Toxicology 249 (2008) 69–74

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Cytotoxicity of combinations of arsenicals on rat urinary bladderurothelial cells in vitro

Merielen G. Nascimentoa,1, Shugo Suzukia, Min Weia,2, Ashish Tiwaria,3, Lora L. Arnolda,Xiufen Lub, X. Chris Leb, Samuel M. Cohena,c,∗

a Department of Pathology and Microbiology and the Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198-3135, United Statesb Department of Environmental Health Sciences, University of Alberta, Edmonton, Alberta T6G 2G3, Canadac Havlik-Wall Professor of Oncology, University of Nebraska Medical Center, Omaha, NE, United States

a r t i c l e i n f o

Article history:

a b s t r a c t

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinking water isy blad moin adof thcytot

Received 4 March 2008Received in revised form 10 April 2008Accepted 14 April 2008Available online 22 April 2008

Keywords:

carcinogenic to the urinarlarsinous acid (DMAIII) aninorganic arsenic in vivoarsenicals. The objectiveergistic toward inducing

ArseniteDimethylarsinic acid (DMAV)Dimethylarsinous acid (DMAIII)Trimethylarsine oxide (TMAO)Urinary bladderUrothelial cell cytotoxicity

but not transformed rat urinarTreatment with the arsenicalsarsenicals used were DMAIII wiCombinations of concentrationone-half or one-quarter the LC5

cells were treated with arsenatby HPLC-ICPMS to determine sone-quarter or one-half the LC5

same as when cells were treateof either arsenical. Treatment wcentration of a second arsenicalof the arsenicals. Quantitation acells have some reductase activof arsenicals in combination wa

1. Introduction

Based on epidemiological data, chronic exposure to high levelsof inorganic arsenic in the drinking water is carcinogenic to the

∗ Corresponding author at: Department of Pathology and Microbiology and theEppley Institute for Cancer Research, Havlik-Wall Professor of Oncology, Universityof Nebraska Medical Center, Omaha, Nebraska 68198-3135, United States.Fax: +1 402 559 9297.

E-mail address: scohen@unmc.edu (S.M. Cohen).1 Present address: Center for the Evaluation of the Environmental Impact on

Human Health (TOXICAM), Department of Pathology, Botucatu Medical School,University of Sao Paulo Estate (UNESP), 18618-000 Botucatu, Sao Paulo, Brazil.

2 Present address: Department of Pathology, Osaka City University Medical School,Osaka 545-8585, Japan.

3 Present address: Department of Chemistry, Indian Institute of Technology,Kanpur 208016, India.

0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2008.04.007

dder of humans. The highly reactive trivalent organic arsenicals dimethy-nomethylarsonous acid (MMAIII) are formed during the metabolism ofdition to the corresponding mono-, di- and trimethylated pentavalent

is study was to determine if combining arsenicals was additive or syn-oxicity in a rat urothelial cell line. The MYP3 cell line, an immortalizedy bladder epithelial cell line, was seeded into appropriate culture wells.was begun 24 h after seeding and continued for 3 days. Combinations ofth arsenite, dimethylarsinic acid (DMAV) or trimethylarsine oxide (TMAO).s used were the LC50, one-quarter or one-half the LC50 of one arsenical with0 of the other arsenical. To determine if MYP3 cells metabolize arsenicals,e, arsenite and MMAV as described above and the medium was analyzedpecies and quantity of arsenicals present. When cells were treated with0 concentration of both arsenicals, the cytotoxicity was approximately thed with half the LC50 concentration or the LC50 concentration, respectively,ith one-quarter the LC50 concentration of one arsenical plus the LC50 con-had similar cytotoxicity as treatment with the LC50 concentration of eithernd speciation of arsenicals in the cell culture medium showed that MYP3ity but the cells do not methylate arsenicals. The effect on the cytotoxicity

s additive rather than synergistic toward a rat urothelial cell line.

© 2008 Elsevier Ireland Ltd. All rights reserved.

urinary bladder of humans (NRC, 1999). Inorganic arsenic existsmainly in the environment as the trivalent arsenite or the pentava-lent arsenate (NRC, 1999). When inorganic arsenic is ingested, itundergoes a series of reductions and oxidative methylations beforebeing excreted in the urine. It has long been thought that thismethylation pathway was a detoxification pathway (Vahter, 1983),but recently it was shown that the highly reactive trivalent organicmetabolites monomethylarsonous acid (MMAIII) and dimethylarsi-nous acid (DMAIII) are formed during the metabolism of inorganicarsenic (Le et al., 2000), and that these trivalent forms of methy-lated arsenicals are highly toxic and may significantly contribute toarsenic carcinogenesis.

Experimental evidence strongly indicates that arsenicals do notbind directly to DNA, and therefore, it is unlikely that arsenic isa DNA-reactive carcinogen (EPA, 2001; Kitchin, 2001; Kenyon andHughes, 2001; Basu et al., 2001). Long-term oral exposure to the

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70 M.G. Nascimento et al. /

organic arsenical dimethylarsinic acid (DMAV) resulted in urinarybladder tumors in rats, with the female more sensitive than themale (Arnold et al., 2006). Short-term experiments with female ratstreated with DMAV showed that its mode of action was defined ascytotoxicity with subsequent regenerative proliferation resultingin hyperplasia and ultimately tumors (Arnold et al., 1999; Cohen etal., 2001). The reactive metabolite DMA III is present in the urineof DMAV-treated rats (Cohen et al., 2002, 2006, 2007). DMAV wasnot carcinogenic to the urinary bladder or other organs in mice in achronic bioassay (Arnold et al., 2006). Recent short-term studies inwhich hyperplasia was observed in the bladder epithelium of ratsand mice following oral administration of high doses of inorganicarsenic (arsenite or arsenate) suggest a similar mode of action in therat bladder for inorganic arsenic (Arnold et al., 2007). Studies haveshown that regardless of the form of arsenic administered, rats bindthe arsenical in the form of DMAIII to an available cysteine present inthe hemoglobin molecule in rat red blood cells that is not present inhumans (Lu et al., 2007), resulting in a slower clearance of arsenicalsin rats than in humans and other species. Rats have another signif-icant difference in arsenic metabolism compared to other species,including humans (Lu et al., 2007). Inorganic arsenic is biomethy-lated by an alternating series of reduction of the pentavalent speciesto the trivalent species followed by oxidative methylation to thepentavalent species. In humans, the biomethylation appears tostop at the pentavalent dimethyl arsenical, DMAV. In rats, however,the biomethylation process proceeds further to the formation oftrimethyl arsenicals, mainly trimethylarsine oxide (TMAO) (Lu etal., 2003) even at low exposures. Efforts have been made to clarifythe mode of action of arsenicals and their metabolites. Despite thesemetabolic differences, the rat model provides an appropriate modelfor investigations of the mode of action for arsenic carcinogenic-ity. The detailed mechanistic effects of arsenic metabolites on theurinary bladder remain unknown in rats as well as in humans, butaccumulating evidence suggests that it involves generation of reac-tive urinary metabolites (trivalent arsenicals), producing urothelialcytotoxicity and regenerative proliferation (Cohen et al., 2001, 2002,2006, 2007).

In vitro experiments have been used to clarify the mechanisticeffects of arsenic metabolites using different cell lines (Gottschalget al., 2006; Kao et al., 2003). Experiments using rat (MYP3) andhuman (1T1) urothelial cell lines showed that DMAIII is cytotoxicto urothelial cells at sub-micromolar concentrations (Cohen et al.,2002). Additionally, trivalent arsenicals (MMAIII and DMAIII) as wellas inorganic arsenic were cytotoxic at concentrations at least 3

orders of magnitude lower than the pentavalent organic arsenicspecies (DMAV, MMAV and TMAO) (Cohen et al., 2002).

Following exposure to inorganic arsenic, either arsenite or arse-nate, a variety of arsenicals, including trivalent species, are presentin the urine secondary to the extensive metabolism that occurs. Itis possible that the mixture of arsenicals in the urine of arsenic-treated rats enhances the urothelial cytotoxicity that is producedgreater than what would be expected for any one of the specificarsenicals present. It is not presently understood which arsenicalsor combination of arsenicals are important for bladder cytotoxic-ity. The objective of this study was to determine the cytotoxicity ofcombinations of various arsenicals on rat urothelial cell lines.

2. Materials and methods

2.1. Chemicals

Sodium arsenate and sodium arsenite were purchased from Sigma (St. Louis,MO). The purity stated by Sigma was 94% for arsenite and 98% for arsenate. DMAV

and MMAV were provided by Luxembourg Industries (Tel-Aviv, Israel). The purityof each was greater than 98.5%. Trimethylarsine oxide (TMAO) was obtained fromTriChemical Laboratories, Inc. (Yamanashi, Japan) with a purity of 98%.

logy 249 (2008) 69–74

MMAIII and DMAIII were synthesized by Dr. William Cullen (University of BritishColumbia, Vancouver, Canada). MMAIII and DMAIII were supplied as the diiodideand monoiodide, respectively. NMR analysis at the University of British Columbiaconfirmed the identity of both chemicals and that the purity of each was at least99%. DMAV purity was confirmed by NMR at UNMC and the other arsenicals wereused without verification of purity. MMAIII and DMAIII were stored in the dark atapproximately 4 ◦C, TMAO was stored in the dark at room temperature, and theother arsenicals were stored desiccated at room temperature. A sample of at least1 g of the test articles, except for TMAO, was retained and stored in the same manner.Due to its extremely high cost, only 0.1 g of TMAO was purchased and therefore nosample was retained.

2.2. Cell line

The MYP3 urinary rat bladder epithelial cell line was provided by Dr. RyoichiOyasu (Northwestern University, Chicago, IL). The MYP3 cell line was obtained froma small nodule that developed in a heterotopically transplanted rat urinary bladderafter treatment with N-methyl-N-nitrosourea (MNU) (Kawamata et al., 1993). Thecell line has retained the characteristics of epithelial cells in culture, expresses ker-atin 5 mRNA, does not exhibit anchorage-independent growth, and does not causethe development of tumors when inoculated subcutaneously in nude mice. The cellswere grown in Ham’s F-12 medium (Gibco-BRL, Grand Island, NY) supplementedwith 10 �M non-essential amino acids, 10 ng/ml epidermal growth factor (EGF),10 �g/ml insulin, 5 �g/ml transferrin, 10% fetal bovine serum, 100 U/ml penicillin,and 100 �g/ml streptomycin (all from Gibco) and 2.7 mg/ml dextrose and 1 �g/mlhydrocortisone (from Sigma, St. Louis, MO). All cells were grown in an atmosphereof 95% air and 5% CO2 at 37 ◦C.

2.3. Experimental design

2.3.1. Experiment 1Cells were seeded at a concentration of 1.0 × 104 cells/24-well plate. Twenty-

four hours later, treatment with various combinations of arsenicals was begun andcontinued for 3 days without changing the medium. In preliminary experiments, itwas determined that seeding with 1.0 × 104 cells/well and treating for 3 days afteran initial 24-h incubation period, resulted in consistent growth in the control wellswithout the control wells reaching 100% confluency prior to the end of treatment(data not shown). Because DMAIII is unstable in culture medium (personal commu-nication from M. Styblo), cells were exposed to a decreasing dose of DMAIII over the3-day treatment period. Cell viability was determined by staining with trypan blueand counting the area of four squares in a hemocytometer. Results were confirmedby counting all nine squares for the combination experiments (data not shown).The percent survivability was calculated as the ratio of cell number in the arsenical-treated cell culture to that in the control culture. The LC50 dose (concentration lethalto 50% of cell population) was determined for each chemical separately and calcu-lated by linear regression analysis of the data using JMP 5.1.1 (SAS Institute Inc., Cary,NC). Concentrations of arsenicals used in the combined arsenical experiments werethe LC50 concentration, one-half or one-quarter of the LC50. Each combination wastested in triplicate. The differences between the treatments and the control werecompared by ANOVA, which, when significant (p < 0.05), was followed by the Tukeytest.

2.3.2. Experiment 2Cells were seeded at a concentration of 1.0 × 104 cells/24-well plate. Twenty-

four hours later, treatment with sodium arsenate, sodium arsenite or MMAV wasbegun and continued for 3 days without a change of medium. Each chemical wastested in triplicate. Cell viability was determined the same as described above. TheLC50 dose was calculated using linear regression analysis of the data in MicrosoftExcel. Quantitation of arsenic species in the medium was used to determine if MYP3cells are able to metabolize arsenicals. An extra plate without seeded cells was pre-pared using the same conditions to clarify if the arsenical activities in the mediumafter 3 days of treatment underwent any spontaneous change. After the treatmentperiod, medium was removed from the wells, frozen in liquid nitrogen, and shippedovernight on dry ice to the University of Alberta, Canada. Quantitative determinationof the arsenic species in the medium was carried out by ion-pair HPLC separation(Le et al., 2000) followed by inductively coupled plasma mass spectrometry (ICPMS)detection (Le et al., 2004; Yuan et al., 2008). Briefly, HPLC separation involved theuse of a reversed phase C18 column (ODS-3, 150 mm × 4.6 mm, 3-�m particle size,Phenomenex, Torrance, CA) and a mobile phase containing 5 mM tetrabutylammo-nium hydroxide, 3 mM malonic acid, and 5% methanol (pH 5.9). An Agilent 7500csoctopole reaction system ICPMS (Agilent Technologies, Japan) was operated at aradio frequency power of 1550 W and an argon carrier gas flow rate of 0.8 L/min.Helium (3.5 mL/min) was used as the collision gas for the octopole reaction modeto reduce isobaric and polyatomic interference. Total arsenic concentrations in themedium were also measured directly using ICPMS after the samples were acidifiedwith 1% HNO3. The standard reference materials (SRM) 1640 Trace Elements in Nat-ural Water and SRM2670 Trace Elements in Urine, both from the National Instituteof Standards and Technology (Gaithersburg, MD), were used for quality control.

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M.G. Nascimento et al. /

3. Results

3.1. Experiment 1

Each arsenical, tested individually at all doses used for thecombination tests, showed that the cytotoxicity increased in a dose-dependent manner (data not shown). The determination of the LC50in MYP3 cells showed that the cytotoxicity of DMAV and TMAOwere in the millimolar range and that the cytotoxicity of arsenite

and DMAIII were in the micromolar range (Fig. 1), similar to ourprevious studies (Cohen et al., 2002).

When one-quarter or one-half of the LC50 for two of the arseni-cal species was combined, the cytotoxicity was increased comparedto the cytotoxicity when the cells were treated separately with thesame dose and same arsenical (data not shown). When one-quarterof the LC50 for two arsenical species was combined, the cytotoxicitywas similar compared to the cytotoxicity when cells were treatedseparately with one-half of the LC50 for either arsenical, showingan additive effect of the arsenicals (Fig. 2). In the same way, com-bination treatments using one-half of the LC50 for both arsenicalshad the same effect as separate treatment with the LC50 dose foreach arsenical (Fig. 2). There was no statistical difference when thecytotoxicity of the combined treatments using one-quarter or one-half of the LC50 for both arsenicals was compared to one-half or tothe entire LC50, respectively, for each arsenical alone, except whenthe cytotoxicity of one-quarter the LC50 doses for TMAO and DMAIII

combined were compared to one-half the LC50 dose of TMAO alone(p < 0.05).

No additive effect was observed when the cells were treatedwith the LC50 of DMAIII combined with one-quarter the LC50 dose

Fig. 1. Dose-dependent effect of each arsenical in MYP3 cells. (A) Arsenite (AsIII); (B) dimoxide (TMAO).

logy 249 (2008) 69–74 71

of arsenite, DMAV or TMAO (Fig. 2). There was also no additive effectwhen one-quarter of the LC50 dose of DMAIII was added to the LC50dose of arsenite, DMAV or TMAO (Fig. 3).

3.2. Experiment 2

Low levels of arsenite present in the medium of arsenate-containing medium incubated with cells but not without cellsshowed that MYP3 cells do have some reductase activity (Table 1).

The presence of very small amounts of arsenate in the mediumof arsenite-containing medium incubated with cells indicatessome spontaneous oxidation of arsenite occurred. However,based on measurement of arsenicals in the medium, there wasno indication that MYP3 cells were able to methylate arseni-cals.

4. Discussion

In a 2-year bioassay, DMAV was carcinogenic to the urinary ratbladder (Arnold et al., 2006). Short-term experiments with femalerats treated with DMAV showed that its mode of action was definedas cytotoxicity with subsequent regenerative proliferation resultingin hyperplasia and ultimately tumors (Arnold et al., 1999; Cohen etal., 2001). The reactive metabolite DMAIII was present in the urine oftreated rats at concentrations that have been shown to be cytotoxicin vitro (Cohen et al., 2002, 2006, 2007). In short-term experi-ments, the major forms of arsenic in the urine of rats treated withDMAV were high concentrations of DMAV itself (approximately66 �M in the urine of rats fed DMAV as 100 ppm of the diet) andTMAO (approximately 73 �M). Co-administration of DMAV with

ethylarsinous acid (DMAIII); (C) dimethylarsinic acid (DMAV); (D) trimethylarsine

72 M.G. Nascimento et al. / Toxicology 249 (2008) 69–74

Fig. 2. Effect of combination with one-quarter or one-half LC50 of arsenical species in MYicant difference between the combination and one arsenical species treatment (p < 0.05)trimethylarsine oxide (TMAO).

DMPS (2,3-dimercaptopropane-1-sulfonic acid), a chelator of triva-lent arsenicals, increased considerably the amount of DMAV anddecreased the TMAO in the urine. In addition, no urothelial hyper-plasia was found in these animals (Cohen et al., 2002). Short-termexperiments with rats treated with DMAV also showed the presenceof DMAIII in fresh void urine at concentrations up to approximately5 �M (Cohen et al., 2002). Studies have shown that regardless of theform of arsenic administered, rats bind the arsenical in the form of

Table 1Speciation and quantitation of arsenicals in arsenic-containing medium incubated with a

Treatment Arsenite (�M) Arsenate (�M)

With cells Without cells With cells Without

Control ND ND ND ND1 �M Arsenite 0.95 ± 0.01 0.97 ± 0.01 0.03 ± 0.00 ND5 �M Arsenate 0.11 ± 0.01 ND 4.98 ± 0.07 5.29 ± 0.05 mM MMAV ND ND ND ND

ND = Not detected.

P3 cells. (A) DMAIII and AsIII; (B) DMAIII and DMAV; (C) DMAIII and TMAO. *Signif-. Arsenite (AsIII), dimethylarsinous acid (DMAIII), dimethylarsinic acid (DMAV) and

DMAIII to an available cysteine present in the hemoglobin moleculein rat red blood cells that is not present in humans or other species(Lu et al., 2007). This provides evidence that the trivalent arsenicalis involved in the cytotoxicity induced by DMAV administration torats.

In vitro studies using the urothelial rat cell line MYP3 showedthat the LC50 for the trivalent arsenicals is in the sub-micromolarrange, with DMAIII being the most cytotoxic. The LC50 for the

nd without MYP3 rat urinary bladder epithelial cells

MMAV (mM) DMAV (mM)

cells With cells Without cells With cells Without cells

ND 0.01 ± 0.00 ND NDND ND ND ND

8 ND ND ND ND4.06 ± 0.01 4.41 ± 0.05 ND ND

M.G. Nascimento et al. / Toxico

Fig. 3. Effect of combination of one-quarter with the entire LC50 of arsenical speciesin MYP3 cells. (A) DMAIII and AsIII; (B) DMAIII and DMAV; (C) DMAIII and TMAO.Arsenite (AsIII), dimethylarsinous acid (DMAIII), dimethylarsinic acid (DMAV) andtrimethylarsine oxide (TMAO).

pentavalent organic arsenicals MMAV, DMAV and TMAO is in themillimolar range, and for arsenate, the LC50 is in the micromolarrange (Cohen et al., 2002) but greater than arsenite. Given the pres-ence of DMAV and TMAO at relatively high concentrations (greaterthan 60 �M) in the presence of cytotoxic concentrations of DMAIII

in the urine of rats orally administered doses of DMAV that pro-duced urinary bladder hyperplasia, the possibility occurred thatother arsenical species could potentiate the DMAIII cytotoxic activ-ity. This could be especially significant at lower exposures to DMAV

which yield urinary concentrations of DMAIII below those produc-ing cytotoxicity (Arnold et al., 2003). Therefore, we examined the

possible effects of combinations of DMAIII with DMAV or TMAO.DMAV and TMAO were also present at high concentrations in theurine of rats treated with arsenite in the drinking water for 1 week,whereas arsenite and arsenate were present at low concentrations(Yoshida et al., 1998). The possible isoform of DMA was not identi-fied in this experiment, so it was unknown how much of the DMAwas in the trivalent or the pentavalent form. In rats treated withDMAV, the proportion of DMAIII and DMAV in the urine was approx-imately 1:60 (Lu et al., 2003). The high cytotoxicity of DMAIII in vitroand the greater proportion of DMAV in the urine of rats treated witharsenite raise the question whether together they could play a rolein the bladder carcinogenicity of arsenicals.

Exposure to inorganic arsenic is carcinogenic for the humanurinary bladder. The presence of arsenite in the urine suggestedthe possibility that arsenite could be involved in urinary blad-der carcinogenesis. Furthermore, arsenite had the second highestconcentration of arsenicals in the urine of humans treated by intra-venous injection with extremely high doses of arsenic trioxide forpromyelocytic anemia (Wang et al., 2004), and MMAIII and DMAIII

were detected in the urine of humans exposed to high levels ofinorganic arsenic (Valenzuela et al., 2005). Low levels of unme-

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tabolized arsenite (approximately 1–2 �M) were also found in theurine of rats treated with arsenite along with other arsenical species(Yoshida et al., 1998). In an in vitro study using rat urothelial cells,it was determined that the effective cytotoxic dose of arsenite isless than 1 �M, although still slightly higher than DMAIII but muchlower than DMAV and TMAO. The presence of arsenite in the urineof arsenite-treated rats at concentrations which were cytotoxic invitro made it important to examine the effect of arsenite combinedwith DMAIII.

Even though DMAIII occurs at very low concentrations in theurine of DMAV-treated rats, the LC50 of DMAIII shows that itis the most cytotoxic of all arsenicals present in the urine ofarsenic-treated rats. The urinary concentration of DMAIII in ratsadministered cytotoxic doses of DMAV was greater than the in vitroconcentration required to achieve cytotoxicity in vitro. If the orallyadministered DMAV was low enough, no DMAIII was detectable inthe urine and no cytotoxicity, regeneration or tumorigenicity wasobserved. Furthermore, humans exposed to high levels of inorganicarsenic in the drinking water excrete urinary concentrations wellabove the cytotoxic level as measured in vitro with rat (MYP3)or human (1T1) cells. Therefore, we used DMAIII as the commonarsenical in all of our combinations. Although the purity of thesodium arsenite used in the studies was slightly lower (94%) thanwe normally use, the arsenite doses were not adjusted since theconcentration of arsenite used to treat the cells was so low thatthe decreased purity had little effect on the actual concentration ofarsenite in the treatment solutions. Combining the arsenical speciesdid not inhibit the cytotoxicity of any of the arsenicals. The com-bination experiments showed that in dose ranges lower than theLC50, the arsenical species tested had an additive effect, i.e., thesechemicals together at low doses were cytotoxic for MYP3 cells witheffects similar to those seen when MYP3 cells were treated withtwice the dose of one of the species by itself. The combinations didnot show any evidence of a synergistic effect.

We also investigated the ability of MYP3 cells to metabolizearsenicals. Measurement of arsenicals in the treatment mediumincubated with or without cells showed no evidence that MYP3cells have the ability to methylate arsenicals, but they do havethe ability to reduce arsenicals as evidenced by the presence ofsmall concentrations of arsenite in the medium incubated with cellstreated with arsenate. Further experiments to measure the concen-tration of arsenicals within the cells will need to be conducted toconclusively determine the methylation status of MYP3 cells.

In vivo, the cytotoxicity of the various arsenicals may be

increased by the cytotoxicity of the downstream pentavalent andtrivalent arsenicals produced by metabolism of the parent arseni-cal. In MYP3 cells, the cytotoxic dose of arsenate is only 4–5 timeshigher than the cytotoxic dose of arsenite indicating the smallamount of arsenite found in arsenate-treated cells did not signifi-cantly affect the cytotoxicity of arsenate. However, trivalent MMAand DMA are approximately 1000 times more cytotoxic than thecorresponding pentavalent arsenical. Therefore, when MYP3 cellsare treated with pentavalent MMA or DMA, the possibility existsthat their cytotoxicity is significantly affected by the presence oftrivalent MMA or DMA which is produced within the cells. Dueto the instability of MMAIII and DMAIII, it is unknown if reductionof MMAV and DMAV to MMAIII and DMAIII, respectively, occurredin the MYP3 cells. To determine if reduction of MMAV and DMAV

to the corresponding trivalent organic arsenical affected the toxic-ity of MMAV and DMAV in vitro, it will be necessary to determinethe ratio between the pentavalent organic arsenicals and the cor-responding trivalent arsenicals within the MYP3 bladder epithelialcells.

Our study showed that the arsenical species tested had anadditive effect when combined at concentrations below the LC50,

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Le, X.C., Lu, X., Ma, M., Cullen, W.R., Aposhian, H.V., Zheng, B., 2000. Speciation of

74 M.G. Nascimento et al. /

suggesting that the urinary concentration of the combined arseni-cals needs to be taken into account in evaluating possible risk. Wewere also able to show that based on measurements of arsenicalsin the medium, there is no evidence that the MYP rat urothelial cellline methylates arsenicals.

Acknowledgements

We gratefully acknowledge Karen Pennington for her technicalassistance and Connie Rosales-Winters for assistance in prepara-tion of this manuscript. M.G. Nascimento’s studies at the Universityof Nebraska Medical Center, Omaha, NE were made possible by afellowship from Fundacao de Amparo a Pesquisa do Estado de SaoPaulo (FAPESP 2005/59817-0), Sao Paulo, Brazil.

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