ORIGINAL RESEARCH

Antioxidant activity and low toxicity of (E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl) pyrrolidin-2-one

Rafael Porto Ineu & Matheus dos Santos &

Olga Soares do Rêgo Barros &

Cristina Wayne Nogueira &

João Batista Teixeira Rocha & Gilson Zeni &Maria Ester Pereira

Received: 22 September 2011 /Accepted: 2 March 2012 /Published online: 13 March 2012# Springer Science+Business Media B.V. 2012

Abstract The aim of the present study was to evalu-ate the potential pharmacological and toxicologicalproperties of (E)-1-(1-(methylthio)-1-(selenopheny)hept-1-en-2-yl) pyrrolidin-2-one (compound 1), anorganoselenium compound. In vitro experimentsshowed that compound 1 presented a reduction in thelipid peroxidation induced by Fe2+ in thiobarbituricacid-reactive species (TBARS) production, and in thegeneration of reactive species caused by Fe2+/malonatein DCFH-DA oxidation. The high dose (500 mg/kg)induced an increase on ALT but not on AST activity.Hepatic, but not cerebral, δ-ALA-D activity from micetreated with 500 mg/kg presented a significant inhibi-tion. Brain catalase activity was significantly inhibitedby 100 mg/kg whereas hepatic catalase activity showeda significant increase at all doses. Hepatic lipid perox-idation was diminished only at lowest dose (100 mg/kg)whereas for brain tissue, all doses induced a significant

reduction in TBARS levels. Brain and liver ascorbicacid contents were increased only at highest dose ofcompound 1. Urea and creatinine levels were not sig-nificantly altered by treatments. This is a promisingcompound with antioxidant activity and low toxicity,suggesting the potential beneficial activity of compound1 against oxidative damage in many parameters studiedin rats and mice.

Keywords Antioxidant . Organoselenium . Oxidativestress . Reactive oxygen species . Selenium . Toxicity

Introduction

Oxidative stress is characterized by a significant in-crease of intracellular oxidizing species concentration,such as reactive oxygen species (ROS) and is oftenaccompanied by the simultaneous loss of antioxidantdefense capacity (Arteel and Sies 2001). ROS provokesevere changes at the cellular level leading to celldeath because of their extreme reactivity. They attackessential cell constituents, such as proteins, lipids, andnucleic acids, leading to the formation of toxic com-pounds (Kahraman et al. 2003). Many diseases anddegenerative processes can be associated with theoverproduction of ROS, including inflammation, brainischemia, mutagenesis, cancer, dementia, and physio-logical aging (Ren et al. 2001).

Cell Biol Toxicol (2012) 28:213–223DOI 10.1007/s10565-012-9217-y

R. P. Ineu :C. W. Nogueira : J. B. T. Rocha :G. Zeni :M. E. Pereira (*)Programa de Pós-Graduação em Bioquímica Toxicológica,Centro de Ciências Naturais e Exatas,Universidade Federal de Santa Maria,97105-900 Santa Maria, RS, Brazile-mail: pereirame@yahoo.com.br

M. dos Santos :O. S. do Rêgo Barros : C. W. Nogueira :J. B. T. Rocha :G. Zeni :M. E. PereiraDepartamento de Química, Centro de Ciências Naturais eExatas, Universidade Federal de Santa Maria,97105-900 Santa Maria, RS, Brazil

Selenium is recognized as an essential element,structural component of several enzymes involved inperoxide decomposition, including glutathione perox-idase (Rotruck et al. 1973; Zaporowska and Zwolak2011) and phospholipids hydroperoxide glutathioneperoxidase (Ursini et al. 1982). To respond to ROSproduction, compounds can be envisaged thatcombine a range of antioxidant activities in onechemically simple molecule. Organoseleniumcompounds have found such wide utility becauseof their effects on an extraordinary number ofvery different reactions, including many carbon–carbon bond formations, under relatively mild re-action conditions. Furthermore, organoseleniumcompounds can usually be used in a wide varietyof functional groups, thus protecting chemistrygroups (Nogueira et al. 2004).

Organoselenium compounds have become attrac-tive synthetic targets because of their chemio, regio,and stereo selective reactions (Moro et al. 2005) andtheir useful biological activity (Nogueira et al. 2004).In fact, a variety of organoselenium compounds withpotential antioxidant activity, including ebselen ana-logs, benzoselenazolinones, diaryl diselenides, selena-mide, and related derivatives have been reported (Sies1993; Saito et al. 1998; Nogueira et al. 2004). Regard-ing toxicological studies, organoselenium compoundsreact with thiol groups from biologically importantmolecules (Nogueira et al. 2004). Accordingly, thiol-containing enzyme, such as δ-aminolevulinate dehy-dratase (δ-ALA-D) that is responsible for the hemesyntheses is inhibited by organoselenium compounds(Barbosa et al. 1998; Maciel et al. 2000; Meotti et al.2003; Nogueira et al. 2003a). The prototype of thisclass of compounds is ebselen, an antioxidant agentwith thiol peroxidase and thioredoxin reductase-likeactivities that has been used with relative success inthe treatment of acute human brain pathologies such asischemia and stroke (Saito et al. 1998; Imai et al.2003).

Diphenyl diselenide (DPDS) is an organoseleniumcompound that shares with ebselen the thiolperoxidase-like activity and other antioxidant proper-ties (Wilson et al. 1989; Rossato et al. 2002a; Meotti etal. 2004; Santos et al. 2005a; Barbosa et al. 2006; deBem et al. 2009). At anti-inflammatory and

antinociceptive doses, this compound has no overttoxicity in mice, rats, or rabbits (Zasso et al.2005; de Bem et al. 2006). Furthermore, DPDShas a protective role in a variety of experimentalmodels associated with the overproduction of freeradicals (Rossato et al. 2002b; Borges et al. 2005;Barbosa et al. 2006; Ineu et al. 2008). Moreover,Plano et al. (2010) demonstrated an antioxidant–proxidant effect of selenium compounds in vitrostudies, like Nogueira and Rocha (2011) thatshowed a toxicology and pharmacology of organo-selenium compounds.

Based on the organoselenium chemistry and pharma-cological properties presented by synthetic organosele-nium compounds, the aim of the present study was toevaluate the potential pharmacological and toxicologi-cal properties of (E)-1-(1-(methylthio)-1-(selenopheny)hept-1-en-2-yl) pyrrolidin-2-one (compound 1), anorganoselenium compound, using in vitro and exvivo experiments, a time that this compound presentedpromising results.

Materials and methods

Animals

Male adult Wistar rats (200–250 g) and male adultSwiss mice (25–35 g, n05–9) from our own breedingcolony were used. The animals were kept in separateanimal rooms on a 12-h light/dark cycle, at a roomtemperature of 22–24 °C, and with free access to waterand food. The animals were used according to theguidelines of the Committee on Care and Use ofExperimental Animal Resources, the Federal Univer-sity of Santa Maria, RS, Brazil. Rats were used for invitro experiments and mice were used for in vivo andex vivo experiments.

Materials

Compound 1 (Fig. 1a) was prepared using the meth-odology described by Barros et al. (2006) and DPDS(Fig. 1b) was prepared by the method previously de-scribed by Paulmier (1986). Analysis of the 1H NMRand 13C NMR spectra showed that the compound 1

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and DPDS presented analytical and spectroscopic datain full agreement with their assigned structures. Thechemical purity of DPDS (99.9 %) was determined byGC/HPLC. Organoselenium compounds were dis-solved in DMSO (dimethylsulfoxide) for in vitro andin soy oil for in vivo exposure. All other reagentswere of analytical grade and obtained from standardcommercial suppliers.

(E)-1-(1-(methylthio)-1-(selenophenyl) hept-1-en-2-yl) pyrrolidin-2-one (1). Yield: (30 %). 1HNMR: CDCl3, 400 MHz, δ(ppm): 7.4–72(m, 5H),3.3(t, 2H, 5.01 Hz), 2.8(s, 3H), 2.3(t, 2H, 5.01 Hz),2.0(q, 2H, 5.01 Hz), 1.9(t, 2H, 7.21 Hz), 1.31–1.22(m,6H), 0.9(t, 3H, 7.21 Hz). 13C NMR: CDCl3, 100 MHz,δ(ppm): 172.2, 131.1, 130.3, 125.2, 117.3, 43.8, 32.9,31.9, 28.8, 28.5, 22.8, 21.6, 18.1, 14.1

In vitro experiments

Tissues preparation

Rats were euthanized by decapitation and liver andbrain were rapidly removed, homogenized in 50 mMTris–HCl, pH 7.4 (1:10, w/v) and centrifuged at2,000×g at 4 °C for 10 min to yield a low-speedsupernatant fraction (S1).

Lipid peroxidation

FeCl2 was used as classical inductor of lipid peroxi-dation. An aliquot of 200 μL of S1 was added to thereaction mixture containing: 10 μM FeCl2 and com-pound 1 at different concentrations (50–500 μM). Af-terwards, the mixture was pre-incubated for 1 h at37 °C. After the pre-incubation, 500 μL thiobarbituricacid (0.8 %), 200 μL SDS (8.1 %), and 500 μL

acetic acid were added to the reaction medium and themixture was incubated for 2 h at 95 °C. The formation oflipid peroxides in the reaction was measured by themethod of Ohkawa et al. (1979) using malondialdehyde(MDA) as an external standard.

Reactive species measurement

Formation of reactive species (RS) was estimatedaccording to a previous report by Ali et al. (1992)and adapted for brain and liver homogenates. Analiquot of S1 was incubated with 10 μL of 2′,7′-dichlorofluorescein diacetate (DCFH-DA; 10 μM)in the presence or the absence of a pro-oxidant(Fe2+/malonate; 0.5 μM/100 μM), compound 1 (1–200 μM) and DPDS (100 μM). The RS levelswere determined by a spectrofluorimetric method.The oxidation of DCFH-DA to fluorescent dichlor-ofluorescein (DCF) is measured for the detectionof intracellular RS. The DCF fluorescence intensityemission was recorded at 525 and 488 nm ofexcitation 30 and 60 min after the addition ofDCFH-DA to the medium.

In view of the in vitro antioxidant properties pre-sented by compound 1 in rats and to gain betterunderstanding of the toxicity of this organoseleniumcompound, some toxicological parameters wereassessed in vivo and ex vivo in mice.

Acute exposure: ex vivo experiments

Mice were treated with a single oral dose, by gavage, ofcompound 1 (100, 250, and 500 mg/kg) or vehicle(1 mL/kg, soy oil). After the compound administration,animals were observed up to 72 h to determine thepotential toxicity of compound 1.

Fig. 1 a Structure of(E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl) pyrrolidin-2-one. bStructure of diphenyldiselenide

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Tissue preparation

After 72 h of exposure, mousewere slightly anesthetizedfor blood collection by heart puncture in tubes contain-ing heparin. Plasma was obtained by centrifugation at2,000×g for 10 min (hemolyzed plasma was discarded).All mice were killed by decapitation and the liver andbrain were quickly removed and homogenized in 10volumes of 50 mM Tris–HCl, pH 7.4. The homogenatewas centrifuged at 2,000×g at 4 °C for 10 min and a lowsupernatant fraction (S1) was used for ex vivo assays.

Lipid peroxidation

The low supernatant fraction (S1) of liver and brainwas used for thiobarbituric acid-reactive species(TBARS) assay according to Ohkawa et al. (1979).Samples were incubated with 500 μL thiobarbituricacid (0.8 %), 200 μL SDS (8.1 %), and 500 μL aceticacid for 2 h at 95 °C. The amount of TBARS producedwas measured at 532 nm (Spectrophotometer U-2001Hitachi), using MDA as an external standard.

δ-ALA-D activity

δ-ALA-D activity from liver and brain was assayed bythe method of Sassa (1982) by measuring the rate ofproduct porphobilinogen (PBG) formation. The reac-tion product was determined using modified Erlich’sreagent at 555 nm (Spectrophotometer U-2001Hitachi). Samples were incubated at 39 °C for 30and 180 min for liver and brain, respectively. Theresults were expressed as nanomoles of PBGformed per hour per milligram of protein.

Ascorbic acid determination

Ascorbic acid determination was performed as de-scribed by Jacques-Silva et al. (2001). The S1 frombrain and liver were precipitated in 10 volumes of cold4% trichloroacetic acid solution. An aliquot (300μL) ofthe sample in a final volume of 1 mL of the solution wasincubated for 3 h at 38 °C; after, 1 mL of H2SO4 65% (v/v) was added to the medium. The reaction product wasdetermined using color reagent containing 4.5 mg/mLdinitrophenyl hydrazine and CuSO4 (0.075 mg/mL) at520 nm (Spectrophotometer U-2001 Hitachi).

Catalase activity

The S1 was assayed spectrophotometrically by themethod of Aebi et al. (1995), which involve monitor-ing the disappearance of H2O2 in the presence of cellhomogenate at 240 nm (Spectrophotometer U-2001Hitachi). The enzymatic activity was expressed aspicomoles of catalase per milligram of protein.

Metabolic parameters

Plasma enzymes aspartate aminotransferase (AST)and alanine amino transferase (ALT) were used asthe biochemical markers for the early acute hepaticdamage and determined by the colorimetric method ofReitman and Frankel (1957). Renal function was ana-lyzed by determining plasma urea (Mackay andMackay 1927) and creatinine levels (Jaffe 1986)(LABTEST, Diagnostic S.A., Minas Gerais, Brazil).

Protein quantification

The S1 was used to measure the protein content by themethod of Lowry et al. (1951) using bovine serumalbumin as the standard.

Statistical analysis

The results are presented as means±SEM. Statisticalanalysis was performed using a one-way analysis ofvariance (ANOVA), followed by LSD test whenappropriate.

Results

In vitro

Effect of compound 1 on lipid peroxidation

The in vitro effect of compound 1 on lipid perox-idation induced by FeCl2 is shown in Fig. 2. In ratbrain, compound 1 at 200 and 400 μM had asignificant effect (p<0.05 by LSD test) in reducinglipid peroxidation to the control level (Fig. 2a). Inhepatic tissue, compound 1 showed a significantreduction in TBARS levels at 400 μM and

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reduced lipid peroxidation levels to the controlvalues at 500 μM (p<0.05 by LSD test; Fig. 2b).

Effect of compound 1 on RS measurement

As can be observed in Fig. 3a, compound 1 de-creased the production of RS induced by Fe2+/malonate in brain (p<0.05 by LSD test) in aconcentration-dependent manner both after 30 and60 min. Interestingly, compound 1 at all concen-trations tested showed a higher effect than DPDS(100 μM). All the significant values were equal orlower than control values. In Fig. 3b, from 1 to200 μM of compound 1 could decrease the rats’liver RS production avoiding the oxidation ofDCFH-DA. Also to liver tissue the compound 1decreased the RS in a concentration-dependentmanner. It is possible to observe that for thistissue, the DPDS was not efficient to diminishthe RS production.

In vivo

Animal’s survival

Mice administrated with 100 or 250 mg/kg of com-pound 1 did not present alterations in the lethal index,whereas the animals treated with highest dose

(500 mg/kg) presented 33 % of death (data notshown).

Ex vivo

Metabolic parameters

One-way ANOVA indicated that plasma urea andcreatinine levels, and plasma AST activity were notchanged by oral administration of compound 1. How-ever, the dose of 500 mg/kg significantly increasedALT activity (p<0.05 by LSD test; Table 1).

δ-ALA-D activity

One-way ANOVA revealed that mice treated with 100and 250 mg/kg of compound 1 had an increased incerebral δ-ALA-D activity (p<0.05 by LSD test),whereas at the highest dose (500 mg/kg) the enzymeactivity was similar to the control value (Fig. 4a).Figure 4b shows that at the highest dose (500 mg/kg), hepatic δ-ALA-D activity was significantlyinhibited in comparison to the control and all othersdoses (p<0.001 by LSD test). DTT did not change theincrease of cerebral δ-ALA-D activity induced by100 mg/kg, and was unable to reactivate the hepaticδ-ALA-D activity inhibition induced by 500 mg/kg ofcompound 1 (data not shown).

Fig. 2 In vitro effects of compound 1 on TBARS production inrat: a brain, b liver. An aliquot of 200 μL of S1 was added to thereaction mixture containing: 10 μM FeCl2 (lipid peroxidationinductor) and compound 1 at different concentrations (50–

500 μM). Data are reported as means±SEM of five independentexperiments. One-way ANOVA/LSD test, different letters rep-resent significant statistical difference among groups (p<0.05)

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Catalase activity

Catalase activity from brain of exposed mice was sig-nificantly inhibited at 100 mg/kg (p<0.05). In contrast,for hepatic tissue, the catalase activity showed a signif-icant increased at all doses tested (p<0.05; Figs. 5a, b).

Lipid peroxidation levels

One-way ANOVA revealed that lipid peroxidationin brain of exposed mice decreased at all doses incomparison to control values (p<0.01 by LSDtest; Fig. 6a); in hepatic tissue, compound 1

Fig. 3 In vitro effects ofcompound 1 on reactivespecies production in rat: abrain, b liver. The S1 wasincubated with 10 μL of2′,7′-dichlorofluoresceindiacetate (10 μM) in thepresence or absence of apro-oxidant (Fe2+/malonate,0.5 μM/100 μM) and com-pound 1 (1–200 μM). Dataare reported as means±SEMof five independent experi-ments. One-way ANOVA/LSD test, different lettersrepresent significant statisti-cal difference among groups(p<0.05)

Table 1 Effects of compound 1 on biochemical parameters

Groups (mg/kg) AST (U/L) ALT (U/L) Urea (mg/dl) Creatinine (mg/dl)

Control 167.05±7.16 114.98±1.74a 26.45±4.84 1.17±0.72

Compound 1 100 187.52±4.41 112.03±2.23a 26.42±1.13 0.97±0.07

Compound 1 250 153.31±14.25 114.65±2.88a 28.12±2.32 1.75±0.27

Compound 1 500 199.46±11.12 125.42±3.96b 23.86±2.53 2.62±0.67

Data are reported as means±SEM of five animals. One-way ANOVA/LSD test, different letters represent significant statisticaldifference among groups (p<0.05)

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diminished TBARS production only at 100 mg/kg(Fig. 6b).

Ascorbic acid determination

One-way ANOVA of ascorbic acid content yielded asignificant result. Compound 1 at 500 mg/kg signifi-cantly increased ascorbic acid levels (p<0.006 byLSD test) in both brain and liver tissues. On the otherhand, ascorbic acid levels were not changed by anyother dose in comparison to control (Fig. 7a, b).

Discussion

The interest in natural and synthetic antioxidant com-pounds that could potentially retard the developmentof some diseases has grown considerably in the scien-tific community in the last decades. Some authors

have proposed that the chemical structures of organo-chalcogens have an important role against oxidativeagents (Tiano et al. 2000). So, the present study dem-onstrated that the compound 1 (E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl) pyrrolidin-2-one hasa potent in vitro antioxidant activity and induces minortoxicological effects at doses lower than 500 mg/kgadministered by oral route.

In vitro experiments showed that the compound 1 isa potent antioxidant agent on lipid peroxidation in-duced by Fe2+ in TBARS production (Fig. 2) ongeneration of reactive species caused by Fe2+/malo-nate in DCFH-DA oxidation (Fig. 3). These antioxi-dant properties were verified in both liver and brainand were concentration-dependent. The results are inaccordance with those obtained using other organo-chalcogens such as dialkyl and diaryl diselenides(Meotti et al. 2004). In this study, the compound 1was more efficient as antioxidant agent on DCFH-DA

Fig. 4 δ-ALA-D activity of mice exposed to compound 1. a brain, b liver. Data are reported as mean±SEM. One-way ANOVA/LSDtest, different letters represent significant statistical difference among groups (p<0.05)

Fig. 5 Catalase activity of mice exposed to compound 1. a brain, b liver. Data are reported as mean±SEM. One-way ANOVA/LSDtest; different letters represent significant statistical difference among groups (p<0.05)

Cell Biol Toxicol (2012) 28:213–223 219

experiment than DPDS which has a great antioxidantproperty when others techniques to determine oxida-tive parameters are used (Nogueira et al. 2004).

Based on the in vitro antioxidant properties ofcompound 1 in rats, we studied the in vivo toxicityof this organoselenium compound on survival and onsome biochemical parameters in mice. For this, thecompound 1 was administered by gavage in a doserange, and it was verified that only the dose of500 mg/kg induced death in around 33 % of micetreated. Considering this result, this organoselenium(compound 1) seems to have similar toxicity to DPDS,since Savegnago et al. (2007) showed that DPDS hasan oral toxicant effect higher than 312 mg/kg in miceand Nogueira et al. (2003b) demonstrated that 65 mg/kg induced seizures and caused 50 % of death wheninjected intraperitoneally in mice. However, the toxic-ity of selenium compounds not only depends on thechemical form and the quantity of the element

consumed, but also on a variety of other factors in-cluding species, age, physiological state, nutrition anddietary interactions, and the route of administration(Nogueira and Rocha 2011). Besides, selenium andsulfur are required and used for many enzymes ascofactors, so we believe that the measure of the quan-tity of these two elements does not correspond to theirdirect action on metabolism, since these can be free orforming organic compounds and their actions can beassociated to the endogenous contents and itsmetabolites.

In order to determine if compound 1 could causerenal damage, we measured plasmatic urea and creat-inine levels. Many reports suggest the enhanced ofserum urea and creatinine levels as indicative of renalinjury (Tanaka-Kagawa et al. 1998; Peixoto andPereira 2007). Regarding increase of urea levels, thisis a water-soluble metabolite produced in the liver thatcan be accumulate in the blood due to alterations on

Fig. 6 Lipid peroxidation (TBARS) of mice exposed to compound 1. a brain, b liver. Data are reported as means±SEM. One-wayANOVA/LSD test, different letters represent significant statistical difference among groups (p<0.01)

Fig. 7 Ascorbic acid content of mice exposed to compound 1. a brain, b liver. Data are reported as means±SEM. One-way ANOVA/LSD test, different letters represent significant statistical difference among groups (p<0.006)

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kidney capacity to excrete it. In addition, measurementof creatinine in the plasma allows to determined renaldysfunction. Our results (Table 1) demonstrated thatthe compound 1 did not change these biochemicalparameters, indicating that the kidney is not a targetfor this organoselenium compound. This result is inaccordance with those verified by Meotti et al. (2003),which demonstrated the absence of DPDS effects onurea and creatinine parameters.

Hepatic injury can be determined by an increase ofplasma aspartate (AST) and alanine (ALT) amino-transferase activities (Devlin 1997). In regard to he-patic toxicity of compound 1, only the highest dose(500 mg/kg) induced an increase of ALT activity(Table 1), the same dose that induced mortality. Thisresult demonstrates that this compound has not hep-atoxic effects. In line with this, Avila et al. (2007)demonstrated that β-organochalcogen did not enhanceserum ALT and AST levels.

δ-ALA-D is an enzyme inhibited in pro-oxidantsituations (Fernandez-Cuartero et al. 1999; Santos etal. 2005a; Peixoto et al. 2007) and is an importantindicator of organochalcogen (Nogueira et al. 2003a)and xenobiotic toxicity (Santos et al. 2005a). Com-pounds that oxidize -SH groups have long been knownas potent δ-ALA-D inhibitors; so the inhibition of δ-ALA-D activity could result in accumulation of itssubstrate, aminolevulinic acid (ALA), which couldcause pro-oxidant effects (Barbosa et al. 1998; Peixotoet al. 2007). In this study, the administration of com-pound 1 enhanced the brain δ-ALA-D activity at 100and 250 mg/kg doses (Fig. 4a). However, only at thehighest dose (500 mg/kg), the hepatic δ-ALA-D ac-tivity was inhibited, supporting low toxicity of thecompound 1. This result agrees with those obtainedby other authors who showed that high doses of orga-chalcogens can be cytotoxic via their ability to cata-lyze the oxidation of thiols and to generate freeradicals (Meotti et al. 2003; Borges et al. 2005). Inaccordance with this, Avila et al. (2006) demonstratedthat organochalcogen compounds inhibited this en-zyme activity in vitro but not in ex vivo experiments.

Catalase (CAT) and peroxidases are the primaryantioxidant defenses against the increase of free radi-cals (Acharya et al. 2004). CAT is one the most im-portant enzymes involved in ameliorating the effectsof oxygen metabolism; this enzyme catalyzes thebreakdown of toxic hydrogen peroxide produced inthe cell to O2 and H2O (Linares et al. 2006). In this

study, the hepatic CAT activity showed increased at alldoses tested whereas the brain CAT activity was di-minished in 100 and 250 mg/kg exposed mice (Fig. 5).This effect of compound 1 exposition occurred simul-taneously to enhance on brain δ-ALA-D activity.These concomitant effects suggest that the lowerALA levels could avoid its auto oxidization and con-sequent formation of H2O2, a CAT substrate, leadingto brain CAT activity decrease. Pearson’s correlation(r200.9078, p00.0472) between CAT and δ-ALA-Dactivity corroborated with this result.

On the other hand, at high dose of compound 1(500 mg/kg) we observed an inhibition of hepatic δ-ALA-D (Fig. 4) and at all doses an increased in CATactivity (Fig. 5); the inhibition of the δ-ALA-D causesan accumulation of ALA, which may auto-oxidize toform reactive oxygen species, such as hydro perox-ides. However, compound 1 increased hepatic CATactivity (Fig. 5b) and ascorbic acid content (Fig. 7)avoiding the possible toxic effect caused by an exces-sive accumulation of hydro peroxides. These effectscorroborate with in vitro results that demonstrated agreat antioxidant property from compound 1 onTBARS (Fig. 2) and RS formation (Fig. 3).

Here, we determined the activity of various bio-chemical parameters that are indicators of oxidativestress. For non-enzymatic antioxidant parameters,TBARS and Vitamin C were some of the examplesthat were measured. In line with this, ours results arein accordance with the literature what reported thatDPDS, an organochalcogen, is a compound that hasa thiol peroxidase-like activity and other antioxidantproperties with relative low toxicity (Wilson et al.1989; Rossato et al. 2002a; Meotti et al. 2004; Santoset al. 2005a, b; Barbosa et al. 2006). In addition,DPDS has a protective role in a variety of experimen-tal models associated with the overproduction of freeradicals (Rossato et al. 2002b; Borges et al. 2005;Barbosa et al. 2006; Ineu et al. 2008). TBARS levelsare assumed to indicate the extent of lipid peroxidationof a given tissue and are strictly linked to oxidativestress found in the organ. Exposure to compound 1caused a marked decrease in TBARS production mea-sured in the brain that was not observed in the liver(Fig. 6b). These results indicate that compound 1 isnot causing a state of oxidative stress in the brain andliver of exposed mice. Vitamin C is always considereda marker of oxidative stress and the reduction of itscontent may indicate an increase in oxidative stress (de

Cell Biol Toxicol (2012) 28:213–223 221

Bem et al. 2006). In the current study, it was possibleto demonstrate that in brain and liver ex vivo experi-ments, ascorbic acid content was normal and increasedonly at highest dose of compound 1 (Fig. 7).

Based on these results presented by compound 1(E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl)pyrrolidin-2-one, we could infer that this is a promis-ing organoselenium compound with great antioxidantactivity and nontoxic levels/concentrations.

Acknowledgment Financial support-provided by FINEP re-search grant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” no. 01.06.0842-00, and by CNPq, CAPES, andFAPERGS no. 10/0005-1 (G.Z.) M.E.P., J.B.T.R., C.W.N., andG.Z. are the recipients of CNPq fellowships.

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