ARTICLE IN PRESS

Pathology – Research and Practice 206 (2010) 357–360

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Pathology – Research and Practice

0344-03

doi:10.1

DOI� Corr

E-m

journal homepage: www.elsevier.de/prp

Short Communication

Diphenyl diselenide behaves differently than ebselen under different pHmedia in rat’s liver preparations

Waseem Hassan �, Mohammad Ibrahim, Joao Batista Teixeira Rocha

Departamento de Quımica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, CEP 97105-900, RS, Brazil

a r t i c l e i n f o

Article history:

Received 24 June 2009

Received in revised form

19 December 2009

Accepted 21 December 2009

Keywords:

pH

Lipid peroxidation

Anti-oxidant

Ebselen

Diphenyl diselenide

38/$ - see front matter & 2010 Elsevier Gmb

016/j.prp.2009.12.009

of original article: 10.1016/j.prp.2008.10.011

esponding author. Tel.: +55 55 3220 8140; fa

ail address: waseem_anw@yahoo.com (W. Ha

a b s t r a c t

The anti-oxidant potential of diphenyl diselenide and ebselen against low pH-mediated peroxidation

processes in rat’s liver preparation are unknown. For this purpose, we have determined anti-oxidant

activities of both compounds at a range of various pH (7.4–5.4) values. Low pH increased the rate of

lipid peroxidation in the absence of Fe+ 2 (20mmol) in liver homogenates. This higher extent of lipid

peroxidation can be explained by the mobilized iron, which may come from reserves where it is weakly

bound. The addition of iron (Fe) chelator desferoxamine (DFO, 1 mmol) to reaction medium completely

inhibited the peroxidation processes at all studied pH values. Diphenyl diselenide (0–100mmol)

significantly protected lipid peroxidation at all studied pH values, while ebselen (0–100mmol) did not

offer any protection. The differences in activities of diphenyl diselenide and ebselen have been

explained with reference to the active selenol formation using their catalytic reactions. The anti-oxidant

potential of diphenyl diselenide is confirmed against acidosis-catalyzed oxidative stress in rat liver

homogenates.

& 2010 Elsevier GmbH. All rights reserved.

Introduction

Organoselenium compounds exhibit potent anti-oxidant activitymediated by their glutathione peroxidase (GPx) mimetic properties.In line with this, ebselen (pz 51; 2-phenyl-1,2-benzisoselenazol-3(2H)-one), a synthetic selenium-containing heterocycle, has beenused experimentally and clinically with success in a variety ofsituations where free radicals are involved. Diphenyl diselenide(DPDS), the simplest diaryl diselenide, is particularly important as apotential anti-oxidant drug because it has been shown to be moreactive as a glutathione peroxidase mimic, less toxic to rodents thanebselen, and also has low toxicity for non-rodent mammals afterlong-term exposure [4,8,11–16].

Based on the redox potential and electrochemistry studied onorganoselenium compounds, we worked on the hypothesis thatthe formation of stable selenolate (Se�1) ions can increase thereducing properties of these moieties on the organochalcogenides,and hypothetically can increase their anti-oxidant properties.However, there are no detailed studies in the literature about thestated issue. Below is a simple reaction that can clarify the issue

R1XXR1+2R2SH-R1X�1+H+ +R1XSR2

where (X=Se).

H. All rights reserved.

x: +55 55 3220 8978.

ssan).

It is apparent from the above reaction that a selenolatemolecule (R1X�1) can be produced with a hydrogen ion. Thisestablishes the basis of the hypothesis, i.e., the more stable this(R1X�1) ion is, the higher will be its anti-oxidant potency. Hence,our work was designed to study any influence of change in pH onthe anti-oxidant effect of (DPDS) and ebselen.

There is also scarcity of data in the literature about thecomparative effect of (DPDS) and ebselen under various patho-physiological conditions in rat’s liver preparations. In this regard,our work was designed to obtain different pH media in vitro andto study the extent of Fe (II)-induced lipid peroxidation, as well asthe anti-oxidant potential of these two organoselenium com-pounds. For this purpose, the influence of pH on (a) either basal orFe (II)-induced lipid peroxidation in rat’s liver homogenate, and(b) on the anti-oxidant potential of diphenyl diselenide andebselen was investigated.

Methodology

Adult male wistar rats (5–7) were anesthetized with ether andkilled by decapitation. The liver was quickly removed, placed onice, and homogenized within 10 min in 10 mmol/l tris/HCl buffer,pH 7.4 (in 10 volume). The homogenates were centrifuged at4000� g at 4 1C for 10 min to yield a low- speed supernatantfraction (S1) that was used immediately for thiobarbituric acid-reactive species TBARS assay [9]. Lipid peroxidation was deter-mined by measuring (TBARS) as described by Ohkawa et al. [9]

ARTICLE IN PRESS

Fig. 1. Effect of pH on basal (shaded bar) or Fe (II)-induced (bar with lines) TBARS

production in rat’s liver homogenate. The values are expressed as nmol of MDA per

gram of tissue. Data are expressed as means7S.E.M. (n=5–7). Different letters

show a significant difference from each pH group, while the asterisk shows a

significant main effect of Fe (II) at po0.05.

Fig. 2. Effect of DPDS (0–100mM) on basal (shaded bar) or Fe (II) (20mM)-induced (bar

are expressed as nmol of MDA per gram of tissue. Data are expressed as means7S.E.M. (

W. Hassan et al. / Pathology – Research and Practice 206 (2010) 357–360358

with minor modifications [11]. Liver homogenate was preparedby homogenization as described above. Aliquots of the S1 (100ml)were incubated for 60 min in a medium containing 10 mM sodiumphosphate buffer of different pH ranging from 5.4 to 7.8 (pH waschecked after the addition of liver supernatant both at thebeginning and at the end of the incubation period) and in thepresence of other reagents at concentrations indicated inthe legends. The mixtures were incubated at 37 1C for 60 min.The reaction was stopped by addition of 0.5 ml of acetic acidbuffer, and lipid peroxidation products were measured by theaddition of 0.5 ml of TBA 0.6% and 0.2 ml of SDS 8.1%. The colorreaction was developed by incubating tubes in boiling water for60 min. TBARS levels were measured at 532 nm using a standardcurve of MDA. The values are expressed in nmol MDA/g of tissue.Where indicated, solutions of FeSO4 (20mM) were made justbefore use in distilled water.

Statistical analysis

The results are expressed as the mean7standard error (SEM).Data were determined statistically by analysis of variance, i.e.,two and three way ANOVA, followed by univariate analysis andDuncan’s multiple range tests when appropriate.

with lines) TBARS production in rat’s liver homogenate at different pH. The values

n=5–7). Different letters show a significant difference from basal group at po0.05.

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W. Hassan et al. / Pathology – Research and Practice 206 (2010) 357–360 359

Results and discussion

The results from the present study revealed that basal andFe(II)—induced TBARS production significantly increased bydecreasing pH, and this effect was more pronounced at pH 5.4and 6.4 (po0.05) (Fig. 1). The pH-dependent TBARS productioncan be explained by the fact that the affinity of transferrin for irondecreases under acidic conditions, leading to dissociation of ironfrom transferrin and other proteins like ferritin and lactoferrin [1].The mobilized iron Fe (II) can interact with enzymatically and/ornon-enzymatically generated superoxide (O2

d–1) (Haber–Weissreaction) and/or hydrogen peroxide (H2O2) (Fenton reaction) [7]producing reactive oxygen species. To support the mechanismand to explore the involvement of Fe (II) especially at low pH, weadded iron chelator, i.e., desferoxamine, DFO (1 mM), in theabsence of extracellular-added iron at acidic values. The additionof DFO inhibited TBARS production at acidic pH values, indicatingthe Fe (II) release and its participation in lipid peroxidationprocesses (data now shown). The acidic pH not only releases ironfrom ‘‘safe’’ sites [1] but also potentiates the pro-oxidant effect ofFe (II) as we observed from a significant increase in TBARSproduction at pH (6.8–5.4) (Fig. 1) (po0.05). These observationsare in accordance with our previous reports that low pH enhancesiron-mediated lipid peroxidation in rat’s tissue homogenate, aswell as phospholipids liposome from egg yolk [5,6].

Fig. 3. Effect of Ebselen (0–100mM) on basal (shaded bar) or Fe (II)-induced (bar with

expressed as nmol of MDA per gram of tissue. Data are expressed as means7S.E.M. (n

DPDS at concentrations of 50 and 100mmol significantly reducedboth basal and Fe (II)-induced TBARS at all studied pH values inliver homogenates (po0.05) (Fig. 2). Surprisingly, ebselen did notoffer any protection against TBARS production in any of the studiedpH in liver preparation (Fig. 3). The difference in the reactivities canbe explained by the fact that in the presence of excess thiol, thediselenide (RSeSeR) (DPDS) is reduced to the selenolate RSe�1 withthe formation of RSSeR (1). Nucleophilic attack of thiol on RSSeRproduces the disulfide and the 2nd selenolate RSe�1 ion

R0SH+RSeSeR 2 R0SSeR+RSe�1+H+ (1)

R0SSeR+R0SH 2 R0SSR0+RSe�1+H+ (2)

The two selenolate molecules produced in the above reactions(1) and (2) can react with H2O2 (organic hydroperoxides)producing selenenic acid reaction (3), which reacts with GSHproducing selenyl sulfide reaction (4) and regenerating a moleculeof diphenyl diselenide reaction (5)

2RSe�1+H2O2 - 2RSeOH (3)

2RSeOH+2GSH - 2RseSG (4)

2RSeSG+2GSH - RSeSeR (5)

lines) TBARS production in rat’s liver homogenate at different pH. The values are

=5–7).

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W. Hassan et al. / Pathology – Research and Practice 206 (2010) 357–360360

When the diselenide bond of diphenyl diselenide is disrupted, twoselenols reaction (1) and (2) can be yielded, differing from ebselenwhere only one selenol molecule is generated, improving thecatalytic reaction that is of particular significance to living cells.These mechanisms support our results that diphenyl diselenidehas higher anti-oxidant potential than ebselen, and we assumethat it can be attributed to selenol formation or to the number ofselenol molecules generated. Several investigators have proposedsome iron-oxygen complexes, such as the ferryl ion [2], perferrylion, [3] and Fe+ 2–O2

d–Fe+ 3 complex [10], for oxidizing species ofFe (II) which can initiate or potentiate the extent of the lipidperoxidation process by producing reactive oxygen species. Aplausible mechanism by which DPDS is conferring protectiveaction against Fe (II)-induced lipid peroxidation in these homo-genates is that DPDS could not only be operating as a free radicalscavenger, but may be interacting with Fe(II) or its oxidized forms.Contrary to our expectations, we did not find any alteration in theanti-oxidant activity (with respect to pH) of DPDS and ebselen inany of the pH values that were reviewed. Data from our laboratoryhave shown that the activity of organochalcogens are tissue-specific, and the activity of diphenyl diselenide is higher in brain,followed by kidney, while in liver homogenates, it offers onlymodest activity [11]. However, we did not observe any enhance-ment in the anti-oxidant potency of the compound with respect topH in rat’s liver homogenate (Fig. 2). However, the resultsobtained from these experiments to date support the notion thatDPDS is a redox-active compound and can protect againstoxidative stress at different pathophysiological pH values.

Acknowledgment

Waseem Hassan is a beneficiary of the TWAS-CNPq DoctoralFellowship Program. The financial support of TWAS, CAPES SAUX,CNPq, VITAE, and FAPERGS is also acknowledged.

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