Dca

DDR

a

A

R

R

8

A

A

K

A

A

A

D

O

S

1

AhoakNctAC

K

1h

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1015–1021

Available online at www.sciencedirect.com

ScienceDirect

journa l homepage: www.e lsev ier .com/ locate /e tap

ifferential influences of various arsenicompounds on antioxidant defense system in livernd kidney of rats

ana Kotyzová ∗, Monika Bludovská, Vladislav Eyblepartment of Pharmacology and Toxicology, Charles University in Prague, Faculty of Medicine in Pilsen, Czechepublic

r t i c l e i n f o

rticle history:

eceived 27 May 2013

eceived in revised form

August 2013

ccepted 4 September 2013

vailable online 12 September 2013

eywords:

rsenite

a b s t r a c t

In this study, oxidative stress-related parameters and As retention were examined in liver

and kidneys of male Wistar rats exposed to arsenic trioxide, sodium arsenite (iAsIII), sodium

arsenate (iAsV), and dimethylarsinic acid (DMAsV) at a single ip dose of 3.8 mg As/kg bw, at

24 h post-exposure. In liver, lipid peroxidation increased in iAsIII-exposed rats, glutathione

peroxidase activity decreased in inorganic arsenic (iAs)-exposed rats, and catalase and

thioredoxin reductase activities decreased significantly in all As-exposed groups. Both As(III)

and As(V) exposure elevated GSH level with no effect on glutathione reductase activity. In

kidneys, catalase activity decreased significantly in iAs-exposed, rats; GSH level, glutathione

reductase and thioredoxin reductase activity decreased in DMAsV-treated, rats. The tissue

rsenate

rsenic trioxide

imethylarsinic acid

xidative stress

elenoenzymes

As retention was higher in kidneys compared to liver and was also higher in As(III)-exposed

compared to As(V)-exposed rats. The results demonstrate similar potency of inorganic As(III)

and As(V) compounds to inhibit/induce antioxidant defense system, with liver being more

vulnerable to acute As(III)- and As(V)-induced oxidative stress.

© 2013 Elsevier B.V. All rights reserved.

are less reactive with tissue constituents and more readily

. Introduction

rsenic is well known to cause acute and chronic adverseealth effects. Exposure to inorganic arsenic (iAs) compoundsccurs in both occupational and environmental settings and isssociated with cancer of skin, lung, urinary bladder, liver, andidneys (Hughes et al., 2011; Jomova et al., 2011; IARC, 2012;aujokas et al., 2013). The mechanism by which iAs inducesancer and other disorders in humans is still unclear. Oxida-

ive stress has been suggested as one of the mechanisms ofs-induced toxicity (Kitchin and Ahmad, 2003; Kitchin andonolly, 2010; Flora, 2011). Mechanistic studies in cultured

∗ Corresponding author at: Department of Pharmacology and Toxicoloarlovarská 48, 301 66 Pilsen, Czech Republic. Tel.: +420 377 593 250; fa

E-mail address: dana.kotyzova@lfp.cuni.cz (D. Kotyzová).382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.etap.2013.09.003

animal and human cells have shown that exposure to vari-ous As compounds results in generation of reactive oxygenspecies (ROS) (Barchowsky et al., 1999; Shi et al., 2004). In lab-oratory animals, ROS are generated during iAs metabolism.In mammals, iAs is metabolized by alternating reductionof As(V) to As(III) and irreversible oxidative methylationby addition of methyl groups from S-adenosyl methionine.Methylation of iAs has been considered the detoxificationmechanism, as the methylated metabolites (MMAsV, DMAsV)

gy, Charles University in Prague, Faculty of Medicine in Pilsen,x: +420 377 593 249.

excreted in the urine (Aposhian, 1997; Vahter, 1999; Thomaset al., 2001). However, further experimental results have doc-umented that the process of methylation leads to formation

p h a

1016 e n v i r o n m e n t a l t o x i c o l o g y a n d

of trivalent methylated intermediates (MMAsIII, DMAsIII) thatare more reactive and carcinogenic than pentavalent com-pounds (Aposhian et al., 2000, 2003; Cohen et al., 2002). Onacute exposure, the ability of As to bind with protein thiolsis likely to play a critical role in detoxification of iAs. Stud-ies in vitro indicate that glutathione (GSH), the major cellularantioxidant, functions as an important detoxifying agent ofarsenicals (Chouchane and Snow, 2001; Styblo et al., 2000;Thomas, 2010). Several in vitro studies examined the effectof As(III) and As(V) compounds on GSH redox status and onthe activity of GSH-related enzymes (Schinella et al., 1996;Chouchane and Snow, 2001; Styblo et al., 1997, 2000; Thomaset al., 2001; Yeh et al., 2002). Arsenicals containing AsIII wereshown to be effective inhibitors of glutathione reductase (GR)in cells.

Various arsenicals have also been tested in vitro asinhibitors of thioredoxin reductase (TrxR), a selenoenzymethat plays a critical role in cellular redox regulation. Highlyreactive cystein and selenocystein residues of TrxR are distincttargets for interaction with As compounds. The inhibition ofTrxR enhances the susceptibility of cells to oxidants and favorsapoptosis. Arsenite and methylated AsIII have been shown toact as extremely potent inhibitors of TrxR in experiments withpurified enzymes and in cultured rat hepatocytes (Lin et al.,1999, 2001). Recent studies have demonstrated that arsenictrioxide (As2O3) can inhibit the active site of TrxR and is nowimplicated for chemotherapy of acute promyelocytic leukemia(Lu et al., 2007; Talbot et al., 2008). Dimethylarsinic acid(DMAsV) is the major metabolite formed after the exposureto iAs in mammals. This compound, used also as an herbicide(cacodylic acid), exerts pro-oxidative properties and has beenshown to act as cancer promoter (Kenyon and Hughes, 2001;Cohen et al., 2006).

Whereas differential influences of As(III) and As(V) com-pounds have been compared in several in vitro studies, similardata from studies in vivo are scarce. To identify the risksof chemical exposure to iAs for humans, observation dataconducted in laboratory animals are required. The presentstudy was designed to evaluate the effects of acute expo-sure to arsenic trioxide (As2O3), sodium arsenite (iAsIII),sodium arsenate (iAsV), and dimethylarsinic acid (DMAsV)on glutathione and thioredoxin systems in relation to otheroxidative stress related parameters and As retention in theliver and kidneys of rats. Selenoenzymes glutathione perox-idase and thioredoxin reductase were of particular interest,because of well-known antagonism between arsenic and sele-nium in many biological systems (Moxon, 1938; Levander,1977).

2. Materials and methods

2.1. Chemicals and reagents

Arsenic trioxide (AsIII2O3; cat.# A-1010), sodium arsenite

(NaAsIIIO2; cat.# S-7400, sodium arsenate (Na2HAsVO4 ·

7H2O; cat.# S-9663), and dimethylarsinic acid [DMAsV;(CH3)2AsO2Na; cat.# C-0250] were purchased from SigmaChemical Co. (St. Louis, MO, USA). All other reagents were ofanalytical grade purity.

r m a c o l o g y 3 6 ( 2 0 1 3 ) 1015–1021

2.2. Animals and experimental design

Adult male Wistar rats (130–140 g b.w.) were obtained fromAnlab (Prague, CZ) and allowed to acclimate for 7 days beforethe experiment. The animals were housed in temperature andhumidity controlled room with a 12 h – light/dark cycle andfree access to standard pellet diet and drinking water. A total of40 animals were randomly assigned to five groups of eight ratsand were treated as follows: group I – control, received onlyphosphate buffered saline solution; group II – arsenic oxide,5 mg/kg of body wt; Group III – sodium arsenite, 6.5 mg/kg ofbody wt; Group IV – sodium arsenate 15.5 mg/kg of body wt;Group V – dimethylarsinic acid sodium salt, 8 mg/kg of bodywt. Stock solution of As2O3 was prepared in 1 N NaOH. Dosingsolutions of arsenicals were prepared in phosphate bufferedsaline (PBS), pH = 7.4 shortly before the experiment. Arseniccompounds were administered intraperitoneally (ip) in a vol-ume of 1 ml/100 g of body wt, as a single dose at equimolardose of arsenic 50 �mol/kg of body wt. Twenty-four hours afterthe treatment, the rats were sacrificed under light ether anes-thesia, and the liver and kidneys were excised immediately.Major part of the tissue sample was kept frozen at −70 ◦C forbiochemical estimations; small part of the tissue was kept at−20 ◦C for wet digestion and analysis of arsenic content.

The experimental treatment protocol was approved by thelocal Animal Care and Use Committee. The investigation con-formed to the Guide for the Care and Use of laboratory Animalspublished by the U.S. National Institute of Health.

2.3. Biochemical analysis

2.3.1. Lipid peroxidationLipid peroxidation (LP) was measured as malondialdehyde(MDA) production formed in the thiobarbituric acid reaction inliver and kidney homogenates as described by Uchiyama andMihara (1978). Briefly, 10% (w/v) liver homogenates (0.25 ml)were mixed with 1.5 ml of 1% H3PO4 and 0.5 ml of thiobarbi-turic acid (0.6%). The tubes were heated for 60 min in boilingwater bath. After cooling in an ice bath, 2 ml of butanol wereadded and the content was mixed vigorously for 20 s. Aftercentrifugation (3000 rpm, 15 min), the absorbance of organiclayer was measured at 520 and 535 nm using spectrometer(Lambda 2 S, Perkin-Elmer Co., USA). The results are expressedin nmol MDA/g of tissue.

2.3.2. Glutathione levelGlutathione (GSH) level was assayed in the deproteinizedsupernatant fraction of liver and kidney homogenates (0.2 g oftissue/8 ml of 0.02 M EDTA) using 5,5′-dithiobis(2-nitrobenzoicacid) (DTNB, Ellman’s reagent) and reading absorption at412 nm (Sedlak and Lindsay, 1968). The results are expressedin �mol GSH/g of tissue.

2.3.3. Antioxidant enzymes activityCatalase (CAT) activity in liver and kidney homogenates was

estimated based on the method of Aebi (1972), by following thedecomposition of H2O2 directly by its decreasing extinction at240 nm. The activity of catalase is expressed as a rate constantof a first order reaction k/g of wet tissue.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1015–1021 1017

Table 1 – Effects of arsenic compounds on the level of lipid peroxidation (LP) and glutathione (GSH) level and on theactivities of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and thioredoxin reductase (TrxR) inthe liver of rats.

Treatment group LP (nmolMDA/g)

CAT (nmolH2O2/min/g)

GSH (�mol/gtissue)

GR (U/g tissue) GPx(�mol/min/g)

TrxR (U/mgprot)

Control 55.9 ± 6.1 53.7 ± 2.6 5.11 ± 0.32 5.69 ± 0.20 22.3 ± 1.8 1.06 ± 0.09Arsenic trioxide 54.0 ± 5.8 38.2 ± 2.4** 6.87 ± 0.62** 5.77 ± 0.47 19.2 ± 1.4** 0.95 ± 0.09*

Arsenite 63.3 ± 8.5* 36.1 ± 1.9** 6.36 ± 0.44** 5.80 ± 0.41 17.7 ± 1.7** 0.85 ± 0.06**

Arsenate 52.7 ± 9.1 37.9 ± 3.1** 5.98 ± 0.29** 5.29 ± 0.53 20.2 ± 1.5* 0.74 ± 0.10**

DMAsV 54.8 ± 8.0 45.0 ± 4.1** 6.33 ± 0.46** 6.10 ± 0.46* 22.1 ± 1.6 0.93 ± 0.10*

Values represent mean ± S.D. for eight animals per group.Significant differences:

awtti3o

it5icGa4

tU(traLet

2

FndaaLt2w

2

Tu

∗ p < 0.05 versus control group.∗∗ p < 0.01 versus control group.

Glutathione peroxidase (GPx) activity was assayed in livernd kidney homogenates by a coupled test system, inhich glutathione reductase is employed for the regenera-

ion of reduced glutathione and butyl hydroperoxide used ashe acceptor substrate (Günzler et al., 1974). The decreasen NADPH concentration was registered photometrically at40 nm. The GPx activity is expressed in �mol NADP+/min/gf tissue.

Glutathione reductase (GR) activity was determined by mon-toring the oxidation of NADPH in the presence of GSSG usinghe method of Smith et al. (1988). The formed GSH reacts with,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and the increasen absorbance at 412 nm was measured. The reaction systemontained 0.1 M phosphate buffer (pH 7.5), 1 mM EDTA, 2 mMSSG and 3 mM DTNB solution. Reactions were started by theddition of 2 mM NADPH and the increase in absorbance at12 nm was measured.

Thioredoxin reductase (TrxR) activity was estimated usinghe Thioredoxin Reductase Assay Kit (Sigma, Saint Louis, MO,SA). The assay is based on the reduction of 5,5′-dithiobis

2-nitrobenzoic) acid (DTNB) with NADPH in the presence ofhioredoxin reductase (Holmgren and Bjornstedt, 1995). Theate of 5-thio-2-nitrobenzoic acid (TNB) formation is measuredt 412 nm (Cary100 Bio UV/VIS spectrometer, Varian, Australiatd.). Measurements were done in the absence and in the pres-nce of a specific thioredoxin reductase inhibitor to subtracthe non-TrxR dependent reduction of DTNB.

.4. Arsenic estimation

or arsenic estimation, a portion (∼100 mg) of liver or kid-ey tissue was decomposed in nitric acid using a microwaveigestion system (Milestone Laboratory Systems, Italy). Totalrsenic content was measured by electrothermal atomicbsorption spectrometry (SpectrAA 220 FS Varian, Australiatd.), using Pd modifier, atomization from platform and inser-ion of a cooling step before atomization (Husáková et al.,007). Arsenic content is expressed in �g/g of tissue weteight. Detection limit for arsenic was 0.02 ppm.

.5. Statistical analysis

he data are presented as means ± SD values. Two tailednpaired Student’s t-test was applied to determine statistical

significance of differences between the means (GraphPad,InStat3). Differences between groups were considered signifi-cant at p values <0.05.

3. Results

3.1. Effect of arsenicals on LP, GSH and antioxidantenzymes in the liver

Table 1 shows changes in glutathione content, lipid peroxi-dation level and antioxidant enzymes activities in the liverat 24 h after arsenicals treatment. A single ip administrationof sodium arsenite caused an increase in hepatic LP (115%of controls, p < 0.05) in comparison with the control groupwhile no alterations were seen in the liver of other As-treatedgroups.

The liver GSH level increased significantly on exposure toeither arsenical (p < 0.01) with maximum increase followingAs2O3 exposure (134% of controls) and minimum increase fol-lowing iAsV administration (117% of controls). The hepatic GRactivity did not change following iAs-treatment and becameslightly increased in DMAsV-treated group (107% of controls,p < 0.05). Conversely, the activity of GPx decreased signifi-cantly on iAs exposure and did not change in DMAsV-exposedrats. The activity of TrxR decreased significantly in all As-treated groups compared to the control group. Exposure toAs2O3 and DMAsV caused a 10% decrease of TrxR activity(p < 0.05), exposure to iAsIII caused a 20% decrease (p < 0.01),and iAsV treatment caused a 30% decrease (p < 0.01) of TrxRactivity. There was also a significant depletion (p < 0.01) ofCAT activity after exposure to all arsenicals tested. LiverCAT activity decreased by 30% in iAs-treated groups and by15% in DMAsV-treated rats in comparison with the controlgroup.

3.2. Effects of arsenicals on LP, GSH and antioxidantenzymes in the kidney

Table 2 demonstrates the changes in renal oxidative stressparameters and antioxidant enzymes activity at 24 h after

acute exposure to tested arsenicals. The level of renal LPremained unchanged except for a slight decrease in iAsIII-treated group (p < 0.05) in comparison with controls. The levelof GSH in the kidneys was not influenced by iAs treatment

1018 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 1015–1021

Table 2 – Effects of arsenic compounds on the level of lipid peroxidation (LP) and glutathione (GSH) level and on theactivities of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and thioredoxin reductase (TrxR) inthe kidneys of rats.

Treatment group MDA (nmol/gtissue)

CAT (nmolH2O2/min/g)

GSH (�mol/gtissue)

GR (U/g tissue) GPx(�mol/min/g)

TR (U/mg prot)

Control 66.4 ± 6.5 11.7 ± 0.9 4.33 ± 0.12 6.90 ± 0.38 10.4 ± 0.5 0.85 ± 0.09Arsenic trioxide 61.7 ± 3.9* 10.1 ± 1.0* 4.23 ± 0.12 6.75 ± 0.45 10.2 ± 0.6 0.87 ± 0.05Arsenite 55.2 ± 3.3** 8.9 ± 0.9** 4.40 ± 0.11 7.06 ± 0.46 10.5 ± 0.7 0.93 ± 0.12Arsenate 61.8 ± 3.9* 9.1 ± 1.0** 4.17 ± 0.20 6.61 ± 0.41 10.3 ± 0.4 0.92 ± 0.07DMAsV 58.8 ± 5.2** 12.3 ± 1.8 4.02 ± 0.16* 6.37 ± 0.44* 10.4 ± 0.7 0.72 ± 0.08*

Values represent mean ± S.D. for eight animals per group.Significant differences:∗ p < 0.05 versus control group.

eters in the liver and kidneys of rats exposed to As2O3, iAs ,

∗∗ p < 0.01 versus control group.

and decreased slightly in DMAs-treated rats (p < 0.05). Simi-larly, the activity of GR and TrxR in kidneys did not changeafter exposure to iAs, however it decreased slightly in DMAV-treated rats (p < 0.05). Conversely, the activity of CAT in thekidneys decreased significantly after exposure to iAs (p < 0.01),and did not change in DMAsV-treated rats. The GPx activityin the kidneys of all As-treated groups exerted no differencecompared to the control group.

3.3. Arsenic concentration in the liver and kidneys

Figs. 1 and 2 illustrate the accumulation of As in the liverand kidneys of rats at 24 h after exposure to different arseni-cals. Acute exposure to arsenicals resulted in a significantincrease of As content in both the liver and the kidneys. In theliver, the highest concentration of As was measured in iAsIII-exposed rats (1.93 ± 0.29), followed by As2O3-exposed group(1.66 ± 0.24), iAsV-group (1.27 ± 0.46) and the lowest As con-centration was measured in DMAsV-exposed rats (0.25 ± 0.05).In the kidneys, the highest concentration of As was estimated

in As2O3-exposed rats (3.97 ± 0.92), followed by iAsIII-exposedgroup (2.52 ± 0.91), and iAsV-exposed group (1.55 ± 0.31). The

Fig. 1 – Arsenic content in the liver of rats exposed toarsenic trioxide, sodium arsenite, sodium arsenate anddimethylarsinic acid at 24 h post-exposure. Arsenicconcentration are in micrograms per gram of wet tissueweight; data represent means ± SD for eight animals pergroup; **p < 0.01 compared to control animals.

lowest As concentration was measured in DMAsV-treatedgroup (0.14 ± 0.09).

4. Discussion

Recent studies have raised further concerns about the role ofoxidative stress in the toxicity of arsenic as a major, natu-rally occurring carcinogen in the environment (Kitchin andConolly, 2010; Jomova et al., 2011; Flora, 2011). The sever-ity of adverse effects of inorganic arsenicals depends on thevalence of As. Trivalent arsenicals exert high reactivity, espe-cially high affinity for thiols. Differential influences of As(III)and As(V) compounds on GSH redox status and antioxidantenzymes have been reported from experiments with purifiedantioxidant enzymes and/or cell lysates (Styblo et al., 1997,2000; Thomas et al., 2001; Chouchane and Snow, 2001; Yehet al., 2002). However, similar data from in vivo experimentsare scarce.

In this study, we evaluate oxidative stress-related param-III

iAsV, and DMAsV. The liver is considered the major site foriAs metabolism; the kidneys represent the major route of As

Fig. 2 – Arsenic content in the kidneys of rats exposed toarsenic trioxide, sodium arsenite, sodium arsenate anddimethylarsinic acid at 24 h post-exposure. Arsenicconcentration are in micrograms per gram of wet tissueweight; data represent means ± SD for eight animals pergroup; **p < 0.01 compared to control animals.

a r m

errWM

hiwr(hra1ltHt(

AbitwcteiGm2icccr

aisaikit&scrbipHAh

ar

e n v i r o n m e n t a l t o x i c o l o g y a n d p h

xcretion. Doses of arsenicals used in the current study cor-espond to the dose of 50 �mol As/kg (3.8 mg As/kg bw) andepresent 2/5 of LD50 estimated for sodium arsenite in male

istar rats at 24 h after ip administration (Ramos et al., 1995;aiti and Chatterjee, 2000).

In the present study, we observed a significant elevation ofepatic GSH level on exposure to any of As compounds admin-

stered, while no change in lipid peroxidation was found,ith the exception of iAsIII treatment. Our observations in

ats are in accordance with the in vitro findings of Yeh et al.2002). Increased hepatic GSH content with no alteration inepatic TBARS was also reported by Schinella et al. (1996) inats treated with iAsV (10 mg As/kg/day) orally for 2 days, andfter a prolonged exposure to iAsIII (3.33 mg As/kg/day, ip) for4 days from the study of Maiti & Chatterjee (2000). Elevatedevels of GSH in the liver following As exposure suggest a coun-eracting mechanism adopted by the system to eliminate As.epatic GSH is used as a reductant for the conversion of As(V)

o As(III); trivalent As is then quickly complexed with GSHThomas, 2010).

Glutathione disulfide (GSSG), formed during reduction ofs(V) to As(III), is reduced to GSH in a reaction catalyzedy GR. Decreased regeneration of GSH from GSSG due tonhibition of GR may affect the intracellular GSH concentra-ion. In reactions with purified yeast GR, arsenic compoundsere demonstrated as potent inhibitors of GR; AsIII-containing

ompounds were shown to act as more potent inhibitors of GRhan AsV-containing arsenicals (Styblo et al., 1997; Thomast al., 2001). In the present study in rats, acute exposure toAs did not affect the activity of GR. Correspondingly, theR activity was not inhibited or activated in in vitro experi-ents in cultured cells (Chouchane and Snow, 2001; Yeh et al.,

002). It is evident that the concentrations of As required tonhibit GR are much higher than physiologically relevant con-entrations. In contrast, the exposure to DMAsV in this studyaused a mild increase in GR activity in the liver with a con-omitant decrease of GR activity in the kidneys of exposedats.

The present study shows a significant decrease in thectivity of scavenging enzymes CAT and GPx in the liver ofAs-exposed rats, suggesting an impaired antioxidant defenseystem. Acute exposure to both AsIII and AsV impaired thebility of tissues to detoxify H2O2 via CAT activity. The decreasen CAT activity was more expressed in the liver than in theidneys of iAs-treated rats. However, after a prolonged admin-

stration of iAsIII at a similar dose, an inverse ratio betweenhe inhibition of hepatic and renal CAT has developed (Maiti

Chatterjee, 2000). Studies addressing the effects of As expo-ure on GPx are scarce. Glutathione peroxidase – an importantomponent of enzymatic antioxidant system, plays a principleole in the reduction of organic hydroperoxides within mem-ranes and lipoproteins in the presence of GSH. In vitro studies

n cells did not indicate a direct interaction between As com-ounds and GPx (Chouchane and Snow, 2001; Yeh et al., 2002).ere we show in rats, that the exposure to both inorganics(III) and As(V) compounds caused a significant decrease ofepatic GPx activity.

Various arsenicals have been previously tested in vitros inhibitors of TrxR, a selenoenzyme, that plays a criticalole in cellular redox regulation. Trivalent arsenic, specifically

a c o l o g y 3 6 ( 2 0 1 3 ) 1015–1021 1019

As2O3, has been shown to inhibit irreversibly mammalianTrxR in vitro and is currently used as a chemotherapeuticagent for acute promyelocytic leukemia (Lu et al., 2007; Talbotet al., 2008). In the present study we show that both AsIII

and AsV compounds inhibit TrxR activity in vivo. Surpris-ingly, iAsV exerted the highest inhibitory effect. This findingsupports the assumption that the extent of TrxR inhibitiondepends on the intracellular concentration of methylated –AsIII intermediates produced in the course of As biomethy-lation (Lin et al., 2001). A metabolic inhibition of seleniummetabolism could also lead to this effect due to reducedcapacity of cells to produce selenoproteins (Talbot et al.,2008).

For both As(III) and As(V) arsenicals, the tissue retentionof As was higher in the kidneys than in the liver. An impor-tant source of As in kidneys could be the uptake of As-GSHconjugates. Arsenic concentrations at 24 h after exposure todifferent As compounds at equimolar As doses were not equal.Lower As concentrations were found in the tissues of iAsV-treated rats. Our observation in rats are in accordance withthe in vitro finding of Yeh et al. (2002), in which lower cellularAs content was found for iAsV treatment of endothelial cellsin comparison with iAsIII treatment at the same concentra-tion. This may result from lower cellular uptake of Na2HAsO4

due to its competition for cellular transport with phosphates(Kenney and Kaplan, 1988).

Low content of As in the tissues of DMAsV treated ratscorresponds to a limited retention of DMAsV in rats. Totalelimination of DMAsV after a single oral or parenteral dosein rodents has been shown to be quite rapid, with major-ity of the dose excreted unmetabolised (Yoshida et al.,1997; Kenyon and Hughes, 2001). However, recent studyof Németi and Gregus (2013) has shown that rats reduceDMAs(V) to DMAs(III) to a significant extent, that the reduc-tion requires GSH, and that the inhibitors of TrxR also inhibitthe DMAs(V) reduction. Decreased level of GSH togetherwith the inhibition of GR and TrxR in the kidneys foundin the present study correlate entirely with the findingsabove.

In summary, our results demonstrate that (i) iAs(III) andiAs(V) compounds have similar potency to inhibit/induceantioxidant defense system on acute exposure in rats; (ii) liveris more sensitive to acute As(III)- and As(V)-induced oxidativestress than kidneys; (iii) both As(III) and As(V) inhibit hepaticGPx and TrxR activity in vivo. The inhibition of hepatic GPx andTrxR activity could be a sign of reduced selenoproteins synthe-sis due to metabolic interaction of arsenic and selenium anddeserves further investigation.

Conflict of interest statement

Nothing declared.

Acknowledgement

This work was supported by the research grant of theMinistry of Education of the Czech Republic (Project No.0021620819).

p h a

r

1020 e n v i r o n m e n t a l t o x i c o l o g y a n d

e f e r e n c e s

Aebi, H., 1972. Catalase. In: Bergmeye, H.U. (Ed.), Methods ofEnzymatic Analysis, vol. 2. Academic Press, New York, pp.673–684.

Aposhian, H.V., 1997. Enzymatic methylation of arsenic speciesand other new approaches to arsenic toxicity. Annu. Rev.Pharmacol. Toxicol. 37, 397–419.

Aposhian, H.V., Gurzau, E.S., Le, X.C., Gurzau, A., Healy, S.M., Lu,X., Ma, M., Yip, L., Zakharyan, R.A., Maiorino, R.M., Dart, R.C.,Tircus, M.G., Gonzalez-Ramirez, D., Morgan, D.L., Avram, D.,Aposhian, M.M., 2000. Occurrence of monomethylarsonousacid in urine of humans exposed to inorganic arsenic. Chem.Res. Toxicol. 13, 693–697.

Aposhian, H.V., Zakharyan, R.A., Avram, M.D., Kopplin, M.J.,Wollenberg, M.L., 2003. Oxidation and detoxification oftrivalent arsenic species. Toxicol. Appl. Pharmacol. 193,1–8.

Barchowsky, A., Klei, L.R., Dudek, E.J., Swartzm, H.M., James, P.E.,1999. Stimulation of reactive oxygen, but not reactive nitrogenspecies, in vascular endothelial cells exposed to low levels ofarsenite. Free Radic. Biol. Med. 27, 1405–1412.

Chouchane, S., Snow, E.T., 2001. In vitro effect of arsenicalcompounds on glutathione-related enzymes. Chem. Res.Toxicol. 14, 517–522.

Cohen, S.M., Arnold, L.L., Uzvolgyi, E., Cano, M., St John, M.,Yamamoto, S., Lu, X., Le, X.C., 2002. Possible role ofdimethylarsinous acid in dimethylarsinic acid-inducedurothelial toxicity and regeneration in the rat. Chem. Res.Toxicol. 15, 1150–1157.

Cohen, S.M., Arnold, L.L., Eldan, M., Lewis, A.S., Beck, B.D., 2006.Methylated arsenicals: the implications of metabolism andcarcinogenicity studies in rodents to human risk assessment.Crit. Rev. Toxicol. 36, 99–133.

Flora, S.J., 2011. Arsenic-induced oxidative stress and itsreversibility. Free Radic. Biol. Med. 51, 257–281.

Günzler, V.A., Kremers, H., Flohe, L., 1974. An improved coupledtest procedure for glutathione peroxidase (EC 1.11.1.9.) inblood. Z. Klin. Chem. Biochem. 12, 444–448.

Holmgren, A., Bjornstedt, M., 1995. Thioredoxin and thioredoxinreductase. Methods Enzymol. 252, 199–208.

Hughes, M.F., Beck, B.D., Chen, Y., Lewis, A.S., Thomas, D.J., 2011.Arsenic exposure and toxicology: a historical perspective.Toxicol. Sci. 123, 305–332.

Husáková, L., Cernohorsky, T., Srámková, J., Vavrusová, L., 2007.Direct determination of arsenic in beer by electrothermalatomic absorption spectrometry with deuterium backgroundcorrection (D2-ET-AAS). Food Chem. 105, 286–292.

IARC (International Agency for Research on Cancer), 2012.Monographs on the Evaluation of Carcinogenic Risks toHumans: Arsenic and arsenic compounds. Volume 100 C,51-94.(http://monographs.iarc.fr/ENG/Monographs/vol100C/).

Jomova, K., Jenisova, Z., Feszterova, M., Baros, S., Liska, J.,Hudecova, D., Rhodes, C.J., Valko, M., 2011. Arsenic: toxicity,oxidative stress and human disease. J. Appl. Toxicol. 31,95–107.

Kenney, L.J., Kaplan, J.H., 1988. Arsenate substitutes forphosphate in the human red cell sodium pump and anionexchanger. J. Biol. Chem. 263, 7954–7960.

Kenyon, E.M., Hughes, M.F., 2001. A concise review of the toxicityand carcinogenicity of dimethylarsinic acid. Toxicology 160,227–236.

Kitchin, K.T., Ahmad, S., 2003. Oxidative stress as a possible modeof action for arsenic carcinogenesis. Toxicol. Lett. 137,

3–13.

Kitchin, K.T., Conolly, R., 2010. Arsenic-induced carcinogenesis –oxidative stress as a possible mode of action and future

r m a c o l o g y 3 6 ( 2 0 1 3 ) 1015–1021

research needs for more biologically based risk assessment.Chem. Res. Toxicol. 23, 327–335.

Levander, O.A., 1977. Metabolic interrelationships betweenarsenic and selenium. Environ. Health Perspect. 19,159–164.

Lin, S., Cullen, W.R., Thomas, D.J., 1999. Methylarsenicals andarsinothiols are potent inhibitors of mouse liver thioredoxinreductase. Chem. Res. Toxicol. 12, 924–930.

Lin, S., Del Razo, L.M., Styblo, M., Wang, C., Cullen, W.R., Thomas,D.J., 2001. Arsenicals inhibit thioredoxin reductase in culturedrat hepatocytes. Chem. Res. Toxicol. 14, 305–311.

Lu, J., Chew, E.H., Holmgren, A., 2007. Targeting thioredoxinreductase is a basis for cancer therapy by arsenic trioxide.Proc. Natl. Acad. Sci. U S A 104, 12288–12293.

Maiti, S., Chatterjee, A.K., 2000. Differential response of cellularantioxidant mechanism of liver and kidney to arsenicexposure and its relation to dietary protein deficiency.Environ. Toxicol. Pharmacol. 8, 227–235.

Moxon, A.L., 1938. The effect of arsenic on the toxicity ofseleniferous grains. Science 88, 81–86.

Naujokas, M.F., Anderson, B., Ahsan, H., Aposhian, H.V., Graziano,J.H., Thompson, C., Suk, W.A., 2013. The broad scope of healtheffects from chronic arsenic exposure: update on a worldwidepublic health problem. Environ. Health Perspect. 121,295–302.

Németi, B., Gregus, Z., 2013. Reduction of dimethylarsinic acid tothe highly toxic dimethylarsinous acid by rats and rat livercytosol. Chem. Res. Toxicol. 26, 432–443.

Ramos, O., Carrizales, L., Yánez, L., Mejía, J., Batres, L., Ortíz, D.,Díaz-Barriga, F., 1995. Arsenic increased lipid peroxidation inrat tissues by a mechanism independent of glutathione levels.Environ. Health Perspect. 103 (Suppl 1), 85–88.

Schinella, G.R., Tournier, H.A., Buschiazzo, H.O., de Buschiazzo,P.M., 1996. Effect of arsenic (V) on the antioxidant defensesystem: in vitro oxidation of rat plasma lipoprotein.Pharmacol. Toxicol. 79, 293–296.

Sedlak, J., Lindsay, R.H., 1968. Estimation of total, proteinboundand nonprotein sulfhydryl groups in tissue with Ellman’sreagent. Analyt. Biochem. 25, 192–205.

Shi, H., Shi, X., Liu, K.J., 2004. Oxidative mechanism of arsenictoxicity and carcinogenesis. Mol. Cell. Biochem. 255, 67–78.

Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay ofglutathione reductase in crude tissue homogenates using5,5′-dithiobis(2-nitrobenzoic acid). Anal. Biochem. 175,408–413.

Styblo, M., Serves, S.V., Cullen, W.R., Thomas, D.J., 1997.Comparative inhibition of yeast glutathione reductase byarsenicals and arsenothiols. Chem. Res. Toxicol. 10, 27–33.

Styblo, M., Del Razo, L.M., Vega, L., Germolec, D.R., LeCluyse, E.L.,Hamilton, G.A., Reed, W., Wang, C., Cullen, W.R., Thomas, D.J.,2000. Comparative toxicity of trivalent and pentavalentinorganic and methylated arsenicals in rat and human cells.Arch. Toxicol. 74, 289–299.

Talbot, S., Nelson, R., Self, W.T., 2008. Arsenic trioxide andauranofin inhibit selenoprotein synthesis: implications forchemotherapy for acute promyelocytic leukaemia. Br. J.Pharmacol. 154, 940–948.

Thomas, D.J., Styblo, M., Lin, S., 2001. The cellular metabolismand systemic toxicity of arsenic. Toxicol. Appl. Pharmacol.176, 127–144.

Thomas, D.J., 2010. Arsenolysis and thiol-dependent arsenatereduction. Toxicol. Sci. 117, 249–252.

Uchiyama, M., Mihara, M., 1978. Determination ofmalondialdehyde precursor in tissues by thiobarbituric acidtest. Analyt. Biochem. 86, 271–278.

Vahter, M., 1999. Methylation of inorganic arsenic in differentmammalian species and population groups. Sci. Prog. 82,69–88.

a r m

Y

e n v i r o n m e n t a l t o x i c o l o g y a n d p h

eh, J.Y., Cheng, L.C., Ou, B.R., Whanger, D.P., Chang, L.W., 2002.

Differential influences of various arsenic compounds onglutathione redox status and antioxidative enzymes inporcine endothelial cells. Cell. Mol. Life Sci. 59,1972–1982.

a c o l o g y 3 6 ( 2 0 1 3 ) 1015–1021 1021

Yoshida, K., Chen, H., Inoue, Y., Wanibuchi, H., Fukushima, S.,

Kuroda, K., Endo, G., 1997. The urinary excretion of arsenicmetabolites after a single oral administration ofdimethylarsinic acid to rats. Arch. Environ. Contam. Toxicol.32, 416–421.