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Chemico-Biological Interactions 190 (2011) 35–44

Contents lists available at ScienceDirect

Chemico-Biological Interactions

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

ydroxyl containing seleno-imine compound exhibits improved anti-oxidantotential and does not inhibit thiol-containing enzymes

aseem Hassana,c,∗, Simone Pintona, Juliana Trevisan da Rochaa, Anna Maria Deobalda,ntonio Luis Bragaa, Cristina Wayne Nogueiraa, Alexandra Susana Latinib, Joao B.T. Rochaa

Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria CEP 97105-900, RS, BrazilLaboratório de Bioenergética e Estresse Oxidativo, Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, BrazilInstitute of Chemical Sciences, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan

r t i c l e i n f o

rticle history:eceived 28 June 2010eceived in revised form 1 December 2010ccepted 10 January 2011vailable online 21 January 2011

eywords:on-bonded interactionsnti-oxidantoxicity

a b s t r a c t

Design and synthesis of organoselenium compounds with high thiol peroxidase (TPx) and low thiol oxi-dase (TOx) activities have been a difficult task and remains a synthetic-activity relationship dilemma. Inthis regard we are reporting for the first time a detail experimental data (both in vitro and in vivo) aboutthe anti-oxidant and toxicological profile of an Imine (–N ) containing organoselenium compound (Com-pound A). The TPx activity of Compound A was significantly higher than diphenyl diselenide (DPDS). BothCompound A and DPDS protected sodium nitropruside (SNP) induced thiobarbituric acid reactive species(TBARS) production in rats tissue homogenate with significantly higher activity observed for CompoundA than DPDS (p < 0.05). The Compound A also exhibited strong antioxidant activity in the DPPH and ABTSradical scavenging assays. This study reveals that an imine group close to selenium atom drasticallyenhances the catalytic activities in the aromatic thiol (PhSH) assay systems. The oxidation of biologicallysignificant thiols reflects the toxicity of the compounds. However, the present data showed that treat-ment with Compound A at 0, 10, 25 or 50 mg/kg was not associated with mortality or body weight loss.Similarly it did not inhibit �-ALA-D and Na+1/K+1 ATPase (sulfhydryl group containing enzymes) activitiesafter acute oral treatment; rather it enhanced non-protein thiols (NPSH) concentration. The CompoundA did not cause any oxidative stress as measured by TBARS production in rat’s tissue preparation. Ourdata also indicate that exposure to Compound A did not affect plasma transaminase activities or levels

of urea and creatinine in rats. Ascorbic acid is always considered a marker of oxidative stress and thereduction of its content may indicate an increase in oxidative stress. Treatment with Compound A didnot alter Ascorbic acid levels in rats. The conducted in vitro and in vivo tests show the versatile therapeuticpotential of this compound in the area of free radical induced damages, will undoubtedly enhance ourunderstanding of the mechanism of model compounds and may ultimately yield insights that result in improved GPx mimics.

. Introduction

Various organoselenium compounds having a directelenium–nitrogen (Se–N) bond have been shown to mimic thective site of glutathione peroxidase (GPx). Among them the most

romising drug is Ebselen (1,2-phenyl- 1,2-benzoisoselenazol--(2H)-one), a heterocyclic compound exhibiting interestinglinical properties in the area of free radical induced damagesnd therapies [1]. Since the discovery that Ebselen mimics the

∗ Corresponding author at: Departamento de Química, Centro de Ciências NaturaisExatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil.

el.: +55 55 3220 8140; fax: +55 55 3220 8978.E-mail address: waseem anw@yahoo.com (W. Hassan).

009-2797/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2011.01.012

© 2011 Elsevier Ireland Ltd. All rights reserved.

hydroperoxides reducing ability of GPx, several groups haveworked towards the synthesis of better GPx mimics. All thesemimics with Se–N bond have been developed with the assumptionthat the enzyme GPx may have a cyclic selenenamide structure inits oxidized form and the peptidic nitrogen atoms may functionsimilarly in the formation of cyclic selenenamide species in thenatural enzyme GPx.

Based on these observations, various organoselenium com-pounds containing heteroatoms, i.e. nitrogen in close proximity tothe selenium has been synthesized in order to study their GPx like

activity. Wilson et al. observed that the inclusion of a strongly basicamino group proximal to the active selenium atom in the moleculeis desirable as it would be expected to catalyze the reaction ofthiols with intermediates like diselenides and selenosulfides. Pre-sumably, the base functions to provide a source of nucleophilic

3 ogical

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6 W. Hassan et al. / Chemico-Biol

hiolate anion. They also observed that the selenosulfide and dise-enide did not react with thiol under neutral conditions. Upon theddition of a strong amine base, however, both gave the seleno-ate and disulfide [2]. Galet et al. worked in the same area and heroved the importance of the Se–N bond for GPx activity. The 1,3-enzoselenazolinones, which contain selenium and nitrogen atoms

n the heterocycle but do not have any direct Se–N bonds wereested for GPx like activity [3]. All 1,3-benzoselenazolinones wereevoid of GPx activity under a variety of conditions. The work byilson et al. to design GPx mimics without direct Se–N bond, alsoet with only limited success [2].To summarize, various organoselenium compounds containing

eteroatoms in close proximity to the selenium have been syn-hesized in order to study their GPx like activity. In this regarde have recently reported the synthesis of a new heterocycliciselenide with imino residue close to selenium, i.e. 2-((1-(2-2-(2-(1-(2-hydroxybenzylideneamino) ethyl) phenyl) diselanyl)henyl) ethylimino) methyl) phenol (Compound A) which showedromising anti-oxidant potential against Fe (II) induced lipid per-xidation under different in vitro patho-physiological conditions4]. However, still there is scarcity of pharmacological and toxico-ogical data about this comparatively new compound. Thus, to get aeeper insight about the potential use of Compound A as a possibleharmacological agent, we determined for the first time its thioleroxidase like activity along with its ability to scavenge free rad-

cals under in vitro conditions. We have also studied whether theompound A can inhibit TBARS productions when incubated withodium nitroprusside (SNP). Similarly the radical scavenging activ-ty was measured by DPPH and ABTS assays. To take a step aheadhe activity of this compound was further compared with diphenyliselenide (DPDS) which we recently showed to posses interestingiological activities under various pathological situations [4–6].

Organoselenium compounds can interact directly with lowolecular thiols, oxidizing them to disulfides as well [7]. Simi-

arly, reduced cysteinyl residues from proteins can also react withhese compounds, which may cause, in the case of the enzymes,he loss of their catalytic activity [8,9]. The mechanism(s) under-ying either the toxic or protective effect of organochalcogens areot completely understood but certainly involves the reaction ofhalcogenides with endogenous thiols [10,11]. The facts that (i) then vitro test indicating high thiol peroxidase (TPx) or anti-oxidantctivity necessarily does not lead to in vivo implications that it wille a safe compound and (ii) a possible high thiol oxidase (TOx)ctivity reduces the interest as it may cause oxidation of biologicalelevant thiol molecules and enzymes, motivated us to study then vivo interaction profile of this compound with thiol groups (–SH).or the purpose we designed an in vivo study, where male adultistar rats were treated with Compound A to get a better under-

tanding of its toxicological aspects. Along with its interaction withon-protein thiols (NPSH) we also provided experimental databout the interaction of Compound A with �-ALA-D and Na+1/K+1

TPase (sulfhydryl containing enzyme). Several other tests haveeen performed to get a detail idea about its in vivo behavior whichould of particular interest in view of its pharmacological and tox-cological potential.

. Materials and methods

.1. Synthesis of Compound A

To a suspension of (R,S)-�-methylbenzylamine(10 mmol) in0 mL of ether at −78 ◦C was added n-BuLi (2,5 M solution inexane, 10 mmol) dropwise. The resulting solution was stirred at78 ◦C for 30 min before TMS (trimethylsilyl chloride) (12 mmol)as added at the same temperature. The reaction mixture was

Interactions 190 (2011) 35–44

allowed to warm to rt naturally and was stirred overnight. Tothis solution at 0 ◦C was added n-BuLi (2,5 M solution in hex-ane, 27 mmol) drop wise. The reaction mixture was allowed toslowly warm to room temperature and stirred for 6 h. Selenium(Se) powder (20 mmol) was added at 0 ◦C, the reaction mixture wasallowed to warm to room temperature naturally and was stirredovernight. NH4Cl saturated solution was added slowly and theresulting solution was oxidized for 2 h. After the usual work up withdiethyl acetate, the product was isolated by column chromatog-raphy (EtOAc/MeOH = 70:30) as a dark yellow oil in 50% of yield[12].

2.2. In vitro assays

2.2.1. Thiol peroxidase activityThe catalytic activity of Compound A (Scheme 2) as a GPx model

enzyme, was evaluated according to Tomoda and Iwaoka method[13] using benzenethiol as a glutathione alternative. The reduc-tion of H2O2 was monitored through the UV absorption increaseat 305 nm, due to diphenyl disulfide formation.

2.2.2. Preparation of tissue homogenate and thiobarbituric acidreactive species (TBARS) assay

Rats were decapitated under mild ether anesthesia and brain,liver and kidneys were rapidly removed, placed on ice and weighed.Tissues were immediately homogenized in cold 10 mM Tris–HCl,pH 7.4 (1/10, w/v) with 10 up-and-down strokes at approximately1200 rev/min in a Teflon-glass homogenizer. The homogenate wascentrifuged for 10 min at 4000 × g to yield a pellet that was dis-carded and a low-speed supernatant (S1). An aliquot of 100 �l ofS1 was incubated for 1 h at 37 ◦C in the presence of both organodise-lenides (final concentrations range of 0–100 �M), with and withoutthe prooxidant, i.e. sodium nitroprusside (SNP) (final concentration10 �M). Productions of TBARS were determined as described bymethod of Ohkawa et al. [14]. Except that the buffer of color reac-tion has a pH of 3.4. The color reaction was developed by adding300 �l 8.1% SDS to S1, followed by sequential addition of 500 �lacetic acid/HCl (pH 3.4) and 500 �l 0.8% of thiobarbituric acid (TBA).This mixture was incubated at 95 ◦C for 1 h.

Malondialdehide (MDA), an end product of fatty acid peroxida-tion, reacts with thiobarbituric acid (TBA) to form a colored complexwhich is measured as nmol of MDA/g of tissue at 532 nm and theabsorbance was compared to that of a standard curve obtainedusing malondialdehyde (MDA).

2.2.3. DPPH radical scavenging assayThe measurement of the (Compound A) scavenging activity

against the radical DPPH• was performed in accordance with Choiet al. [15]. Briefly, 85 �M DPPH• was added to a medium containingdifferent (Compound A) concentrations. The medium was incu-bated for 30 min at room temperature. The decrease in absorbancemeasured at 518 nm depicted the scavenging activity of the (Com-pound A) against DPPH•. Ascorbic acid was used as positive controlto determine the maximal decrease in DPPH• absorbance. The val-ues are expressed in percentage of inhibition of DPPH• absorbance(% inhibition) in relation to the control values without the (Com-pound A) (ascorbic acid maximal inhibition was considered 100%of inhibition).

2.2.4. ABTS radical scavenging assay

The determination of the ABTS radical scavenging effect of Com-

pound A was performed according to the method of Re et al. [16],with some modifications. Initially, the ABTS radical was generatedby reacting 7 mM ABTS solution in water with 140 mM potassiumpersulfate in the dark for 12–16 h. In the day of the assay, the

ogical Interactions 190 (2011) 35–44 37

piBAdsbta

2

2

((wtl

2

aw8wM

2

mpeoomfi

2

tTaT3iouNtp[

2

m(tmo((

2

b

Fig. 1. Thiol peroxidase like activity of DPDS and Compound A (at 10 �M) in thepresence of hydrogen peroxide. The difference in peroxidase activity was calculated

W. Hassan et al. / Chemico-Biol

re-formed ABTS radical solution was diluted 1:88 (1 ml ABTS rad-cal plus (+) 87 ml 10 mM potassium phosphate buffer, pH 7.0).riefly, ABTS radical was added to a medium containing compound(10–50 �M). The media were incubated for 30 min at 25 ◦C. The

ecrease in absorbance was measured at 734 nm, depicting thecavenging activity of Compound A against ABTS radical. Ascor-ic acid (10–50 �M) was used as a positive control to determinehe maximal decrease in ABTS absorbance. Results are expresseds percentage (%age) of the blank (without compound A).

.3. In vivo assays

.3.1. Animal’s treatmentRats were divided into four groups, daily weighted and treated

orally) for 3 days (at the interval of 24 h) with compound A at 0canola oil) 10, 25 and 50 mg/kg. 24 h after the last injection animalsere anesthetized to the collection of blood via heart puncture and

hen killed by decapitation. All the experiments were conducted ateast four times and similar results were obtained.

.3.2. Thiobarbituric acid reactive species (TBARS) levelsTBARS levels, a measure of lipid peroxidation, were determined

s described by Ohkawa et al. [14]. An aliquot of S1 was incubatedith 0.8% thiobarbituric acid (TBA), acetic acid buffer pH 3.4 and

.1% sodium dodecil sulphate at 95 ◦C for 2 h. The color reactionas measured at 532 nm. TBARS levels were expressed as nmolDA (malondialdehyde)/g of tissue.

.3.3. ı-Aminolevulinate dehydratase (ı-ALA-D) activityCerebral, hepatic and renal �-ALA-D activity was assayed by the

ethod of Sassa [17] by measuring the rate of product, i.e. por-hobilinogen (PBG) formation (expressed as nmol PBG/mgptn/h)xcept that 1 M potassium phosphate buffer, pH 6.8 and 12 mMf aminolevulinic acid (ALA) were used. Incubations were carriedut for 30 min at 39 ◦C. The reaction product was determined usingodified Ehrlich’s reagent at 555 nm, with a molar absorption coef-

cient of 6.1 × 104 M−1 for the Ehrlich–porphobilinogen salt.

.3.4. Na+1/K+1-ATPase activityImmediately after the sacrifice, the kidneys were removed and

he homogenate was prepared in 0.05 M Tris–HCl buffer (pH 7.4).he homogenate was centrifuged at 4000 × g at 4 ◦C for 10 minnd supernatant was used for assay of protein Na+1/K+1-ATPase.he reaction mixture for Na+1/K+1-ATPase activity assay containedmM MgCl, 125 mM NaCl, 20 mM KCl and 50 mM Tris–HCl, pH 7.4,

n a final volume of 500 �l. The reaction was initiated by additionf ATP to a final concentration of 3.0 mM. Controls were carried outnder the same conditions with the addition of 0.1 mM ouabain.a+1/K+1-ATPase activity was calculated as nmol Pi/mg ptn/min by

he difference between the two assays. Released inorganic phos-hate (Pi) was measured by the method of Fiske and Subbarow18].

.3.5. Nonprotein thiols (NPSH) contentCerebral, hepatic and renal NPSH levels were determined by the

ethod of Ellman [19]. A sample of supernatant (500 �L) was mixed1:1) with 10% trichloroacetic acid (500 �L). After centrifugation,he protein pellet was discarded and free –SH groups were deter-

ined as nmol/g tissue in a clear supernatant. An aliquot (100 �L)f supernatant was added in a 1 M potassium phosphate buffer850 �L), pH 7.4, and 10 mM 5,5′-dithio-bis(2-nitrobenzoic acid)

DTNB) (50 �L). The color reaction was measured at 412 nm.

.3.6. Protein determinationProtein was measured by the method of Bradford [20] using

ovine serum albumin as standard.

at different times range from 0 s to 300 s at 30 s intervals. Data are the means offive to seven independent experiments done in different days. Data are expressedas mean ± SEM.

2.3.7. Ascorbic acid levelsHepatic and renal ascorbic acid (AA) determination was per-

formed as described by Jacques-Silva et al. [21]. Protein (liver) wasprecipitated in 10 V of a cold 4% trichloroacetic acid solution. Analiquot of homogenized sample (300 mL), in a final volume of 1 mLof the solution, was incubated at 38 ◦C for 3 h, then 1 mL H2SO4 65%(v/v) was added to the medium. The reaction product was deter-mined as �g AA/g tissue using color reagent containing 4.5 mg/mLdinitrophenyl hydrazine and CuSO4 (0.075 mg/mL).

2.3.8. Hepatic markers of damagePlasma enzymes, i.e. aspartate aminotransferase (AST) and ala-

nine aminotransferase (ALT) were used as the biochemical markersfor the early acute hepatic damage according to Reitman andFrankel [22], using a commercial Kit (LABTEST, Diagnostica S.A.,Minas Gerais, Brazil) and expressed as U/dl.

2.3.9. Renal markers of damageRenal function was analyzed using a commercial Kit (LABTEST,

Diagnostica S.A., Minas Gerais, Brazil) by determining plasma urea[23] and creatinine [24] and expressed as mg/dl.Statistical analysis

The results are expressed as the mean ± standard error (SEM).Data were analyzed statistically by analysis of variance (Two-wayANOVA), followed by univariate analysis and Duncan’s multiplerange test when appropriate.

3. Results

3.1. In vitro

Both compounds, i.e. DPDS and Compound A displayed thiolperoxidase like (TPx) activity. Two way ANOVA revealed signifi-cantly (p < 0.05) higher (TPx) for Compound A (Fig. 1). Two wayANOVA of cerebral, renal and hepatic TBARS production indicated

a significant main effect of SNP and DPDS/(Compound A) (p < 0.05).It is possible to observe that SNP caused statistically significantincrease in TBARS production while both DPDS and Compound Areduced it in all tested homogenates (Fig. 2). The Compound A alsoexhibited strong antioxidant activity in the DPPH and ABTS radical

38 W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44

Fig. 2. Effect of DPDS and Compound A on low-speed supernatant (S1) from brain, kidney and liver against TBARS production. Sodium nitropruside (SNP) at 20 �M was usedas pro-oxidants to induce TBARS production. TBARS are expressed as nmol of MDA/g of tissue. Data are presented as mean ± SEM (n = 5). Asterisks represent significant effectas compared with control.

Fig. 3. DPPH• radical scavenging activity of DPDS and Compound A. The values are expressed in percentage of inhibition of DPPH• absorbance (% inhibition) in relation tothe control values without the (Compound A) (ascorbic acid maximal inhibition was considered 100% of inhibition). Data are presented as mean ± SEM (n = 5) p < 0.05 fromrespective control. Asterisk represents significant effect as compared with control.

W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44 39

F expreD repres

st(

3

acrt(Ncbiaew

4

sttt

ig. 4. ABTS• radical scavenging activity of DPDS and Compound A. The values areata are presented as mean ± SEM (n = 5) p < 0.05 from respective control. Asterisk

cavenging assay for compound A. DPDS did not show any protec-ion against radical production at none of the tested concentrationsFigs. 3 and 4).

.2. In vivo results

After acute treatment with Compound A, the cerebral, hepaticnd renal TBARS levels were not changed in all treated groups whenompared to control group as shown in (Fig. 5). Two-way ANOVAevealed that compound A significantly increased NPSH concentra-ion at the highest tested concentration in all tested homogenatesFig. 6). d-ALA-D (from rats’ brain, liver and kidney) (Fig. 7) anda+K+ ATPase activity was not affected by the acute treatment withompound A at all doses administered (Fig. 8). The levels of ascor-ic acid were not altered by the treatment with the compound A

n liver and kidney (Fig. 9). AST and ALT plasmatic activities, Ureand Creatinine levels were not increased after exposure to differ-nt doses of Compound A, indicating that hepatic and renal damageas not detected (Fig. 10).

. Discussion

Based on observation that the active site of GPx may involveome interactions with other amino acid residues that would alterhe enzyme’s activity. Wendel and coworkers [25] reported thathe catalytically active selenocysteine residue in GPx is located athe N-terminal end of helix a1. It was proposed that the amino acid

Fig. 5. Effect of Compound A on TBARS formation at different doses

ssed in percentage of inhibition in relation to control without tested compounds.ents significant effect as compared with control.

residues near the selenium atom might interact weakly with theselenium, which would certainly stabilize the active site selenolateand enhance its nucleophilic reactivity. This phenomenon is repli-cated in our results, which indicates that Compound A has a veryhigh thiol peroxidase activity than DPDS. We can draw two possi-ble conclusions as to the effects of an intramolecular imino moietyin a selenium catalyst: (i) The effects of selenium and the basicimino nitrogen on the GPx activity are remarkably cooperative forH2O2 reduction. (ii) The selenium as an active center catalyses thereduction of H2O2 by interacting with the proximate basic iminonitrogen.

The basic question in here is why Compound A has better thiolperoxidase activity than DPDS? We can explain this issue by assum-ing a hypothetical cycle (Scheme 1).

The initial step is the reaction of Compound A with phenylthiol (PhSH) to produce selenol (SeH)/selenolate (Se−1). This is afeasible reaction because of hypervalent Se· · ·N interaction whichassist via N–H intermolecular weak hydrogen bonding. This is animportant step in the catalytic cycle of these molecules and is differ-ent than normal diselenides because of the unavailability of thesenon-bonded interactions, i.e. DPDS. Literature data [26] about sim-ilar compounds reveals that both theory (MO calculations) and

experiments (77Se-NMR) suggest that the proximate nitrogen baseactivates the selenol intermediate into corresponding selenolateanion, which should play a key role in accelerating the catalyticcycle [26]. Further oxidation of the Compound A with H2O2 pro-duces selenenic acid, which then rapidly undergoes bimolecular

. Data are reported as mean ± SEM for four rats in each group.

40 W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44

Fig. 6. Effect of Compound A on no-protein thiol concentration at the highest tested concentration. Data are reported as mean ± SEM for four rats in each group. Asterisksrepresent significant effect as compared with control.

F ptn/ha

dr

o

Fn

ig. 7. Effect of Compound A on the activity of �-ALA-D (expressed as nmol PBG/mgs mean ± SEM for four rats in each group.

isplacement at the selenium atom with two molecules of RSH to

egenerate the initial compound (Scheme 1).

(Scheme 2)The experimental data of thiol peroxidase indeed reflects these

bservations and after comparing it with diphenyl diselenide

ig. 8. Effect of Compound A on renal Na+1/K+1-ATPase. Results are expressed asmol Pi/mg ptn/min. Data are reported as mean ± SEM for four rats in each group.

) at different doses. PBG is used as porphobilinogen abbreviation. Data are reported

where there are no Se· · ·N non-bonded interactions shows lowerthiol peroxidase activity (Fig. 1). Our observation also reflects theresults obtained by Tomoda and coworker [13]. He studied theeffect of an amino group on the antioxidant activity of GPx by usingvarious model compounds. The initial reduction rates of H2O2 werestudied by using (PhSH) as a glutathione alternative. The presenceof these amino moieties enhanced thiol peroxidase activities.

The detailed mechanistic studies on similar GPx mimicsrevealed the fact that the Se· · ·N intramolecular nonbonded interac-tions may (i) activate the Se–Se bond towards an oxidative cleavage,(ii) stabilise the selenenic acid form of the catalyst against fur-ther oxidation, (iii) enhance the nucleophilic attack of thiol atsulfur rather than selenium, and (iv) activate the selenol intermedi-ate towards oxidation through conversion into its conjugate base,selenolate. In addition, the amine may serve to deprotonate thethiol sulfhydryl group and thus provide a high local concentrationof nucleophilic thiolate anion [26].

The synthetic nitrogen-centered DPPH radical is not biologicallyrelevant but DPPH• assay is often used to evaluate the ability ofantioxidant to scavenge free radicals which are known to be a majorfactor in biological damages caused by oxidative stress (Fig. 3)demonstrates DPPH• scavenging activity, expressed in percents,

caused by different concentrations of both compounds.

The results demonstrate that Compound A has the ability to neu-tralize the DPPH• radical or quench free radicals in the form of ABTS•

radicals (Fig. 4). Thus, it could prevent or decrease the damage ina human body caused by free radicals, which, according to Valko

W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44 41

conc

[D

l

apwSiahf

Fig. 9. Effect of Compound A on ascorbic acid level at the highest tested

27] attack biological macromolecules such as lipids, proteins andNA.

In this study, we demonstrated that SNP, a NO donor, can induceipid peroxidation in vitro, in rat’s tissue homogenates (Fig. 2).

The result presented in Fig. 2 indicated that both Compound And DPDS exerted an antioxidant effect on in vitro SNP induced lipideroxidation in rat’s tissue homogenate. A plausible mechanism byhich these compounds are conferring protective action against

NP induced lipid peroxidation is its thiol peroxidase-like activ-ty (Fig. 1). However, the compound A showed significantly highernti-oxidant potential than DPDS which could be attributed to itsigher thiol peroxidase like activity. Another possible explanation

or SNP induced TBARS production could be the direct interac-

Fig. 10. Effect of Compound A on toxicological parameters after 3-doses of acut

entration. Data are reported as mean ± SEM for four rats in each group.

tion between these compounds with SNP or its derivatives or evenquenching free radicals (shown by effective DPPH (Fig. 3) andABTS radical scavenging assays (Fig. 4)) which may have resultedfrom lipid peroxidation chain reaction caused by NO released fromSNP.

Data from our laboratory has shown that organochalcogens canbe extremely toxic with a single dose and with a short intervention[1]. The synthesis of this compound is reported very recently [4]

and since there was no toxicological data (acute) available for thiscompound we extend the idea and reported for the 1st time itsacute toxicological data.

Our study focused on in vitro/in vivo and inhibitory effect ofcompound A in rat’s tissue preparation. We have evaluated the

e treatment. Data are reported as mean ± SEM for four rats in each group.

42 W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44

DPDS

RSeSeRR'SH2

R'SSR'

(1)

(4)

(3)

(2)

RSe2-1

RSeOH2

H2O2

R'SH2

RSeSG2

R'SH2

Se)2

N

OH

Se)2

N

OH

H

Se)2

N

OH

Se)2

N

OH

SG

2RSH

RSSR

H2O2

H2O

2RSH

2RSH

ACompound

chem

io

addnoed

clawolholtoitss

Scheme 1. Comparative TPx s

nhibitory effect of compound A against SNP mediated lipid per-xidation at a range of concentrations.

Data concerning compound A distribution in tissues and bloodfter exposure/treatment are lacking in the literature. However,ata from our laboratory have shown comparative deposition ofiphenyl diselenide, i.e. an organoselenium analogous in liver, kid-ey, and brain of mice [28]. Although not directly transferable fromne element to the other, the levels of selenium found in liver afterxposure to acute and chronic relatively high doses of diphenyliselenide were found to be close to 100 �m.

One aspect that deserves to be discussed here is the facts thatompound A exhibited significant in vitro antioxidant activity atow micromolar range (5–10 �M for the case of TBARS inhibitionnd as a mimetic of Gpx). Although it is difficult to predict whatould be the exact in vivo doses needed to attain these in vitro levels

f compound A, based on previous study [28] with diphenyl dise-enide (where toxic doses were associated with liver concentrationsigher that 100 �M), we decided here to determine the in vivo effectf compound A using a range of dose that was about 10–50 timesower than that used for diphenyl diselenide. Our premise was thathe choice of these doses could be associated with non-toxic levelsf compound A in hepatic and cerebral and renal tissues. In fact, the

n vivo results support that the deposition of compound A was notoxic. However, we cannot ascertain that the levels could be con-idered similar to that causing antioxidant activity in vitro. Indirectupport to an antioxidant (and possibly similar concentrations to

Se

N

Se

N

OH

HO

Scheme 2. Molecular structure of compound A.

OH

e for Compound A and DPDS.

that needed to inhibit TBARS and to display thiol peroxidase-likeactivity) was indicated by an increase in NPSH after treatment withthe highest dose compound A in liver, kidney and brain.

The levels of TBARS, an earlier marker of lipid peroxidation, werenot changed after oral exposure to (compound A) in rat’s brain, liverand kidney (Fig. 5). These data suggest that this compound does notcause any oxidative stress in rat’s tissue after acute treatment.

�-Aminolevulinate dehydratase (�-ALA-D) is a sulfhydryl con-taining enzyme that is inhibited by a variety of sulfhydrylreagents [29–31]. This enzyme catalyses the condensation of two�-aminolevulinic acid (ALA) molecules with the formation ofporphobilinogen, which is a heme precursor [32]. Consequently,�-ALA-D inhibition may impair heme biosynthesis [33] and canresult in the accumulation of ALA, which may affect the aerobicmetabolism and may have some prooxidant activity [34]. Data fromour laboratory have shown that �-ALA-D activity is significantlyinhibited by diphenyl diselenide [1]. In contrast results from thisstudy indicate that Compound A did not alter the enzyme activity(Fig. 7) from different organs indicating a fact that in contrast todiphenyl diselenide �-ALA-D it is not an in vivo molecular targetfor Compound A.

There are several reports which have demonstrated the impor-tance of thiol groups for Na+1/K+1-ATPase catalysis. In fact, –SHgroups of this enzyme is highly susceptible to oxidizing agents[35–37]. Recently DPDS was reported to inhibit the cerebralNa+1/K+1-ATPase in a concentration dependent manner and thatthe inhibition may occur through a change in the crucial thiolgroups of this enzyme [38]. In significant contrast to DPDS, com-pound A at the highest tested concentration did not inhibit theNa+1/K+1-ATPase activity (Fig. 8). We speculate that the lack of toxi-city of Compound A in the ex vivo �-ALA-D and Na/K ATPase assay isrelated to its weak ability to oxidize thiols groups. This observationwas further strengthened by the fact that compound A did not alternon-protein thiol content in fact it improved NPSH concentration

(Fig. 6).

In order to determine if treatment with Compound A couldcause hepatic damage, we measured plasmatic activities of ASTand ALT. These transaminases are located in different sub-celullar spaces in hepatocytes, and they are typically released

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nto the blood after exposure to hepatotoxic chemical agents39].

Rats exposed to Compound A had markers of hepatic and renalunction within the normal range (Fig. 10). Corroborating withhese data, acute exposure to high doses of DPDS did not affectlasma transaminase activities or levels of urea and creatinine inodents [40]. Furthermore, previous study has reported that rab-its chronically exposed to DPDS supplemented diet have levelsf markers of hepatic and renal functions within the normal range41].

Ascorbic acid is always considered a marker of oxidative stressnd the reduction of its content may indicate an increase in oxida-ive stress [21]. Exposure to Compound A did not alter vitamin Cevels in liver and kidney (Fig. 9). Taken together, these data sug-est that acute exposure to disubstituted diaryl diselenides did notnduce oxidative stress in rats.

. Conclusion

The in vitro data presented here provides concrete experimen-al data for the first time about this organoselenium compound,aving high thiol peroxidase activity, strong anti-oxidant poten-ial against SNP induced lipid peroxidation and radical scavengingctivity (more than DPDS). Apart from that we have shown fromur in vivo data that acute exposure of compound A did not causeny enzyme inhibition. The fact that this compound causes low oregligible toxicity in rats indicates that Compound A is a promis-

ng candidate for future pharmacological studies. The present dataolidifies the position of Compound A as a potential candidate drugo be tested against a variety of neurodegenerative diseases or freeadical induced damages.

onflict of interest

There is no conflict of interest in the conduct and reporting ofesearch (e.g., financial interests in a test or procedure, funding byharmaceutical companies for drug research).

cknowledgments

The authors are grateful for the financial support of TWAS andNPq. Waseem Hassan is a beneficiary of the TWAS CNPq doc-oral fellowship program. J.B.T. Rocha gratefully acknowledges thenancial support of CAPES SAUX, CNPq, VITAE and FAPERGS.

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