ( o - gsao ) was toxic and this was proposed to result from increased 
 accumulation of the drug in cells , including normal cells due to loss of multidrug 
 resistance efflux .
consequently , o - gsao is unlikely to be of much further 
 interest as an antineoplastic agent , whereas the clinical efficacy of p - gsao is 
 eagerly awaited .
as(iii ) 
 as the anhydrous form of as(oh)3 ( trisenox , cell therapeutics , seattle , wash , 
  usa ) received 
 fda approval in 2000 as a chemotherapeutic agent for the treatment of apl . 
 acute promyelocytic leukemia ( apl )
is associated with reciprocal and balanced 
 chromosomal translocations always involving the retinoic acid receptor alpha 
 ( raralpha ) gene on chromosome 17 and variable partner genes on distinct 
 chromosomes .
raralpha fuses to the promyeloctyic leukemia ( pml ) gene in the 
 majority of apl cases ( reviewed in ) .
arsenic trioxide is particularly 
 effective at killing apl cells and this was proposed to be the direct result of 
 its ability to induce the relocalization and degradation of the nuclear body 
 protein pml , as well as the degradation of pml - raralpha 
 in apl cells [ 119122 ] .
however , 
 this seems unlikely to be the main mechanism of action for arsenic trioxide 
 given that no differences in sensitivity to growth inhibition and killing by 
 apoptosis have been observed between wild - type and pml 
 cells .
arsenic 
 trioxide as a single agent has provided 86% complete hematologic remission with 
 minimal toxicity in apl patients , equal to any of the current standards 
 of care for treating apl , including the combination of all - trans retinoic acid 
 ( atra ) plus chemotherapy . this 
 raises the question why apl cells are very sensitive to arsenic - containing 
 compounds like arsenic trioxide .
it would appear that the reason is because apl 
 cells express the transmembrane transporter protein , aquaglyceroporin 9 ( aqp9 ) 
 involved in arsenic uptake   at much 
 higher levels in apl cells than in other leukemic cell types and that 
 correlates with arsenite sensitivity .
in this regard , it is worth noting that 
 aquaglyceroporins aqp7 and aqp9 are present in normal cell types . 

interestingly , aqp9 is primarily expressed 
 in human lung , liver , and leukocytes   and this may help explain arsenic 
 toxicity , given that liver is one of the main organs affected .
the fact that aqp9 provides 
 apl cancer cell specificity with high response rates suggests that if arsenic - containing 
 compounds could be targeted for specific delivery into cancer cells , then they 
 would represent outstanding agents for killing these cells .
however , further 
 modifications will be required to provide suitable drug targeting for improved 
 delivery of arsenic - containing compounds to cancer cells .
vicinal thiols 
 located in key enzymes and proteins provide targets for reaction with arsenic - containing 
 compounds , particularly organic derivatives such as phenylarsenic - containing 
 compounds that favour intramolecular cross - linking between adjacent thiols .
intriguingly , 
 most of the key intracellular targets for this reaction have been identified to 
 include the main redox regulatory systems in the mitochondria , including thioredoxin 
 and peroxiredoxin systems and the adenine nucleotide transporter , all of whose 
 function is adversely affected .
the net result is the activation of several 
 independent pathways including ros production to facilitate the induction of 
 apoptosis .
one main pathway operates via the opening of the mptp , the other via 
 activation of ask1 kinase , and the jnk / bid / bax pathway of channel formation in 
 mom . in the case of apl , cell 
 selectivity for sensitive responses to these drugs
low mdr levels 
 present in dividing endothelial cells also provides selective targeting by 
 specially substituted phenylarsenic - containing compounds like p - gsao , leading 
 to decreased blood supply into tumors , with some toxicity to cancer cells , but 
 little toxicity on normal cells .
hence , a combination of selective delivery and 
 retention provides the necessary targeting of arsenic - containing compounds to 
 tumors and provides scope for additional modifications to be made to enhance 
 the antineoplastic activity of arsenic - containing compounds , given their range 
 of actions and efficiency in killing cancer cells .
 arsenic - based compounds have become accepted agents for cancer therapy providing high rates of remission of some cancers such as acute promyelocytic leukemia ( apl ) . 
 the mechanisms by which arsenic - containing compounds kill cells and 
 reasons for selective killing of only certain types of cancer cells such as apls have recently been delineated . 
 this knowledge 
 was gained in parallel with increasing understanding and awareness of the importance of intracellular redox systems and 
 regulation of the production of reactive oxygen species ( ros ) by controlling mitochondrial function . 
 many of the targets for 
 the arsenic - containing compounds are mitochondrial proteins involved in regulating the production of ros . 
 inhibition of these 
 proteins by disulfide linkage of vicinal thiol groups often leads to increased production of ros and induction of apoptotic 
 signalling pathways . 
 sensitivity or resistance to the actions of arsenic - containing compounds on cancer cells and normal 
 cells depends on the levels of transport systems for their uptake or efflux from the cells as well as their redox defence 
 mechanisms . 
 the exact mechanisms of arsenic toxicity as well as its anticancer properties are likely to be related and these 
 aspects of arsenic metabolism are covered in this review . 
 greater understanding of the mechanisms of action of arsenic will 
 help determine the risks of human exposure to this chemical . 
 novel organic arsenic - containing compounds and the lessons 
 learned from studying their selective sensitivity in targeting dividing endothelial cells to inhibit angiogenesis raise the future 
 possibility for designing better targeted antineoplastic arsenic - containing compounds with less toxicity to normal cells . 
1. ARSENIC AND ARSENIC-CONTAINING 
	COMPOUNDS IN THE NATURAL ENVIRONMENT OR AS A RESULT OF HUMAN APPLICATION
2. METHYLATED FORMS OF ARSENIC SHOW  INCREASED CYTOTOXICITY INSIDE
CELLS
3. CELLULAR ACTIONS OF ARSENIC-CONTAINING 
	 COMPOUNDS
4. THE ADENINE NUCLEOTIDE TRANSPORTER (ANT): A CRITICAL TARGET OF ARSENIC-CONTAINING COMPOUNDS IN THE MITOCHONDRIA

5. MODIFIED ORGANIC ARSENIC STRUCTURES  WITH INCREASED POTENCY AS
ANTINEOPLASTIC AGENTS

6. TARGETING OF CANCER CELLS: SELECTIVE UPTAKE 
	AND DELIVERY INTO SPECIFIC TYPES OF CANCER CELLS
7. CONCLUSIONS