Hydroxyl Containing Seleno-Imine Compound Exhibits Improved Anti-Oxidant Potential and Does Not Inhibit Thiol-Containing Enzymes

Chemico-Biological Interactions 190 (2011) 35–44

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Chemico-Biological Interactions

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Hydroxyl containing seleno-imine compound exhibits improved anti-oxidant potential and does not inhibit thiol-containing enzymes

Waseem Hassan a,c,∗, Simone Pinton a, Juliana Trevisan da Rocha a, Anna Maria Deobald a, Antonio Luis Braga a, Cristina Wayne Nogueira a, Alexandra Susana Latini b, Joao B.T. Rocha a

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

article info abstract

Article history: Design and synthesis of organoselenium compounds with high thiol peroxidase (TPx) and low thiol oxi- Received 28 June 2010 dase (TOx) activities have been a difficult task and remains a synthetic-activity relationship dilemma. In Received in revised form 1 December 2010 this regard we are reporting for the first time a detail experimental data (both in vitro and in vivo) about Accepted 10 January 2011 the anti-oxidant and toxicological profile of an Imine (–N ) containing organoselenium compound (Com- Available online 21 January 2011 pound A). The TPx activity of Compound A was significantly higher than diphenyl diselenide (DPDS). Both Compound A and DPDS protected sodium nitropruside (SNP) induced thiobarbituric acid reactive species Keywords: (TBARS) production in rats tissue homogenate with significantly higher activity observed for Compound Non-bonded interactions Anti-oxidant A than DPDS (p < 0.05). The Compound A also exhibited strong antioxidant activity in the DPPH and ABTS Toxicity radical scavenging assays. This study reveals that an imine group close to selenium atom drastically enhances the catalytic activities in the aromatic thiol (PhSH) assay systems. The oxidation of biologically significant thiols reflects the toxicity of the compounds. However, the present data showed that treatment 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) activities after acute oral treatment; rather it enhanced non-protein thiols (NPSH) concentration. The Compound A did not cause any oxidative stress as measured by TBARS production in rat’s tissue preparation. Our data 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 the reduction of its content may indicate an increase in oxidative stress. Treatment with Compound A did not alter Ascorbic acid levels in rats. The conducted in vitro and in vivo tests show the versatile therapeutic potential of this compound in the area of free radical induced damages, will undoubtedly enhance our understanding of the mechanism of model compounds and may ultimately yield insights that result in improved GPx mimics. © 2011 Elsevier Ireland Ltd. Open access under the Elsevier OA license.

1. Introduction hydroperoxides reducing ability of GPx, several groups have worked towards the synthesis of better GPx mimics. All these Various organoselenium compounds having a direct mimics with Se–N bond have been developed with the assumption selenium–nitrogen (Se–N) bond have been shown to mimic the that the enzyme GPx may have a cyclic selenenamide structure in active site of glutathione peroxidase (GPx). Among them the most its oxidized form and the peptidic nitrogen atoms may function promising drug is Ebselen (1,2-phenyl- 1,2-benzoisoselenazol- similarly in the formation of cyclic selenenamide species in the 3-(2H)-one), a heterocyclic compound exhibiting interesting natural enzyme GPx. clinical properties in the area of free radical induced damages Based on these observations, various organoselenium com- and therapies [1]. Since the discovery that Ebselen mimics the pounds containing heteroatoms, i.e. nitrogen in close proximity to the selenium has been synthesized in order to study their GPx like activity. Wilson et al. observed that the inclusion of a strongly basic amino group proximal to the active selenium atom in the molecule ∗ Corresponding author at: Departamento de Química, Centro de Ciências Naturais is desirable as it would be expected to catalyze the reaction of e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. Tel.: +55 55 3220 8140; fax: +55 55 3220 8978. thiols with intermediates like diselenides and selenosulﬁdes. Pre- E-mail address: waseem [email protected] (W. Hassan). sumably, the base functions to provide a source of nucleophilic

0009-2797 © 2011 Elsevier Ireland Ltd. Open access under the Elsevier OA license. doi:10.1016/j.cbi.2011.01.012 36 W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44 thiolate anion. They also observed that the selenosulfide and dise- allowed to warm to rt naturally and was stirred overnight. To lenide did not react with thiol under neutral conditions. Upon the this solution at 0 ◦C was added n-BuLi (2,5 M solution in hex- addition of a strong amine base, however, both gave the seleno- ane, 27 mmol) drop wise. The reaction mixture was allowed to late and disulfide [2]. Galet et al. worked in the same area and he slowly warm to room temperature and stirred for 6 h. Selenium proved the importance of the Se–N bond for GPx activity. The 1,3- (Se) powder (20 mmol) was added at 0 ◦C, the reaction mixture was benzoselenazolinones, which contain selenium and nitrogen atoms allowed to warm to room temperature naturally and was stirred in the heterocycle but do not have any direct Se–N bonds were overnight. NH4Cl saturated solution was added slowly and the tested for GPx like activity [3]. All 1,3-benzoselenazolinones were resulting solution was oxidized for 2 h. After the usual work up with devoid of GPx activity under a variety of conditions. The work by diethyl acetate, the product was isolated by column chromatog- Wilson et al. to design GPx mimics without direct Se–N bond, also raphy (EtOAc/MeOH = 70:30) as a dark yellow oil in 50% of yield met with only limited success [2]. [12]. To summarize, various organoselenium compounds containing heteroatoms in close proximity to the selenium have been syn- 2.2. In vitro assays thesized in order to study their GPx like activity. In this regard we have recently reported the synthesis of a new heterocyclic 2.2.1. Thiol peroxidase activity diselenide with imino residue close to selenium, i.e. 2-((1-(2- The catalytic activity of Compound A (Scheme 2) as a GPx model (2-(2-(1-(2-hydroxybenzylideneamino) ethyl) phenyl) diselanyl) enzyme, was evaluated according to Tomoda and Iwaoka method phenyl) ethylimino) methyl) phenol (Compound A) which showed [13] using benzenethiol as a glutathione alternative. The reduc- promising anti-oxidant potential against Fe (II) induced lipid per- tion of H2O2 was monitored through the UV absorption increase oxidation under different in vitro patho-physiological conditions at 305 nm, due to diphenyl disulfide formation. [4]. However, still there is scarcity of pharmacological and toxicological data about this comparatively new compound. Thus, to get a 2.2.2. Preparation of tissue homogenate and thiobarbituric acid deeper insight about the potential use of Compound A as a possible reactive species (TBARS) assay pharmacological agent, we determined for the first time its thiol Rats were decapitated under mild ether anesthesia and brain, peroxidase like activity along with its ability to scavenge free rad- liver and kidneys were rapidly removed, placed on ice and weighed. icals under in vitro conditions. We have also studied whether the Tissues were immediately homogenized in cold 10 mM Tris–HCl, compound A can inhibit TBARS productions when incubated with pH 7.4 (1/10, w/v) with 10 up-and-down strokes at approximately sodium nitroprusside (SNP). Similarly the radical scavenging activ- 1200 rev/min in a Teflon-glass homogenizer. The homogenate was ity was measured by DPPH and ABTS assays. To take a step ahead centrifuged for 10 min at 4000 × g to yield a pellet that was dis- the activity of this compound was further compared with diphenyl carded and a low-speed supernatant (S1). An aliquot of 100 ␮lof diselenide (DPDS) which we recently showed to posses interesting S1 was incubated for 1 h at 37 ◦C in the presence of both organodise- biological activities under various pathological situations [4–6]. lenides (final concentrations range of 0–100 ␮M), with and without Organoselenium compounds can interact directly with low the prooxidant, i.e. sodium nitroprusside (SNP) (final concentration molecular thiols, oxidizing them to disulfides as well [7]. Simi- 10 ␮M). Productions of TBARS were determined as described by larly, reduced cysteinyl residues from proteins can also react with method of Ohkawa et al. [14]. Except that the buffer of color reac- these compounds, which may cause, in the case of the enzymes, tion has a pH of 3.4. The color reaction was developed by adding the loss of their catalytic activity [8,9]. The mechanism(s) under- 300 ␮l 8.1% SDS to S1, followed by sequential addition of 500 ␮l lying either the toxic or protective effect of organochalcogens are acetic acid/HCl (pH 3.4) and 500 ␮l 0.8% of thiobarbituric acid (TBA). not completely understood but certainly involves the reaction of This mixture was incubated at 95 ◦C for 1 h. chalcogenides with endogenous thiols [10,11]. The facts that (i) the Malondialdehide (MDA), an end product of fatty acid peroxida- in vitro test indicating high thiol peroxidase (TPx) or anti-oxidant tion, reacts with thiobarbituric acid (TBA) to form a colored complex activity necessarily does not lead to in vivo implications that it will which is measured as nmol of MDA/g of tissue at 532 nm and the be a safe compound and (ii) a possible high thiol oxidase (TOx) absorbance was compared to that of a standard curve obtained activity reduces the interest as it may cause oxidation of biological using malondialdehyde (MDA). relevant thiol molecules and enzymes, motivated us to study the in vivo interaction profile of this compound with thiol groups (–SH). For the purpose we designed an in vivo study, where male adult 2.2.3. DPPH radical scavenging assay wistar rats were treated with Compound A to get a better under- The measurement of the (Compound A) scavenging activity • standing of its toxicological aspects. Along with its interaction with against the radical DPPH was performed in accordance with Choi ␮ • non-protein thiols (NPSH) we also provided experimental data et al. [15]. Briefly, 85 M DPPH was added to a medium containing about the interaction of Compound A with ␣-ALA-D and Na+1/K+1 different (Compound A) concentrations. The medium was incu- ATPase (sulfhydryl containing enzyme). Several other tests have bated for 30 min at room temperature. The decrease in absorbance been performed to get a detail idea about its in vivo behavior which measured at 518 nm depicted the scavenging activity of the (Com- • could of particular interest in view of its pharmacological and tox- pound A) against DPPH . Ascorbic acid was used as positive control • icological potential. to determine the maximal decrease in DPPH absorbance. The values are expressed in percentage of inhibition of DPPH• absorbance (% inhibition) in relation to the control values without the (Com- 2. Materials and methods pound A) (ascorbic acid maximal inhibition was considered 100% of inhibition). 2.1. Synthesis of Compound A 2.2.4. ABTS radical scavenging assay To a suspension of (R,S)-␣-methylbenzylamine(10 mmol) in The determination of the ABTS radical scavenging effect of Com- 40 mL of ether at −78 ◦C was added n-BuLi (2,5 M solution in pound A was performed according to the method of Re et al. [16], hexane, 10 mmol) dropwise. The resulting solution was stirred at with some modifications. Initially, the ABTS radical was generated −78 ◦C for 30 min before TMS (trimethylsilyl chloride) (12 mmol) by reacting 7 mM ABTS solution in water with 140 mM potassium was added at the same temperature. The reaction mixture was persulfate in the dark for 12–16 h. In the day of the assay, the W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44 37 pre-formed ABTS radical solution was diluted 1:88 (1 ml ABTS radical plus (+) 87 ml 10 mM potassium phosphate buffer, pH 7.0). Briefly, ABTS radical was added to a medium containing compound A (10–50 ␮M). The media were incubated for 30 min at 25 ◦C. The decrease in absorbance was measured at 734 nm, depicting the scavenging activity of Compound A against ABTS radical. Ascor- bic acid (10–50 ␮M) was used as a positive control to determine the maximal decrease in ABTS absorbance. Results are expressed as percentage (%age) of the blank (without compound A).

2.3. In vivo assays

2.3.1. Animal’s treatment Rats were divided into four groups, daily weighted and treated (orally) for 3 days (at the interval of 24 h) with compound A at 0 (canola oil) 10, 25 and 50 mg/kg. 24 h after the last injection animals were anesthetized to the collection of blood via heart puncture and then killed by decapitation. All the experiments were conducted at least four times and similar results were obtained.

2.3.2. Thiobarbituric acid reactive species (TBARS) levels Fig. 1. Thiol peroxidase like activity of DPDS and Compound A (at 10 ␮M) in the TBARS levels, a measure of lipid peroxidation, were determined presence of hydrogen peroxide. The difference in peroxidase activity was calculated as described by Ohkawa et al. [14]. An aliquot of S1 was incubated at different times range from 0 s to 300 s at 30 s intervals. Data are the means of five to seven independent experiments done in different days. Data are expressed with 0.8% thiobarbituric acid (TBA), acetic acid buffer pH 3.4 and as mean ± SEM. 8.1% sodium dodecil sulphate at 95 ◦C for 2 h. The color reaction was measured at 532 nm. TBARS levels were expressed as nmol MDA (malondialdehyde)/g of tissue. 2.3.7. Ascorbic acid levels Hepatic and renal ascorbic acid (AA) determination was per- ı ı 2.3.3. -Aminolevulinate dehydratase ( -ALA-D) activity formed as described by Jacques-Silva et al. [21]. Protein (liver) was ␣ Cerebral, hepatic and renal -ALA-D activity was assayed by the precipitated in 10 V of a cold 4% trichloroacetic acid solution. An method of Sassa [17] by measuring the rate of product, i.e. por- aliquot of homogenized sample (300 mL), in a final volume of 1 mL phobilinogen (PBG) formation (expressed as nmol PBG/mgptn/h) ◦ of the solution, was incubated at 38 C for 3 h, then 1 mL H2SO4 65% except that 1 M potassium phosphate buffer, pH 6.8 and 12 mM (v/v) was added to the medium. The reaction product was deter- of aminolevulinic acid (ALA) were used. Incubations were carried mined as ␮g AA/g tissue using color reagent containing 4.5 mg/mL out for 30 min at 39 ◦C. The reaction product was determined using dinitrophenyl hydrazine and CuSO4 (0.075 mg/mL). modified Ehrlich’s reagent at 555 nm, with a molar absorption coef- ficient of 6.1 × 104 M−1 for the Ehrlich–porphobilinogen salt. 2.3.8. Hepatic markers of damage Plasma enzymes, i.e. aspartate aminotransferase (AST) and ala- 2.3.4. Na+1/K+1-ATPase activity nine aminotransferase (ALT) were used as the biochemical markers Immediately after the sacrifice, the kidneys were removed and for the early acute hepatic damage according to Reitman and the homogenate was prepared in 0.05 M Tris–HCl buffer (pH 7.4). Frankel [22], using a commercial Kit (LABTEST, Diagnostica S.A., The homogenate was centrifuged at 4000 × g at 4 ◦C for 10 min Minas Gerais, Brazil) and expressed as U/dl. and supernatant was used for assay of protein Na+1/K+1-ATPase. The reaction mixture for Na+1/K+1-ATPase activity assay contained 3 mM MgCl, 125 mM NaCl, 20 mM KCl and 50 mM Tris–HCl, pH 7.4, 2.3.9. Renal markers of damage in a final volume of 500 ␮l. The reaction was initiated by addition Renal function was analyzed using a commercial Kit (LABTEST, of ATP to a final concentration of 3.0 mM. Controls were carried out Diagnostica S.A., Minas Gerais, Brazil) by determining plasma urea under the same conditions with the addition of 0.1 mM ouabain. [23] and creatinine [24] and expressed as mg/dl.Statistical analysis ± Na+1/K+1-ATPase activity was calculated as nmol Pi/mg ptn/min by The results are expressed as the mean standard error (SEM). the difference between the two assays. Released inorganic phos- Data were analyzed statistically by analysis of variance (Two-way phate (Pi) was measured by the method of Fiske and Subbarow ANOVA), followed by univariate analysis and Duncan’s multiple [18]. range test when appropriate.

2.3.5. Nonprotein thiols (NPSH) content 3. Results Cerebral, hepatic and renal NPSH levels were determined by the method of Ellman [19]. A sample of supernatant (500 ␮L) was mixed 3.1. In vitro (1:1) with 10% trichloroacetic acid (500 ␮L). After centrifugation, the protein pellet was discarded and free –SH groups were deter- Both compounds, i.e. DPDS and Compound A displayed thiol mined as nmol/g tissue in a clear supernatant. An aliquot (100 ␮L) peroxidase like (TPx) activity. Two way ANOVA revealed signifi- of supernatant was added in a 1 M potassium phosphate buffer cantly (p < 0.05) higher (TPx) for Compound A (Fig. 1). Two way (850 ␮L), pH 7.4, and 10 mM 5,5-dithio-bis(2-nitrobenzoic acid) ANOVA of cerebral, renal and hepatic TBARS production indicated (DTNB) (50 ␮L). The color reaction was measured at 412 nm. a significant main effect of SNP and DPDS/(Compound A) (p < 0.05). It is possible to observe that SNP caused statistically significant 2.3.6. Protein determination increase in TBARS production while both DPDS and Compound A Protein was measured by the method of Bradford [20] using reduced it in all tested homogenates (Fig. 2). The Compound A also bovine serum albumin as standard. exhibited 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 used as 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 effect as 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 to the 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 from respective control. Asterisk represents signiﬁcant effect as compared with control. W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44 39

Fig. 4. ABTS• radical scavenging activity of DPDS and Compound A. The values are expressed in percentage of inhibition in relation to control without tested compounds. Data are presented as mean ± SEM (n =5)p < 0.05 from respective control. Asterisk represents signiﬁcant effect as compared with control. scavenging assay for compound A. DPDS did not show any protec- residues near the selenium atom might interact weakly with the tion against radical production at none of the tested concentrations selenium, which would certainly stabilize the active site selenolate (Figs. 3 and 4). and enhance its nucleophilic reactivity. This phenomenon is repli- cated in our results, which indicates that Compound A has a very 3.2. In vivo results high thiol peroxidase activity than DPDS. We can draw two possible conclusions as to the effects of an intramolecular imino moiety After acute treatment with Compound A, the cerebral, hepatic in a selenium catalyst: (i) The effects of selenium and the basic and renal TBARS levels were not changed in all treated groups when imino nitrogen on the GPx activity are remarkably cooperative for compared to control group as shown in (Fig. 5). Two-way ANOVA H2O2 reduction. (ii) The selenium as an active center catalyses the revealed that compound A signiﬁcantly increased NPSH concentra- reduction of H2O2 by interacting with the proximate basic imino tion at the highest tested concentration in all tested homogenates nitrogen. (Fig. 6). d-ALA-D (from rats’ brain, liver and kidney) (Fig. 7) and The basic question in here is why Compound A has better thiol Na+K+ ATPase activity was not affected by the acute treatment with peroxidase activity than DPDS? We can explain this issue by assum- compound A at all doses administered (Fig. 8). The levels of ascor- ing a hypothetical cycle (Scheme 1). bic acid were not altered by the treatment with the compound A The initial step is the reaction of Compound A with phenyl − in liver and kidney (Fig. 9). AST and ALT plasmatic activities, Urea thiol (PhSH) to produce selenol (SeH)/selenolate (Se 1). This is a and Creatinine levels were not increased after exposure to differ- feasible reaction because of hypervalent Se···N interaction which ent doses of Compound A, indicating that hepatic and renal damage assist via N–H intermolecular weak hydrogen bonding. This is an was not detected (Fig. 10). important step in the catalytic cycle of these molecules and is different than normal diselenides because of the unavailability of these 4. Discussion non-bonded interactions, i.e. DPDS. Literature data [26] about similar compounds reveals that both theory (MO calculations) and Based on observation that the active site of GPx may involve experiments (77Se-NMR) suggest that the proximate nitrogen base some interactions with other amino acid residues that would alter activates the selenol intermediate into corresponding selenolate the enzyme’s activity. Wendel and coworkers [25] reported that anion, which should play a key role in accelerating the catalytic the catalytically active selenocysteine residue in GPx is located at cycle [26]. Further oxidation of the Compound A with H2O2 pro- the N-terminal end of helix a1. It was proposed that the amino acid duces selenenic acid, which then rapidly undergoes bimolecular

Fig. 5. Effect of Compound A on TBARS formation at different doses. 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. Asterisks represent signiﬁcant effect as compared with control.

Fig. 7. Effect of Compound A on the activity of ␣-ALA-D (expressed as nmol PBG/mgptn/h) at different doses. PBG is used as porphobilinogen abbreviation. Data are reported as mean ± SEM for four rats in each group. displacement at the selenium atom with two molecules of RSH to where there are no Se···N non-bonded interactions shows lower regenerate the initial compound (Scheme 1). thiol peroxidase activity (Fig. 1). Our observation also reﬂects the (Scheme 2) results obtained by Tomoda and coworker [13]. He studied the The experimental data of thiol peroxidase indeed reﬂects these effect of an amino group on the antioxidant activity of GPx by using observations and after comparing it with diphenyl diselenide various model compounds. The initial reduction rates of H2O2 were studied by using (PhSH) as a glutathione alternative. The presence of these amino moieties enhanced thiol peroxidase activities. The detailed mechanistic studies on similar GPx mimics revealed the fact that the Se···N intramolecular nonbonded interactions may (i) activate the Se–Se bond towards an oxidative cleavage, (ii) stabilise the selenenic acid form of the catalyst against further oxidation, (iii) enhance the nucleophilic attack of thiol at sulfur rather than selenium, and (iv) activate the selenol intermediate towards oxidation through conversion into its conjugate base, selenolate. In addition, the amine may serve to deprotonate the thiol sulfhydryl group and thus provide a high local concentration of nucleophilic thiolate anion [26]. The synthetic nitrogen-centered DPPH radical is not biologically relevant but DPPH• assay is often used to evaluate the ability of antioxidant to scavenge free radicals which are known to be a major factor 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•

Fig. 8. Effect of Compound A on renal Na+1/K+1-ATPase. Results are expressed as radicals (Fig. 4). Thus, it could prevent or decrease the damage in nmol Pi/mg ptn/min. Data are reported as mean ± SEM for four rats in each group. a human body caused by free radicals, which, according to Valko W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44 41

Fig. 9. Effect of Compound A on ascorbic acid level at the highest tested concentration. Data are reported as mean ± SEM for four rats in each group.

[27] attack biological macromolecules such as lipids, proteins and tion between these compounds with SNP or its derivatives or even DNA. quenching free radicals (shown by effective DPPH (Fig. 3) and In this study, we demonstrated that SNP, a NO donor, can induce ABTS radical scavenging assays (Fig. 4)) which may have resulted lipid peroxidation in vitro, in rat’s tissue homogenates (Fig. 2). from lipid peroxidation chain reaction caused by NO released from The result presented in Fig. 2 indicated that both Compound A SNP. and DPDS exerted an antioxidant effect on in vitro SNP induced lipid Data from our laboratory has shown that organochalcogens can peroxidation in rat’s tissue homogenate. A plausible mechanism by be extremely toxic with a single dose and with a short intervention which these compounds are conferring protective action against [1]. The synthesis of this compound is reported very recently [4] SNP induced lipid peroxidation is its thiol peroxidase-like activ- and since there was no toxicological data (acute) available for this ity (Fig. 1). However, the compound A showed signiﬁcantly higher compound we extend the idea and reported for the 1st time its anti-oxidant potential than DPDS which could be attributed to its acute toxicological data. higher thiol peroxidase like activity. Another possible explanation Our study focused on in vitro/in vivo and inhibitory effect of for SNP induced TBARS production could be the direct interac- compound A in rat’s tissue preparation. We have evaluated the

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

Compound A

Se) DPDS 2 2RSH 2 R'SH N RSeSeR H S R (1) 2 RSSR 2 R'SH R'SSR' OH

Se)2 Se)2 2 RSeSG H 2 RSe-1 N N (4) (2)

OH OH H2O2

2 OH R H2O2 2 R'SH S Se)2 2 RSeOH H (3) N H2O

Scheme 1. Comparative TPx scheme for Compound A and DPDS. inhibitory effect of compound A against SNP mediated lipid per- that needed to inhibit TBARS and to display thiol peroxidase-like oxidation at a range of concentrations. activity) was indicated by an increase in NPSH after treatment with Data concerning compound A distribution in tissues and blood the highest dose compound A in liver, kidney and brain. after exposure/treatment are lacking in the literature. However, The levels of TBARS, an earlier marker of lipid peroxidation, were data from our laboratory have shown comparative deposition of not changed after oral exposure to (compound A) in rat’s brain, liver diphenyl diselenide, i.e. an organoselenium analogous in liver, kid- and kidney (Fig. 5). These data suggest that this compound does not ney, and brain of mice [28]. Although not directly transferable from cause any oxidative stress in rat’s tissue after acute treatment. one element to the other, the levels of selenium found in liver after ␦-Aminolevulinate dehydratase (␦-ALA-D) is a sulfhydryl con- exposure to acute and chronic relatively high doses of diphenyl taining enzyme that is inhibited by a variety of sulfhydryl diselenide were found to be close to 100 ␮m. reagents [29–31]. This enzyme catalyses the condensation of two One aspect that deserves to be discussed here is the facts that ␦-aminolevulinic acid (ALA) molecules with the formation of compound A exhibited significant in vitro antioxidant activity at porphobilinogen, which is a heme precursor [32]. Consequently, low micromolar range (5–10 ␮M for the case of TBARS inhibition ␦-ALA-D inhibition may impair heme biosynthesis [33] and can and as a mimetic of Gpx). Although it is difficult to predict what result in the accumulation of ALA, which may affect the aerobic would be the exact in vivo doses needed to attain these in vitro levels metabolism and may have some prooxidant activity [34]. Data from of compound A, based on previous study [28] with diphenyl dise- our laboratory have shown that ␦-ALA-D activity is significantly lenide (where toxic doses were associated with liver concentrations inhibited by diphenyl diselenide [1]. In contrast results from this higher that 100 ␮M), we decided here to determine the in vivo effect study indicate that Compound A did not alter the enzyme activity of compound A using a range of dose that was about 10–50 times (Fig. 7) from different organs indicating a fact that in contrast to lower than that used for diphenyl diselenide. Our premise was that diphenyl diselenide ␦-ALA-D it is not an in vivo molecular target the choice of these doses could be associated with non-toxic levels for Compound A. of compound A in hepatic and cerebral and renal tissues. In fact, the There are several reports which have demonstrated the impor- in vivo results support that the deposition of compound A was not tance of thiol groups for Na+1/K+1-ATPase catalysis. In fact, –SH toxic. However, we cannot ascertain that the levels could be con- groups of this enzyme is highly susceptible to oxidizing agents sidered similar to that causing antioxidant activity in vitro. Indirect [35–37]. Recently DPDS was reported to inhibit the cerebral support to an antioxidant (and possibly similar concentrations to Na+1/K+1-ATPase in a concentration dependent manner and that the inhibition may occur through a change in the crucial thiol groups of this enzyme [38]. In significant contrast to DPDS, compound A at the highest tested concentration did not inhibit the Na+1/K+1-ATPase activity (Fig. 8). We speculate that the lack of toxi- OH Se Se city of Compound A in the ex vivo ␦-ALA-D and Na/K ATPase assay is related to its weak ability to oxidize thiols groups. This observation N was further strengthened by the fact that compound A did not alter N non-protein thiol content in fact it improved NPSH concentration HO (Fig. 6). In order to determine if treatment with Compound A could cause hepatic damage, we measured plasmatic activities of AST and ALT. These transaminases are located in different sub- Scheme 2. Molecular structure of compound A. celullar spaces in hepatocytes, and they are typically released W. Hassan et al. / Chemico-Biological Interactions 190 (2011) 35–44 43 into the blood after exposure to hepatotoxic chemical agents [8] N. Gupta, T.D. Porter, Inhibition of human squalene monooxygenase by sele- [39]. nium compounds, J. Biochem. Mol. Toxicol. 16 (2001) 18–23. [9] N.B.V. Barbosa, J.B.T. Rocha, G. Zeni, T. Emanuelli, M.C. Beque, A.L. Braga, Rats exposed to Compound A had markers of hepatic and renal Effect of organic forms of selenium on delta-aminolevulinate dehydratase from function within the normal range (Fig. 10). Corroborating with liver, kidney, and brain of adult rats, Toxicol. Appl. Pharmacol. 149 (1998) these data, acute exposure to high doses of DPDS did not affect 243–253. [10] E.N Maciel, R.C. Bolzan, A.L. Braga, J.B.T. Rocha, Diphenyl diselenide and plasma transaminase activities or levels of urea and creatinine in diphenyl ditelluride differentially affect ␦-aminolevulinate dehydratase from rodents [40]. Furthermore, previous study has reported that rab- liver, kidney, and brain of mice, J. Biochem. Mol. Toxicol. 14 (2000) bits chronically exposed to DPDS supplemented diet have levels 310–319. of markers of hepatic and renal functions within the normal range [11] A. Daiber, M. Zhou, M. 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