Cytotoxicity of combinations of arsenicals on rat urinary bladder urothelial cells in vitro
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Toxicology 249 (2008) 6974
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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:Received 4 March 2008Received in revised form 10 April 2008Accepted 14 April 2008Available online 22 April 2008
Keywords:ArseniteDimethylarsinic acid (DMAV)Dimethylarsinous acid (DMAIII)Trimethylarsine oxide (TMAO)Urinary bladderUrothelial cell cytotoxicity
a b s t r a c t
Based on epidemiological data, chronic exposure to high levels of inocarcinogenic to the urinary bladder of humans. The highly reactivelarsinous acid (DMAIII) and monomethylarsonous acid (MMAIII) arinorganic arsenic in vivo in addition to the corresponding mono-arsenicals. The objective of this study was to determine if combinergistic toward inducing cytotoxicity in a rat urothelial cell line. Tbut not transformed rat urinary bladder epithelial cell line, was seTreatment with the arsenicals was begun 24h after seeding and coarsenicals usedwere DMAIII with arsenite, dimethylarsinic acid (DM
r or orminebed als prls, th
same as when cells were treated with half the LC50 concentration or the LC50 concentration, respectively,of either arsenical. Treatment with one-quarter the LC50 concentration of one arsenical plus the LC50 con-centration of a second arsenical had similar cytotoxicity as treatmentwith the LC50 concentration of eitherof the arsenicals. Quantitation and speciation of arsenicals in the cell culture medium showed that MYP3cells have some reductase activity but the cells do not methylate arsenicals. The effect on the cytotoxicityof arsenicals in combination was additive rather than synergistic toward a rat urothelial cell line.
Based on epidemiological dataof inorganic arsenic in the drink
Corresponding author at: DepartmenEppley Institute for Cancer Research, Havof Nebraska Medical Center, Omaha, NebrFax: +1 402 559 9297.
E-mail address: email@example.com (S.1 Present address: Center for the Eval
Human Health (TOXICAM), Department oUniversity of Sao Paulo Estate (UNESP), 182 Present address: Department of Pathol
Osaka 545-8585, Japan.3 Present address: Department of Chem
Kanpur 208016, India.
0300-483X/$ see front matter 2008 Edoi:10.1016/j.tox.2008.04.007 2008 Elsevier Ireland Ltd. All rights reserved.
, chronic exposure to high levelsing water is carcinogenic to the
t of Pathology and Microbiology and thelik-Wall Professor of Oncology, Universityaska 68198-3135, United States.
M. Cohen).uation of the Environmental Impact onf Pathology, Botucatu Medical School,618-000 Botucatu, Sao Paulo, Brazil.ogy, Osaka City UniversityMedical School,
istry, Indian Institute of Technology,
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 oxidativemethylations beforebeing excreted in the urine. It has long been thought that thismethylation pathway was a detoxication pathway (Vahter, 1983),but recently it was shown that the highly reactive trivalent organicmetabolitesmonomethylarsonous 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 andmay signicantly 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
lsevier Ireland Ltd. All rights reserved.Combinations of concentrations usedwere the LC50, one-quarteone-half or one-quarter the LC50 of the other arsenical. To detecells were treated with arsenate, arsenite and MMAV as describy HPLC-ICPMS to determine species and quantity of arsenicaone-quarter or one-half the LC50 concentration of both arsenicarganic arsenic in the drinkingwater istrivalent organic arsenicals dimethy-e formed during the metabolism of, di- and trimethylated pentavalenting arsenicals was additive or syn-he MYP3 cell line, an immortalizededed into appropriate culture wells.ntinued for 3 days. Combinations ofAV) or trimethylarsine oxide (TMAO).ne-half the LC50 of one arsenicalwithif MYP3 cells metabolize arsenicals,bove and the medium was analyzedesent. When cells were treated withe cytotoxicity was approximately the
70 M.G. Nascimento et al. / Toxicology 249 (2008) 6974
organic arsenical dimethylarsinic acid (DMAV) resulted in urinarybladder tumors in rats, with the female more sensitive than themale (Arnold et al., 2006). Short-termexperimentswith female ratstreated with DMAV showed that its mode of action was dened 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 similarmodeof action in therat bladder for inorganic arsenic (Arnold et al., 2007). Studies haveshown that regardless of the formof arsenic administered, rats bindthearsenical in the formofDMAIII to anavailable cysteinepresent inthe hemoglobinmolecule in rat red blood cells that is not present inhumans (Luet al., 2007), resulting ina slower clearanceof arsenicalsin rats than in humans and other species. Rats have another signif-icant difference in arsenic metabincluding humans (Lu et al., 2007latedbyanalternating seriesof redto the trivalent species followedpentavalent species. In humansstop at the pentavalent dimethyl athe biomethylation process proctrimethyl arsenicals, mainly trimal., 2003) even at low exposures.themodeofactionof arsenicals anmetabolic differences, the ratmodfor investigations of the mode ofity. The detailed mechanistic effeurinary bladder remain unknownaccumulating evidence suggests ttive urinarymetabolites (trivalencytotoxicity and regenerativeprol2006, 2007).
In vitro experiments have beeeffects of arsenic metabolites usiet al., 2006; Kao et al., 2003). Exhuman (1T1) urothelial cell linesto urothelial cells at sub-microm2002). Additionally, trivalent arseas inorganic arsenic were cytotoorders of magnitude lower thanspecies (DMAV, MMAV and TMAO
Following exposure to inorgannate, a variety of arsenicals, includin the urine secondary to the extis possible that the mixture of atreated rats enhances the urothegreater than what would be exparsenicals present. It is not preseor combination of arsenicals areity. The objective of this study wacombinations of various arsenica
2. Materials and methods
Sodium arsenate and sodium arsenitMO). The purity stated by Sigma was 94%and MMAV were provided by Luxembouof each was greater than 98.5%. TrimethyTriChemical Laboratories, Inc. (Yamanash
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 Columbiaconrmed the identity of both chemicals and that the purity of each was at least99%. DMAV purity was conrmed by NMR at UNMC and the other arsenicals wereused without verication 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 samemanner.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 tumorswhen inoculated subcutaneously in nudemice. The cellswere grown in Hams F-12 medium (Gibco-BRL, Grand Island, NY) supplementedwith 10M non-essential amino acids, 10ng/ml epidermal growth factor (EGF),
in, 10%m Gibis, MO
rationious cing th1.01sulted100%unstaexposy wases in ae comated atrol curminethe ded in tone-qbetwsigni
rationdiumthouts detear regs in thls. Anclarift anythewyofAlm wasely col., 200(ODSobile, andgilentnd anhe colnterfely usince maents
ersbuolism compared to other species,). Inorganic arsenic is biomethy-uctionof thepentavalent speciesby oxidative methylation to the
, the biomethylation appears torsenical, DMAV. In rats, however,eeds further to the formation ofethylarsine oxide (TMAO) (Lu etEfforts have been made to clarifyd theirmetabolites.Despite theseel provides an appropriatemodelaction for arsenic carcinogenic-cts of arsenic metabolites on thein rats as well as in humans, buthat it involves generation of reac-t arsenicals), producing urothelialiferation (Cohenet al., 2001, 2002,
n used to clarify the mechanisticng different cell lines (Gottschalgperiments using rat (MYP3) andshowed that DMAIII is cytotoxic
olar concentrations (Cohen et al.,nicals (MMAIII andDMAIII) aswellxic at concentrations at least 3the pentavalent organic arsenic) (Cohen et al., 2002).ic arsenic, either arsenite or arse-ing trivalent species, are present
ensive metabolism that occurs. Itrsenicals in the urine of arsenic-lial cytotoxicity that is producedected for any one of the specicntly understood which arsenicalsimportant for bladder cytotoxic-s to determine the cytotoxicity ofls on rat urothelial cell lines.
e were purchased from Sigma (St. Louis,for arsenite and 98% for arsenate. DMAV
rg Industries (Tel-Aviv, Israel). The puritylarsine oxide (TMAO) was obtained fromi, Japan) with a purity of 98%.
10g/ml insulin, 5g/ml transferrand 100g/ml streptomycin (all frohydrocortisone (from Sigma, St. Louof 95% air and 5% CO2 at 37 C.
2.3. Experimental design
2.3.1. Experiment 1Cells were seeded at a concent
four hours later, treatment with varcontinued for 3 days without changwas determined that seeding withan initial 24-h incubation period, rewithout the control wells reaching(data not shown). Because DMAIII isnication from M. Styblo), cells were3-day treatment period. Cell viabilitand counting the area of four squarby counting all nine squares for thThe percent survivability was calcultreated cell culture to that in the conto 50% of cell population) was detelated by linear regression analysis ofNC). Concentrations of arsenicals usthe LC50 concentration, one-half ortested in triplicate. The differencescompared by ANOVA, which, whentest.
2.3.2. Experiment 2Cells were seeded at a concent
four hours later, treatment with sobegun and continued for 3 days witested in triplicate. Cell viability waLC50 dose was calculated using lineExcel. Quantitation of arsenic speciecells are able to metabolize arsenicapared using the same conditions toafter 3 days of treatment underwenperiod, mediumwas removed fromovernight ondry ice to theUniversitof the arsenic species in the mediu(Le et al., 2000) followed by inductivdetection (Le et al., 2004; Yuan et ause of a reversed phase C18 columnPhenomenex, Torrance, CA) and a mnium hydroxide, 3mM malonic acidoctopole reaction system ICPMS (Aradio frequency power of 1550W aHelium (3.5mL/min) was used as tto reduce isobaric and polyatomic imedium were also measured directwith 1% HNO3. The standard referenural Water and SRM2670 Trace Elemof Standards and Technology (Gaithfetal bovine serum, 100U/ml penicillin,co) and 2.7mg/ml dextrose and 1g/ml). All cells were grown in an atmosphere
of 1.0104 cells/24-well plate. Twenty-ombinations of arsenicals was begun ande medium. In preliminary experiments, it04 cells/well and treating for 3 days afterin consistent growth in the control wellsconuency prior to the end of treatmentble in culture medium (personal commu-ed to a decreasing dose of DMAIII over thedetermined by staining with trypan bluehemocytometer. Results were conrmedbination experiments (data not shown).s the ratio of cell number in the arsenical-lture. The LC50 dose (concentration lethal
d for each chemical separately and calcu-ata using JMP 5.1.1 (SAS Institute Inc., Cary,he combined arsenical experiments wereuarter of the LC50. Each combination waseen the treatments and the control werecant (p
M.G. Nascimento et al. / Toxicology 249 (2008) 6974 71
3.1. Experiment 1
Each arsenical, tested individually at all doses used for thecombination tests, showed that the cytotoxicity increased inadose-dependentmanner (datanot shown). Thedeterminationof the LC50in MYP3 cells showed that the cytotoxicity of DMAV and TMAOwere in the millimolar range and that the cytotoxicity of arseniteand 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 specieswas combined, the cytotoxicitywas 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 specieswas 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 cytotoxici...