Toxicology and Applied Pharmacology 262 (2012) 185–193

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Toxicology and Applied Pharmacology

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Identification of an S-adenosylmethionine (SAM) dependent arsenicmethyltransferase in Danio rerio

Mohamad Hamdi a, Masafumi Yoshinaga b, Charles Packianathan b, Jie Qin b, Janell Hallauer a,Joseph R. McDermott a, Hung-Chi Yang c, Kan-Jen Tsai d, Zijuan Liu a,⁎a Department of Biological Sciences, Oakland University, Rochester, MI 48309, USAb Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, FL33199, USAc Department of Medical Biotechnology and Laboratory Sciences, Chang-Gung University, Tao-Yuan, Kwei-San 333, Taiwand School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan

Abbreviations: SAM, S-adenosylmethionine; MMAMMAV, monomethylarsonic acid; DMAV, dimethylarsinic aICP-MS, inductively-coupled plasma spectrometry; GSH, g⁎ Corresponding author at: Department of Biological

2200 Squirrel Rd., Rochester, MI 48309, USA. Fax: +1 2E-mail address: liu2345@oakland.edu (Z. Liu).

0041-008X/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.taap.2012.04.035

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 March 2012Revised 10 April 2012Accepted 27 April 2012Available online 7 May 2012

Keywords:ZebrafishArsenicArseniteSeleniteMethylationSAMGSHHPLC–ICP-MSMMAIII

MMAV

DMAV

Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity.Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can bedetected in tissues and excretions. Herewe report that zebrafish exposed to arsenite (AsIII) produces organic arsen-icals, including MMAIII, MMAV and DMAV with characteristic tissue ratios, demonstrating that an arsenic methyla-tion pathway exists in zebrafish. Inmammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenicmethyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequencealignment.Western blotting analysis showed that As3mtwas universally expressed in zebrafish tissues. Prominentexpression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mtwas expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated thatAs3mtmethylated AsIII to DMAV as an end product and producedMMAIII andMMAV as intermediates. The activityof As3mt was inhibited by elevated concentrations of the substrate AsIII as well as the metalloid selenite, which is awell-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of ei-ther Cys160 or Cys210,which corresponds to conserved cysteine residues inAS3MThomologs, suggesting that theyare involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodentorthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable ver-tebrate model for understanding arsenic-associated diseases in humans.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Arsenic is an environmental toxicant, a carcinogen as well as a thera-peutic drug (de The and Chen, 2010; Kitchin and Conolly, 2010; Tseng,2008).Mechanisms of arsenicmetabolismhave been extensively studied.It has been found that trivalent arsenite (AsIII) and pentavalent arsenate(AsV) uptake are facilitated by aquaglyceroporins (Hamdi et al., 2009;Liu et al., 2002) and phosphate transporters (Beene et al., 2011; Bun-yaet al., 1996) in prokaryotes and eukaryotes. The adventitious uptake andaccumulation of arsenic via nutrient transporters drive the evolution ofa variety of arsenic detoxification pathways. One of the major pathwaysis methylation, which has been studied inmicrobes, plants andmammals(Drobna et al., 2009; Qin et al., 2006; Ye et al., 2012). Methylation of

III, monomethylarsonous acid;cid; β-ME, β-mercaptoethanol;lutathione.sciences, Oakland University,48 370 4225.

rights reserved.

inorganic arsenicals produces different organic forms, which varyin ease of excretion, reactivity and carcinogenicity. In human, arsenicmethylation produces dimethylarsinic acid (DMAV) as a major prod-uct along with other species, including monomethylarsonous acid(MMAIII), monomethylarsonic acid (MMAV), dimethylarsonousacid (DMAIII) and trimethylarsine oxide (TMAO) (Styblo et al.,2002). All inorganic and methylated arsenicals can be detected inurine with different ratios depending on individual methylation effi-ciencies (Katsoyiannis et al., 2007; Styblo et al., 2002). Methylationhas been viewed as a detoxification process for a long time since pen-tavalent arsenic species are less toxic than inorganic arsenic (Stybloet al., 2002). However, the trivalent intermediates, MMAIII andDMAIII, which can also be produced and excreted as intermediates,aremore toxic than inorganic AsV or AsIII, whichmeans under certaincircumstances methylation activity can exacerbate arsenic toxicity(Drobna et al., 2005; Hirano et al., 2004; Petrick et al., 2000). There-fore, arsenic methylation serves as one of the decisive factors to de-termine the overall arsenic induced malignancy (Rehman andNaranmandura, 2012). However, it is not well understood what cau-ses interindividual variation in methylation efficiencies and in what

186 M. Hamdi et al. / Toxicology and Applied Pharmacology 262 (2012) 185–193

situations arsenic methylation is beneficial for human health (Chunget al., 2009; Kwong et al., 2010; Wnek et al., 2010).

The bonafide arsenicmethyltransferase, AS3MT,was isolated from ratliver, the major site for arsenic methylation (Thomas et al., 2004; Tseng,2007). Currently, several AS3MT homologs have been identified frommi-crobes to plants, each with a similar function in arsenic methylation(Meng et al., 2011; Qin et al., 2006). Based on sequence alignment, twoAS3MT homologs were identified in zebrafish. In this study, a closer ho-molog, as3mtwas cloned and the enzyme function in arsenicmethylationwas studied using purified recombinant protein.

The AsIII methylation by AS3MT is proposed to have two rounds of re-action; each round includes oxidativemethylation followed by reduction.Thefirst round reactionproducesMMAVwhich is then reduced toMMAIII,followed by a second round of methylation to DMAV (Marapakala et al.,2012). Some microbial homologs undergo a third round of methylationto TMA (trimethylarsinic acid), a gaseous form of arsenic (Qin et al.,2009), or the oxidized form of TMAO (trimethylarsine oxide). It hasbeen shown that intracellular glutathione (GSH) level regulates the for-mation of TMA and possibly serves as a broad determinant of the patternand extent of formation of arsenicmetabolites (Waters et al., 2004). How-ever, it is not clear which reaction step is rate-limiting. The detailedmechanisms are still lacking and questions such as whether toxic triva-lent intermediates are formed by AS3MT and which conditions favortheir accumulation remain unanswered. Currently reported biochemicalexperiments were done in conditions fostering a complete reactionusing high enzyme concentrations and prolonged reaction times, thusthe trivalent intermediate, MMAIII, has not been detected. In this study,under a well controlled condition dependent on SAM and a reduc-tant (β-mercaptoethanol, β-ME), we demonstrated that As3mt un-dergoes two rounds of step-wise methylation, with initial product ofmono-methylated MMAIII and MMAV, followed by the second andfinal rounds of step with product of DMAV.

The lack of a three dimensional protein structure of AS3MT homologsprevented the complete elucidation of enzymatic mechanisms such asinitial substrate binding and connection between different catalytic cy-cles. Currently, conserved cysteines have been identified in the sequencesof a wide range of AS3MT homologs, and mutation of these cysteines ledto the loss of zAs3mt function (Song et al., 2010). By homology modelingof As3mt using a recently available structure of an algal ArsM without(PDB ID 3P7E) and with bound AsIII (PDB ID 3QNH), we identified twoconserved residues, C160 and C210, that we suggest are required for ini-tial AsIII binding.

Zebrafish is an emerging model organism in toxicological studieswith advantages in ease of toxicant exposure, high embryo availabil-ity and amenability of genetic modification. Additionally, zebrafishgenome has conserved genes closely related to arsenic metabolismin humans thus providing an ideal animal model for investigatingmechanisms of pathogenesis induced by arsenic exposure. In thisstudy, we used zebrafish model to understand further the bio-methylation of arsenic both in vivo and in vitro.

The methylation of arsenic in humans is influenced by many fac-tors. For example, the individual difference, such as Body Mass Index(BMI), is found to be associated with altered arsenic methylation(Gomez-Rubio et al., 2011). Environmental factors, including thetypes of arsenic exposure and availability of micronutrients, for ex-ample selenium, may lead to variation in arsenic methylation. Thepresence of selenium will interfere with arsenic methylation given itsgeneral chemical properties similar to arsenic and its potential to inhibitAS3MT (Song et al., 2010). Epidemiological studies demonstrated thatlow serum selenium is associated with increased MMA in urine (Basu etal., 2011). In the current study we found that selenium, in the form ofselenite, is a potent inhibitor for As3mt function and is predicted to com-pete with AsIII for its initial binding. Since trivalent arsenite is also a clin-ically approved drug for leukemia treatment (Antman, 2001), factorsmodulating As3mt function will be correlated to the interindividual dif-ference in arsenic pathology and pharmacology. Detailed understanding

of the enzymatic mechanisms of As3mt will promote application ofzebrafish in the screening of efficient pharmacological intervention inthe methylation process. The analogy of arsenic metabolism in zebrafishvalidates the use of this model in future arsenic toxicology and carcino-genesis studies.

Materials and methods

Zebrafish husbandry and arsenic exposure. All studies involvingzebrafish have been approved by Oakland University IACUC. ZebrafishZF-1 line was maintained in an automatic 14:10 light:dark cycle at28 °C. Static waterborne arsenic exposure was carried out in adultzebrafish using 300 ppb or 5 ppm sodium arsenite for 120 h. After treat-ment, zebrafish were rinsed for 1 h and euthanized with 0.3 mM tricaine(MS-222 (Ethyl 3-Aminobenzoate Methanesulfonate), Sigma). Tissueswere isolated and pooled (30 fish tissues used as one sample) and massof each sample was determined. All tissue samples were homogenizedin phosphate buffered saline (PBS buffer, pH 7.4) and adjusted to a finalconcentration of 100 mg wet tissue/ml buffer, and then centrifuged at13,000×g for 10 min. The resulting supernatants were filtered with aMicrocon Ultracel YM-3 centrifugal filter (Millipore, MA) for speciationanalysis by HPLC–ICP-MS (Hamdi et al., 2009).

Chemicals. The MMAIII was obtained through Dr. Styblo's lab (NorthCarolina University). All other chemicals were obtained through com-mercial sources at analytical grade.

Cloning of zebrafish as3mt and site-directed mutagenesis. Zebrafishas3mt was cloned from a cDNA mixture synthesized from mRNA thathas been extracted from whole zebrafish, and amplified using a pairof PCR primers with BglII and XhoI restriction sites (forward: 5′-GCAGATCTATGGCACCACGTCCAAAGCAGG-3′ and reverse: 5′-GCCTCGAGTCTATAAAGATGTTGCCTTCAG-3′). The amplified as3mtwas initial-ly cloned into pGEMT-easy (Promega) and a second round of PCR isapplied to add 6XHis tag, followed by subcloning into pMAL-cX2 expres-sion vector (NEB) (Liu et al., 2006). The resulting plasmid pMal-as3mtwas transformed into Escherichia coli BL21 (NEB) for overexpressionand purification. The mutants of C165S and C210S were created bysite-directed mutagenesis (Stratagene) using following primers:C165S forward: 5′-GATATTATCATA TCAAATTCTGTGGTGAATCTG-3′;reverse: 5′-CAGATTCACCACAGAATTTGATATGATAATATC-3′; C210Sforward: 5′-CTTTATGGGGCGAGAGCCTCAGTGGAGCATTG-3′; reverse: 5′-CAATGCTCCACTGAGGCTCTCGCCCCATAAAG-3′. Both the WT gene andthe mutants were verified by nucleotide sequencing.

Overexpression and purification of As3mt and mutants in E. coli. TheE. coli strain BL21 carrying pMAL-as3mt1 was grown in LB medium sup-plied with ampicillin at 37 °C to an OD600 0.6–1.0, followed by inductionwith 0.6 mM IPTG for 8 h. The overnight culture was harvested by centri-fugation and washed. The cells were resuspended in buffer A (50 mMMOPS, pH 7.5, 20% (wt./vol.) glycerol, 0.5 M NaCl, 20 mM imidazole,and 10mM β-ME) and lysed by a single pass through French-press at20,000 psi. Membranes and unbroken cells were removed by ultracentri-fugation. The supernatant was loaded at a flow rate of 0.5 ml/min onto aNi(II)-NTA column (QIAGEN) preequilibrated with buffer A. After wash-ing with the same buffer, protein was eluted with 60 ml of buffer B(50 mMMOPS, pH 7.5, 20% (wt./vol.) glycerol, 0.5 MNaCl, 200 mM imid-azole, and 10 mM β-ME). Fractions containing zAs3mt were pooled andloaded onto the Amylose column (NEB) preequilibrated with buffer C(200 mM NaCl, 20 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM sodiumazide, 10 mM β-ME) and washed. The zAs3mt protein was eluted withbuffer D (200 mM NaCl, 20 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM so-dium azide, 10 mM β-ME, 10 mM maltose). The zAs3mt fractions wereconcentrated using a 30-kDa-cutoff Amicon Ultrafilter (Millipore). Pro-tein concentrations were determined by Absorbance Assay (280 nm).The purity of As3mt1 was determined by SDS gel electrophoresis.

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SDS-PAGE and western immunoblotting. Protein samples or zebrafishtissue samples (males and females) were processed at 100 °C for 5 minand loaded on 12% SDS-PAGE gel, followed by coomassie brilliant bluestaining. A custom raised primary polyclonal antibody (Abmart) ofzAs3mt was applied in the western-blotting at 1:1000 dilution. Banddensity was quantified using the ImageJ software after scanning. Meanvalue and standard errors were calculated using SigmaPlot 10.0.

Analysis of enzymatic reaction using purified As3mt. The in vitro re-action mixture contained 10 mM Tris–HCl (pH 7.4), 0.1 M NaCl,0.5 mM EDTA, 5 mM β-ME, 1 mM SAM, 1 mM GSH, 100 μM sodiumarsenite (AsIII) and 1 μM zAs3mt, unless other concentrations are in-dicated. Reaction mixtures were incubated at 37 °C for indicatedtimes and the methylation reaction was stopped by filtration with acut off column. A negative control was performed in the absence ofzAs3mt from the above mixture. Inhibitors were added at indicatedconcentrations at the beginning of the reaction.

Arsenic binding assay. Sodium arsenite at indicated concentrationswas incubated with 2 μM purified WT or mutant proteins. After 30 minthemixture was passed through spin columns (Micro Bio-Spin 6, Biorad)to remove the unbound free arsenic. The protein bound arsenic was elut-ed and quantified by inductively coupled plasma-mass spectrometer(ICP-MS, ELAN 9000, PerkinElmer).

Structural modeling of zAs3mt. The 0.63 Å resolution structureCyanidio ArsM (CmArsM, 1–323 residues, PDB ID 3P7E) was used as areference to construct a C-terminal truncated (1–299 residues) As3mtmodel. The two sequences shared 43.85% of identity and 285 residueswere aligned.

Arsenic speciation by HPLC–ICP-MS. The speciation of samples fromcell lysate, enzymatic reaction mixtures or the zebrafish tissues wasachieved using ICP-MS coupled with HPLC (Series 2000, PerkinElmer) inthe front end. For arsenic samples generated from the enzymatic reactionthe reactionwas halted by removing As3mt from the reaction samples bycentrifugation using a 10-kDa-cutoff Amicon Ultrafilter (Millipore). Thefiltrate was then separated and analyzed by HPLC–ICP-MS using areverse-phase C18 column (Jupiter 300) eluted isocraticallywith amobilephase (3 mM malonic acid, 5 mM tetrabutylammonium hydroxide, and5% methanol, pH 5.6, with a flow rate of 1.0 ml/min). Arsenic standardswere purchased from commercial suppliers.

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Fig. 1. Arsenic methylation in zebrafish tissues. Adult zebrafish were exposed to sodium arspeciation assays using HPLC–ICP-MS. Each sample contains a pool of thirty tissues. Speciesresent 5 ppm and the lower plots represent 300 ppb AsIII treated samples.

Results

Zebrafishmethylate inorganic AsIII toMMAII, MMAV andDMAV under arsenicexposure

The arsenic biomethylation profile was examined in zebrafish(males and females) and compared under arsenic exposure. Two ar-senic concentrations, 300 ppb and 5 ppm, were applied for 120 h tomodel two different levels of non-lethal doses. The pattern of arsenicmethylation was determined and compared in different tissues. Ourresults showed that, compared with acute exposure, little methylatedarsenic can be detected following sublethal exposure, with AsIII as thedominant species in most tissues (Fig. 1). When 5 ppmwas applied, asignificant amount of methylated arsenic species appeared. DMAV

was found in brain, gill, and liver while MMAV was detected in all tis-sues at a relatively low level. The intestine showed a significant amountof MMAIII, and several unknown components, possibly involved the ar-senic methylation through microbial activity. An unknown peak notcorresponding to any arsenic standards that eluted after DMAV wasdetected in almost all tissues and is hypothesized to be some type ofAs-GSH conjugate or a metabolite of an uncharacterized pathway. In alltissues the inorganic form of AsIII persisted in many tissues as the dom-inant species, indicating that the biomethylation in these tissues cannotbe efficiently processed. Factors accounting for differences of arsenic re-tention patterns in specific tissuesmay be collectively determined by thecombined activity of biomethylation, uptake and efflux. We noticed thatskin displayed aminimal arsenic accumulation but with high ratio of bio-methylation. Since the arsenic transporters that facilitate AsIII uptakewere identified in skin (Hamdi et al., 2009),we predict skin could have ei-ther efficient extrusion systems or is able to metabolize arsenic via un-known mechanisms. During all AsIII exposures the other importantinorganic arsenical, AsV, was not detected in any tissues. Our previousstudies show that no AsV is detected when zebrafish were exposed toAsV, indicating that efficient reducing systems exist in zebrafish (Beeneet al., 2011).

The As3mt is extensively expressed in zebrafish tissues

The expression of As3mtwas ubiquitous in zebrafish tissues (Fig. 2). Incontrast to mammalian AS3MT which is mainly expressed in liver,zebrafish As3mt is a broadly expressed protein detected in brain, eye,gill, intestine, liver, muscle and skin. Quantitative analysis showed that

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Fig. 2. Expression of As3mt in different zebrafish tissues determined by western-blotting.Zebrafish tissue was isolated and homogenized. All samples were suspended in PBS bufferat final concentration of 200mgwet tissue/ml.Western blottingwas performed using a cus-tom raised antibody (Abmart). To quantify the expression of As3mt in different tissues, acommercial antibody that reacts with pan-actin (alpha-actin and beta-actin) was appliedas a control. Lower panel: quantification of As3mt protein expression from western-blotting analysis. Three replicates were obtained from western-blotting and quantified byImageJ after scanning. Mean value and standard errors were derived with SigmaPlot 10.0.

188 M. Hamdi et al. / Toxicology and Applied Pharmacology 262 (2012) 185–193

zAs3mt is mostly expressed in liver, muscle and intestine. As a result, ar-senicmethylation occurred inmany tissues andmethylated arsenic prod-ucts were detected (Fig. 1). However, in tissues such as muscle withsubstantial As3mt expression, no methylated species were detected. It is

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possible that the activity of this enzyme is sensitive to tissue microenvi-ronments, such as an absence of essential cofactors.

Purified As3mt catalyzed step-wise methylation of AsIII, producing DMAV

as the end product and MMAIII and MMAV as intermediates

His-tagged andMBP-tagged As3mt were purified as soluble recombi-nant proteins at approximately 80 kDa following Nickle-column andMaltose columnpurification (Fig. 3A). The enzymatic activitywas assayedin buffer D, which contains SAM and β-ME, with an enzyme:substratemolar ratio of 1:100. The reactionwasmonitored in a time‐course fashionin order to detect step-wise formation and release of intermediates andproducts. The presence of MMAV and MMAIII was detected in the firstround of methylation, followed by a second round of methylation toDMAV (Fig. 3). Our results showed that DMAV is the final product andMMAV and MMAIII are plausible intermediates. These in vitro results areconsistent with our observations in vivo. DMAV is also the dominant bio-methylation product detected inmammals and the final product formostAS3MT homologs, showing that zebrafish and mammals have similar ar-senic biomethylation pathways. MMAIII is rarely observed in tissues, pos-sibly due to the rate of second round of methylation following MMAIII

formation which is faster than the first round. To our knowledge, this isthefirst experimental evidence showing the existence ofMMAIII interme-diate in an in vitro system using a purified arsenic methyltransferase.

Increased substrate concentration inhibits As3mt function

As AS3MT catalyzes multiple steps in arsenic methylation it is antici-pated that themethylation patternmight be different according to variedsubstrate concentrations. To address this question in vitro, the effect ofsubstrate doses on zAs3mt activity was investigated by applying variousconcentrations of AsIII concentrations in the enzymatic reaction mixture.As shown in Fig. 4, with an AsIII concentration range from 25 μM to

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200 μM the As3mt activity declined. As3mt completely lost methylationability at an AsIII concentration of 200 μM.

Selenite is an inhibitor for As3mt

Selenium has been used to antagonize arsenic toxicity. In this studywe found that selenium, in the form of selenite, inhibited As3MT activityefficiently. Addition of sodium selenite at 1:10molar ratiowith AsIII in thereaction mixture completely abolished the arsenic methylation (Fig. 5).

Mutation of two cysteines (C160 and C210) leads to a loss of As3mt functionand arsenic binding

In this studywedirectly tested if two cysteines (Fig. 6A) are involvedin AsIII binding. The C165S and C210S mutants of As3mt were createdand ectopically expressed in E. coli. The arsenic methylation activity ofthese mutants was studied and compared with the wild type (WT)As3mt. The cysteine mutation in these sites completely abolished thefunction of zAs3mt and no methylated arsenic species were produced(Fig. 6B). To further determine if these cysteines are required for sub-strate AsIII binding a direct assaywas performed to investigate the prop-erties of C165S and C210S in arsenic binding. Our results showed thatWT As3mt binds AsIII substrates and saturated at an approximately1:1 stoichiometry ratio, suggesting one AsIII binding site in the enzyme.However, both C160S and C210S mutants lost capability to bind AsIII,again indicating these two residues are required for substrate binding(Fig. 6C). In support of this hypothesis, a structure model was con-structed based on a thermophilic alga Cyanidioschyzon sp. 5508 ArsM.As suggested in As3mt structure, the cysteines C160 and C210 are inclose proximity and form a reasonable AsIII binding site (Fig. 6D).

Discussion

Due to the abundance of arsenic in the environment and diversity ofmechanisms through which arsenic causes toxicity, it is important to

understand the regulation of chemical metabolism, distribution, andbodily clearance of arsenic. The methylation of arsenic is one of thekey variables and thus detailed understanding of mechanism of arsenicmethyltransferases is crucial. Despite the possibility of other enzymesthat catalyze arsenic biomethylation, such as N-6 adenine-specificDNAmethyltransferase (N6AMT1), which was recently found to meth-ylateMMAIII to DMAV (Ren et al., 2011), AS3MT is considered themajorenzyme in arsenic biomethylation. This is consistently supported bymany independent clinical studies which link AS3MT variants with al-tered profiles and ratios of arsenic methylated species (Engstrom etal., 2011; Valenzuela et al., 2009). It is clear that AS3MT transforms ar-senic into less toxic DMAV which is more easily cleared from the body.However, it remains an open question whether AS3MT is responsiblefor the formation of more toxic intermediates such as MMAIII. Thisstudy showed zAs3mt transforms AsIII to DMAV similar to mammalianAS3MThomologs (Fig. 3). The absence of significantMMAIII in zebrafishtissue after arsenic exposure can be explained throughMMAIII not beingrate limiting in the second round under these exposure conditions.

190 M. Hamdi et al. / Toxicology and Applied Pharmacology 262 (2012) 185–193

Fig. 6. Cys165 and Cys210 are predicted AsIII binding sites. A). Sequence alignment of hsAS3MT, zAs3mt and CmArsM. Three conserved cysteines were indicated in the figure (boxed).B). Mutants of C160S and C210S were unable to methylate AsIII. Upper panel: cytosol from E. coli expressing mutants C160S and C210S with IPTG induction. Lower panel: arsenicspeciation in the culture medium of WT, negative control, mutants C160S and C210S, respectively after IPTG induction. Inside the figure showed the As3mt expression in E. coliafter IPTG induction. C). C160S and C210S were unable to bind AsIII. Upper panel, WT, C160S and C210S were purified as recombinant proteins. Lower panel, binding assay ofzAs3mt WT, C160S and C210S indicate that mutation of cysteines at 160 and 210 disabled the AsIII binding (plotted with SigmaPlot 10.0 with a hyperbolic curve). D). Structuremodel of zebrafish As3mt. The 0.63 Å CmArsM (1–323 residues) structure was used to construct As3mt model (1–299 residues). Cys160 and Cys210 were labeled in yellow color.Two addition functional domains, SAM binding domain and C-terminal domain were indicated in the structure.

191M. Hamdi et al. / Toxicology and Applied Pharmacology 262 (2012) 185–193

The unique nature of arsenic speciation may influence the pathogen-esis of certain diseases such as cancer. The carcinogenic potential of arse-nic is related to the sum of total arsenicals, including endogenouslygenerated organic species. Therefore, function of AS3MT is a key factorin arsenic-induced carcinogenesis by affecting arsenic metabolism andbodily retention. This assumption is corroborated by a correlation be-tween arsenicmethylation and cancer incidence in epidemiological stud-ies carried out in arsenic contaminated areas (Kwong et al., 2010; Wneket al., 2010). Moreover, it has been shown by independent reports thatAS3MTpolymorphisms influence arsenicmetabolismand individual can-cer risk (Agusa et al., 2009; Kojima et al., 2009; Valenzuela et al., 2009).Analysis of arsenic speciation in urine shows that the concentration ofmonomethylated species is highly correlated with increased cancer risk(Chung et al., 2009).

Possible carcinogenic mechanisms include arsenic induced oxida-tive and DNA damages (Kojima et al., 2009) and pathogenesis via thegeneration of reactive oxygen species (ROS) (Jomova et al., 2011). Al-though all forms of inorganic arsenic as well as certain organic formshave been shown to generate ROS, the magnitude of arsenic inducedoxidative stress is highly speciation dependent (Hughes, 2009). Arsenicalso deregulates oncogenic signaling pathways and this is also likely tobe speciation dependent. Given that arsenic biomethylation via AS3MTactivity produces intermediates with different capacities in elevatingoxidative stress and ROS, it is reasonable to assume that AS3MT activitymay link to overall arsenic-induced carcinogenesis via controlling OSduring arsenic exposure. Thus, the biologic action of AS3MT is predictedto play important roles in arsenic induced oxidative stress, DNA damageand ultimately, carcinogenesis. It follows that factors that modify AS3MT

192 M. Hamdi et al. / Toxicology and Applied Pharmacology 262 (2012) 185–193

activity will affect overall arsenic-induced carcinogenesis. Therefore it iscritical to elucidate the fundamental mechanisms of arsenic-inducedmethylation by AS3MT, and investigate the factors that may modify theenzymatic activities.

The function of AS3MT in arsenic methylation raises the question ofhow AS3MT produces methylated trivalent species and under whichconditions the more toxic intermediates exist. It has been difficult tomeasure the activity of AS3MT in real-time. Some trivalent arsenic,such asMMAIII, can be detected in human urine; however, this plausibleintermediate has not been observed using purified enzyme. Possiblereasons for the lack of such results are: 1) most studies of methylationactivity using purified human or rodent AS3MT were performed withhigh enzyme:substrate ratios, which fosters a complete reaction, butnot detectable quantities of intermediates, i.e., the trivalent methylatedspecies (Song et al., 2011); 2) until recently, no three dimensional struc-tures were available, which precluded structure–function analysis andelucidation of the catalytic mechanism. In our studies, using a purifiedzebrafish As3mt, we studied the time‐course of arsenic methylation,and demonstrated the existence of trivalent and pentavalent arsenic in-termediatesMMAIII andMMAV,whichwere formed in the first round ofthe cycle. The reaction ends up with DMAV and the third round of meth-ylation is not achieved, therefore TMA gas is not detected (data notshown). These in vitro results are highly consistentwith the arsenic distri-bution in vivo, with all these intermediates and end products detectedunder AsIII exposure. In rodents, the dominant product of AsIII bio-methylation is DMAV, with a small portion of MMAV, which has beendemonstrated in tissue culture and knockout mice (Drobna et al., 2004).The arsenic cellular retention is related to both biomethylation andmem-brane transport of substrates and products. For example, in human hepa-tocytes, DMAV is readily extruded while MMAV is mostly retained(Drobna et al., 2004). Our results in tissues of AsIII exposed zebrafish in-dicated that MMAV and DMAV could be detected in many tissues withdifferent patterns which resemble those in rodents and humans. Inter-estingly, an uncharacterized arsenic compound which is not consistentwith any known arsenic methylated compounds was identified in alltissue samples. Further studies are required to determine whether thisarsenic species resulted from a different metabolic pathway otherthan biomethylation.

The AS3MT catalytic mechanisms were previously studied usingbiochemical approaches (Song et al., 2010) but the detailed mecha-nisms such as AsIII binding, and the continuous catalysis of two cyclesof reactions have not been elucidated in AS3MT. By site-directed muta-genesis, function of conserved and non-conserved cysteines were in-vestigated in human AS3MT where a cysteine, C72, is shown to becritical for the reaction and hypothesized to be involved in the initialsubstrate binding (Song et al., 2011). However, in our study usingAs3mt, we suggest that C165 and C210 form a substrate binding site.Single mutation of these cysteines disabled the As3mt function as wellas binding ability to arsenic. Therefore the C165 and C210may togetherform a coordinating site for arsenic binding. The coordinated arrange-ment of these two cysteines was indicated in the structure model ofAs3mt in this study (Fig. 6D). In this model, these two cysteines areclose to each other and form a pocket for arsenic binding. Single muta-tion of C65S produced indistinguishable levels of AsIII binding to WT(data not shown), indicating that its importance comes from a role incatalytic activity following binding. Results using CmArsM orthologshow that C74 (C65 in zebrafish As3mt) is required only for the secondround of methylation but not the first round of methylation, while bothCys174 and Cys224 are involved in the entire reaction. Taken togetherour results are consistent with the information derived from CmArsMstructure, showing that AsIII is bound by Cys160 and C210 (Cys174and Cys224 in CmArsM)while Cys65 (Cys72 in CmArsM)moves towardthe other two AsIII-binding cysteine residues when SAM is bound. Themodel also discloses a SAM binding domain and a C-terminal domainwith an unknown function. More studies are required to investigatethe in depth coordination of the two rounds of reaction.

The function of AS3MT is affected by several factors, such as diva-lent metals zinc, copper, mercury and metalloid selenite (Song et al.,2010). Through different mechanisms these compounds all reactwith coordinated thiols. We report here that selenite is an efficient in-hibitor for zAs3mt function. Given both its similar molecular structureto AsIII and ability to bind vicinal thiols, it is postulated that SeIV iscompeting for same binding sites as arsenic, similar to the binding ofcysteines in zinc finger structure (Larabee et al., 2002).We also showedthat elevated substrate concentration completely abolished the As3mtactivity in the MMA or DMA production. Currently the basis is notclear and there are several possibilities: 1) increased substrate will af-fect the initial binding and change the appropriate position of substrateand cofactors. 2) Increased substrate is toxic for the enzyme bymodify-ing the overall enzyme conformation.More experiments are required toinvestigate the precise mechanisms of substrate inhibition in zAs3mt.

The in vitro activity of As3mt andothermammalianAS3MT is relative-ly low, which requires a higher enzyme:substrate ratio. Possible explana-tions for the reduced activity are that there are unknown cofactors absentfrom the in vitro system, such as endogenous reducing systems. The ele-vated AsIII retention in tissues indicates that the in vivo activity of AS3MTis also limited. Froman evolutionary perspective, As3mt function requiresSAM,which requires ATPduring synthesis. On the other hand, it is reason-able that zebrafish or mammals do not require a highly efficient enzymeto metabolize commonly occurring low levels of environmental arsenic.An additional detoxification pathway, such as MRP-mediated GSH conju-gate efflux can provide an alternative detoxification pathway (Liu et al.,2001).

Currently there are no studies on the regulation of As3mt and fur-ther studies are needed to understand the regulation of As3mt ex-pression along with the role of As3mt in a long-term chronic arsenicexposure. The zebrafish model can be well adapted to these purposesby the availability of as3mt transgenesis, along with the advantage inconducting high throughput chemical exposure studies and dose re-sponsive toxicity studies with a large number of samples applied.Zebrafish can be an ideal animal model for translational researchnot only to understand the role of As3mt in overall arsenic-inducedcarcinogenesis but also to facilitate therapeutic drug discovery fortreatment and prevention for arsenic related malignancy via pharma-cological intervention (Amatruda and Patton, 2008; Lam et al., 2006;Nguyen et al., 2011).

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by NIH ES016856 to Zijuan Liu and NIHGM55425 to Barry P. Rosen (Florida International University). We ap-preciate Dr. Barry P. Rosen's critical review of this manuscript.

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