Chemico-Biological Interactions 190 (2011) 35–44 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Contents lists available at ScienceDirect provided by Elsevier - Publisher Connector Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 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 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) 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 toxico- logical 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.