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Journal of Trace Elements in Medicine and Biology 23 (2009) 324–333

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TOXICOLOGY

Involvement of non-enzymatic antioxidant defenses in the protective effect

of diphenyl diselenide on testicular damage induced by cadmium in mice

Ricardo Brandaoa, Francielli Weber Santosb, Renata Oliveiraa, Silvane Souza Romanc,Cristina Wayne Nogueiraa,�

aDepartamento de Quımica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria,

Santa Maria, CEP 97105-900, RS, BrazilbCentro de Ciencias da Saude de Uruguaiana, Universidade Federal do Pampa, UNIPAMPA, Uruguaiana,

CEP 97500-009, RS, BrazilcDepartamento de Ciencias da Saude, Universidade Regional Integrada do Alto Uruguai e das Missoes,

Campus de Erechim, CEP 99700-000, Erechim, RS, Brazil

Received 20 June 2008; accepted 21 June 2009

Abstract

The involvement of non-enzymatic antioxidant defenses in the protective effect of diphenyl diselenide (PhSe)2 ontesticular damage caused by cadmium in mice was investigated. Mice received a single dose of CdCl2 (5mg/kg,intraperitoneally). Thirty minutes after the CdCl2 injection, they received a single oral dose of (PhSe)2 (400 mmol/kg).Twenty-four hours after CdCl2 administration, blood samples were collected and mice were killed and had their testesdissected. Parameters in plasma (aspartate (AST) and alanine (ALT) aminotransferases and lactato dehydrogenase(LDH) activities as well as creatinine levels) were determined. The activity of d-aminolevulinate dehydratase (d-ALA-D), the levels of thiobarbituric acid-reactive substances (TBARS), ascorbic acid and nonprotein thiols (NPSH) andhistological analysis were determined in collected samples. Results demonstrated that (PhSe)2 protected againsttoxicity induced by CdCl2 on d-ALA-D activity, ascorbic acid and NPSH levels. (PhSe)2 protected against the increasein plasma AST, ALT and LDH activities caused by CdCl2. Testes of mice exposed to CdCl2 showed markedhistopathological alterations that were ameliorated by administration of (PhSe)2. (PhSe)2 protected against toxicityinduced by CdCl2 in testes of mice. Ascorbic acid and NPSH, non-enzymatic antioxidant defenses, are involved in theprotective effect of (PhSe)2 against testicular damage caused by CdCl2 in mice.r 2009 Elsevier GmbH. All rights reserved.

Keywords: Cadmium; Ascorbic acid; Testicular damage; Selenium; NPSH

Introduction

Cadmium is a nonessential element widely used inindustry that is also found as an important side

ee front matter r 2009 Elsevier GmbH. All rights reserved.

mb.2009.06.006

ing author. Tel.: +55 55 3220 8140;

0 8978.

ess: criswn@quimica.ufsm.br (C.W. Nogueira).

contaminant in agricultural products [1]. Cadmiumcontamination of soil and water has raised concernbecause this metal is bio-accumulated in the upper levelsof the food chain, including humans, in which itsbiological half-life is about 20 years [1]. Social practices,such as cigarette smoking, increase cadmium intake inhumans [2]. Given its current rate of release to theenvironment, cadmium content in the human body is

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likely to increase in the future [1]. This, in turn, couldlead to a higher incidence of cadmium-related diseasesincluding osteoporosis, nephrotoxicity, pulmonary em-physema and liver dysfunction [3–5].

The principal toxic endpoints of cadmium areconsidered to be renal dysfunction, hepatic injury,reproductive damage and cancer [6,7]. In male experi-mental animals, cadmium has been shown to affecttestes weight, to cause histopathological lesions, whichresult in reduced sperm counts and impaired spermmotility, and to adversely affect fertility [8–10]. Variousmechanisms have been suggested to explain cadmium-induced cellular toxicity. The alteration in antioxidantdefense systems and the production of reactive oxygenspecies (ROS) have been related to the toxicity inducedby cadmium [11]. In accordance, Oteiza et al. [12] havedemonstrated that ROS are involved in cadmium-induced testicular damage. ROS enhance lipid perox-idation, deplete antioxidant system, cause DNA damageas well as alter gene expression and apoptosis [13]. Yanget al. [14] have reported that cadmium induces DNAsingle-strand breaks in Leydig cells in vitro. Moreover,cadmium has a profound effect on sex organ weight,which is the primary indicator of possible alteration inandrogen status [15]. Cadmium can also directly inhibitprimary Leydig cell steroidogenic function, reducingtestosterone levels [16]. Evidence in the literature hasshown an increase in cadmium accumulation in thehypothalamus, pituitary and testis and a decrease inplasma levels of follicle-stimulating hormone in rats,suggesting a possible effect of cadmium on thehypothalamic–pituitary–testicular axis [17].

Many defense mechanisms are implicated in amelior-ating cadmium-induced oxidative damage. Amongthem, antioxidants such as ascorbic acid, a-tocopheroland selenium play a role as free-radical scavengers[18,19]. Accordingly, it was reported that testes could beprotected from the toxic effects of cadmium remarkablyby mainly antioxidant treatments [20].

In this way, diphenyl diselenide (PhSe)2, an organo-selenium compound, was as effective in restoringoxidative damage induced by cadmium in testes of miceas the chelating compounds [9,10]. (PhSe)2 is anantioxidant studied by our research group [21] andothers [22]. Besides antioxidant activity, differentpharmacological properties have been attributed to(PhSe)2, among them antiulcer [23], anti-inflammatory,antinociceptive [24] and hepato-protection [25].

In the present study the involvement of non-enzy-matic antioxidant defenses in the protective effect of(PhSe)2, administered by oral route, on testiculardamage induced by cadmium in mice was investigated.Thereby, we evaluated the effect of cadmium on d-ALA-D activity, lipid peroxidation, ascorbic acid and NPSHlevels in testes of mice. Biochemical parameters thatindicate tissue damage such as plasma AST, ALT and

LDH activities, creatinine levels as well as histologicalanalysis were performed.

Materials and methods

Chemicals

Cadmium chloride (CdCl2) was obtained from Merck(Darmstadt, Germany). d-Aminolevulinic acid (d-ALA)and p-dimethylaminobenzaldehyde were purchasedfrom Sigma (St. Louis, MO, USA). Diphenyl diselenide(PhSe)2 was prepared according to Paulmier [26].Analysis of the 1H NMR and 13C NMR spectra showedanalytical and spectroscopic data in full agreement withits assigned structure. The chemical purity of (PhSe)2(99.9%) was determined by GC/HPLC. All otherchemicals were of analytical grade and obtained fromstandard commercial suppliers. (PhSe)2 was dissolved incanola oil.

Animals

Male adult Swiss albino mice (30–35 g) from our ownbreeding colony were used. The animals were kept inseparate animal rooms, on a 12 h light/dark cycle, at atemperature of 2272 1C, with free access to food andwater. This study was approved by the Ethics andAnimal Welfare Committee of Universidade Federal deSanta Maria.

Exposure

Mice received a single injection of CdCl2 intraper-itoneally (i.p.) (5mg/kg body weight) (dissolved in salineat 0.5mg/mL) [9,10]. Thirty minutes after the injectionof CdCl2, mice received a single oral dose of 400 mmol/kg (PhSe)2 (dissolved in canola oil) [24].

The protocol of mice treatment is given as follows:Group 1: saline (i.p.)+canola oil (p.o.)Group 2: 5.0mg/kg CdCl2 (i.p)+canola oil (p.o.)Group 3: saline (i.p.)+400 mmol/kg (PhSe)2 (p.o.)Group 4: 5.0mg/kg CdCl2 (i.p)+400 mmol/kg (PhSe)2

(p.o.)Twenty-four hours after CdCl2 injection, the blood

samples were collected directly from the ventricle of theheart in anesthetized animals. Subsequently, mice wereeuthanized and testes were removed. The tissue sampleswere homogenized in 50mM Tris/HCl, pH 7.5 (1/10,w/v) and centrifuged at 2400g for 15min. The low-speedsupernatants (S1) were separated and used for biochem-ical analyses.

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Lipid peroxidation

TBARS levels, a measure of lipid peroxidation, weredetermined as described by Ohkawa et al. [27]. Analiquot (200 mL) of S1 was incubated at 95 1C for 2 h.The absorbance was read at 532 nm and the dataexpressed as nmol malondialdehyde (MDA)/mg protein.

Nonprotein thiols (NPSH)

NPSH levels were determined by the method ofEllman [28]. This method is simple, fast, direct and anideal method for determining the content of �SH inpure protein solution. To determine NPSH of testes,an aliquot of S1 (500 mL) was mixed (1:1) with 10%trichloroacetic acid. After centrifugation, an aliquot(200 mL) of supernatant was added in a 1M potassiumphosphate buffer (750 mL), pH 7.4 and 10mM 5,50-dithio-bis (2-nitrobenzoic acid) (DTNB) (50 mL). Thecolor reaction was measured at 412 nm. NPSH levelswere expressed as mmol NPSH/g tissue.

Ascorbic acid

Ascorbic acid determination was performed asdescribed by Jacques-Silva et al. [29]. Protein (testes)was precipitated in 10 volumes of a cold 4% trichlor-oacetic acid solution. An aliquot of sample (300 mL) in afinal volume of 1mL of the solution was incubated at38 1C for 3 h, then 1mL H2SO4 65% (v/v) was added tothe medium. The reaction product was determined usingcolor reagent containing 4.5mg/mL dinitrophenyl hy-drazine and CuSO4 (0.075mg/mL) and the dataexpressed as mg ascorbic acid/g tissue.

Catalase activity

Catalase activity was assayed spectrophotometricallyby the method of Aebi [30], which involves monitoringthe disappearance of H2O2 in the S1 presence at 240 nm.Enzymatic reaction was initiated by adding an aliquot of20 mL of S1 and the substrate (H2O2) to a concentrationof 0.3mM in a medium containing 50mM phosphatebuffer, pH 7.0. The enzymatic activity was expressed inUnits (one Unit decomposes 1 mmol of H2O2/min at pH7 at 25 1C).

d-ALA-D activity

Persuasive evidence has indicated that d-ALA-D isextremely sensitive to the presence of pro-oxidant agents[31], which oxidize SH groups essential for enzymeactivity. d-ALA-D activity was assayed according to themethod of Sassa [32] by measuring the rate of productporphobilinogen (PBG) formation except that 45mM

sodium phosphate buffer and 2.2mM d-ALA were used.An aliquot of 50 mL of S1 was incubated at 37 1C for 2 h.The reaction product was determined using modifiedEhrlich’s reagent at 555 nm. d-ALA-D activity wasexpressed as nmol PBG/mg protein/h.

AST, ALT and LDH activities and creatinine levels

Plasma AST and ALT activities were determinedusing commercial Kits (LABTEST, Diagnostica S.A.,Minas Gerais, Brazil) and the data expressed asInternational Units IU/L.

Plasma LDH activity was monitored spectrophoto-metrically by the rate of increase in absorbance at340 nm at 30 1C resulting from formation of NADH[33]. The assay medium contained 21mM glycine-KOHbuffer (pH 10), 0.25mM NAD+ and 25 mL of plasma.The mixture was pre-incubated for 3min, and thereaction was started by adding neutralized lactic acid(pH 6.8) to provide a final concentration of 50mM. Thereaction was linear for up to 2min. Plasma LDHactivity was expressed as mmol/mL/min.

Renal function was analysed using a commercial Kit(LABTEST, Diagnostica S.A., Minas Gerais, Brazil) bydetermining plasma creatinine.

Histological analysis

At euthanasia, all mice were subjected to a thoroughnecropsy evaluation. Histological analysis was per-formed from three animals per group. Picture of oneanimal per group was randomly selected from thephotos of three animals per group. Organ weight fortestes was recorded, and tissues were saved and fixed in10% formalin. For light microscopy examination,tissues were embedded in paraffin, sectioned at 5 mmand stained with hematoxylin and eosin.

Protein determination

Protein was measured by the method of Bradford [34]using bovine serum albumin as standard.

Statistical analysis

Data are expressed as mean7S.E.M. Statisticalanalysis was performed to compare treated group withrespective control groups using two-way analysis ofvariance (ANOVA), followed by Duncan’s multiplerange test when appropriate. Main effects are presentedonly when the higher second order interaction was non-significant. Values of po0.05 were considered statisti-cally significant.

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0

2

4

6

8

10

µmol

/g ti

ssue

a

ab

R. Brandao et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 324–333 327

Results

Lipid peroxidation

Two-way ANOVA of testicular TBARS data yieldeda significant main effect of CdCl2 (po0.001) and (PhSe)2(po0.028) (Fig. 1). Post hoc comparisons demonstratedthat mice exposed to CdCl2 showed an increase (about5.0 times) in TBARS levels when compared to those ofthe control group. (PhSe)2 was ineffective in ameliorat-ing the increase in TBARS levels caused by CdCl2 intestes of mice.

Control CdCl2 (PhSe)2 CdCl2 + (PhSe)2

Fig. 2. Effect of (PhSe)2 on testicular NPSH levels of mice

exposed to CdCl2. Data are reported as mean7S.E.M. from

six to eight animals per group. aDenotes po0.05 as compared

to the control group (two-way ANOVA/Duncan). bDenotes

po0.05 as compared to the CdCl2 group (two-way ANOVA/

Duncan).

224

280

NPSH levels

A significant CdCl2� (PhSe)2 interaction on testicularNPSH levels was observed (F1,35 ¼ 8.36, po0.006)(Fig. 2). CdCl2 exposure caused a reduction (�59%) inNPSH levels in testes when compared to those found inthe control group. (PhSe)2 was effective in amelioratingNPSH levels reduced by CdCl2 exposure, but the levelsof NPSH reached only 54% of control levels.

0

56

112

168

µg A

A/g

tiss

ue

a

ab

Control CdCl2 (PhSe)2 CdCl2 + (PhSe)2

Fig. 3. Effect of (PhSe)2 on testicular ascorbic acid levels of

mice exposed to CdCl2. Data are reported as mean7S.E.M.

from six to eight animals per group. aDenotes po0.05 as

Ascorbic acid levels

Two-way ANOVA of ascorbic acid data yielded asignificant CdCl2� (PhSe)2 interaction (F1,36 ¼ 11.26,po0.002) (Fig. 3). Post hoc comparisons demonstratedthat mice exposed to CdCl2 presented a reduction(�62%) in ascorbic acid levels when compared withthose of the control group. (PhSe)2 administration waspartially effective in ameliorating the reduction ofascorbic acid levels caused by CdCl2.

0

25

50

75

100

125

nmol

MD

A/m

g pr

otei

n

aa

Control CdCl2 (PhSe)2 CdCl2 + (PhSe)2

Fig. 1. Effect of (PhSe)2 on testicular TBARS levels of mice

exposed to CdCl2. Data are reported as mean7S.E.M. from

six to eight animals per group. aDenotes po0.05 as compared

to the control group (two-way ANOVA/Duncan).

compared to the control group (two-way ANOVA/Duncan).bDenotes po0.05 as compared to the CdCl2 group (two-way

ANOVA/Duncan).

Catalase activity

Catalase activity remained unaltered in mice exposedto CdCl2 or (PhSe)2 (data not shown).

d-ALA-D activity

Two-way ANOVA of d-ALA-D activity yielded asignificant CdCl2� (PhSe)2 interaction (F1,37 ¼ 5.69,po0.022) (Fig. 4). Post hoc comparisons demonstratedthat mice exposed to CdCl2 presented a decrease(�46%) in d-ALA-D activity when compared to thoseof the control group. Oral treatment with (PhSe)2

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protected against inhibition of d-ALA-D caused byCdCl2 in testes of mice.

AST, ALT and LDH activities

A significant main effect of CdCl2 (po0.001) and(PhSe)2 (po0.037) in plasma AST activity was found(Table 1). Results demonstrated that CdCl2 exposurecaused an increase (�83%) in the enzyme activity whencompared to the control group. (PhSe)2 reduced ASTactivity increased by CdCl2 exposure, but the activity ofAST reached 152% of control levels.

Two-way ANOVA of plasma ALT activity yielded asignificant main effect of CdCl2 (po0.004) and (PhSe)2(po0.009) (Table 1). Mice exposed to CdCl2 presentedan increase (�55%) in ALT activity when comparedwith those of the control group. (PhSe)2 stopped theincrease in ALT activity induced by CdCl2 in testes ofmice.

Two-way ANOVA of plasma LDH activity showed asignificant main effect of CdCl2 (po0.001) and (PhSe)2(po0.013) (Table 1). Mice exposed to CdCl2 showed anincrease (�80%) in LDH activity when compared to

0

4

8

12

16

20

nmol

PB

G/m

g pr

otei

n/ho

ur

a

b

Control CdCl2 (PhSe)2 CdCl2 + (PhSe)2

Fig. 4. Effect of (PhSe)2 on testicular d-ALA-D activity of

mice exposed to CdCl2. Data are reported as mean7S.E.M.

from six to eight animals per group. aDenotes po0.05 as

compared to the control group (two-way ANOVA/Duncan).bDenotes po0.05 as compared to the CdCl2 group (two-way

ANOVA/Duncan).

Table 1. Effect of (PhSe)2 on AST, ALT and LDH activities in pl

AST (IU/L)

Control 93.0673.66

CdCl2 169.8877.68a

(PhSe)2 87.5775.76

CdCl2+(PhSe)2 141.08711.66a,b

Data are mean7S.E.M. from six to eight animals in each group.aDenotes po0.05 as compared to the control group (two-way ANOVA/DbDenotes po0.05 as compared to the CdCl2 group (two-way ANOVA/Du

those of the control group. (PhSe)2 ameliorated theincrease in LDH activity caused by CdCl2 in testes ofmice.

Creatinine levels

Creatinine levels remained unaltered in mice exposedto CdCl2 or (PhSe)2 (data not shown).

Histological evaluation

Testes from animals belonging to the control and(PhSe)2 groups revealed a normal aspect of theseminiferous tubules (Figs. 5A and B, respectively).Normal features of spermatogenesis, spermatogonia andspermatid were found in testes of mice from control and(PhSe)2 groups (Figs. 6A and B, respectively).

Testes of mice exposed to CdCl2 showed intenseinterstitial edema and marked vascular congestion(Fig. 5C). The evaluation of the seminiferous tubulesof mice from the CdCl2 group showed spermatogonia,spermatocite, spermatid and Leydig cells with intensecytoplasmic eosinophilia and the presence of erythro-cytes (Figs. 6C and D).

Evaluation of testes from mice exposed to CdCl2 and(PhSe)2 showed the seminiferous tubules with histologicalcharacteristics more preserved than those from the CdCl2group. Tubules displayed moderate edema (Fig. 5D) andmoderate cytoplasmic eosinophilia of spermatogonia,spermatocite, spermatids and Leydig cells (Fig. 6E).

Discussion

Cadmium is an important heavy metal widely used inNiCd batteries, metal plating, pigments, plastics andalloys [35]. This metal stimulates free-radical produc-tion, resulting in oxidative deterioration of lipids,proteins and DNA, as well as initiating variouspathological conditions in humans and animals [36].Therefore, protection against the acute actions ofcadmium can be achieved through the antioxidantsystems [37].

asma of mice exposed to cadmium.

ALT (IU/L) LDH (mmol/ml/min)

29.3972.71 219.45723.8

45.5374.26a 395.19730.06a

19.8671.31 188.64724.62

30.8576.46b 266.57729.78b

uncan).

ncan).

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Fig. 5. Microscopic analysis of mouse testicular structure. (A) Control group shows normal testicular structure, with normal aspect

of the seminiferous tubules (H&E, 100� ), (B) (PhSe)2 group shows normal testicular structure (H&E, 100� ), (C) CdCl2 group

shows intense interstitial edema (white arrow) and marked vascular congestion (black arrow) (H&E, 100� ), (D) CdCl2 and (PhSe)2group shows tubules with histological structure more preserved than the CdCl2 group, with moderate edema (white arrow) (H&E,

100� ).

R. Brandao et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 324–333 329

In the present study, we demonstrate the involvementof non-enzymatic antioxidant defenses (NPSH andascorbic acid) in the protective effect of (PhSe)2,administered by oral route, on testicular damageinduced by cadmium in mice. On the one hand, the factthat catalase was not modified by exposure to (PhSe)2could reinforce the supposition that the mechanisminvolved in its protective effect is, mainly, by acting inthe non-enzymatic antioxidant defense system. On theother hand, the potential synergistic contribution ofenzymatic antioxidant defenses in the protective effect of(PhSe)2 should be considered. This contribution issupported by the following reasons: (i) catalase is onlyone of the many antioxidant enzymes; (ii) selenium is anessential element for antioxidant selenoenzymes, such asglutathione peroxidase and thioredoxin reductase; and(iii) (PhSe)2 has been reported to possess glutathioneperoxidase-like activity in vitro [21] and thereforemay exert the protective effects through its enzymaticaction.

The testicular damage was evidenced in this study byan increase in lipid peroxidation, an inhibition of ALA-D activity, a reduction in NPSH and ascorbic acidlevels, markers of oxidative stress, and it was confirmedby marked alterations in histopathology. Testes of miceexposed to CdCl2 showed intense interstitial edema,accentuated vascular congestion and spermatogonia,

spermatocite and spermatids, cells responsible forspermatogenesis, with intense cytoplasmic eosinophilia[38]. Moreover, mice exposed to CdCl2 showed anintense cytoplasmic eosinophilia in Leydig cells, whichare responsible for testosterone secretion [39]. Theeosinophilic cytoplasm may indicate a future necrosisbecause it is a sign that the cell fails to make the meiosisand, consequently, does not produce sperm fromspermatids [40]. The presence of erythrocytes in testesof mice from the CdCl2 group suggests a hemorrhagicprocess. Therapy with (PhSe)2 was effective in amelior-ating histological alterations caused by CdCl2 in testesof mice.

Stohs et al. [13] have reported that cadmium exposureincreases oxidative stress through the generation ofnoxious radicals such as superoxide anion radicals,hydroxyl radicals, nitric oxide and hydrogen peroxide.These radicals are transitory due to their high chemicalreactivity and thus can stimulate lipid peroxidation anddeleterious modification of complex lipoprotein assem-blies in biomembranes and cellular dysfunction [13]. Inaccordance, the authors have demonstrated that animalsexposed to cadmium showed increased lipid peroxida-tion in different tissues [10,41]. As pointed out in theintroduction section, another mechanism involved incadmium toxicity is the reduction in non-enzymaticantioxidant defenses [41].

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**

*

Fig. 6. Microscopic analysis of a detailed structure of mouse seminiferous tubules. (A) Control group shows normal seminiferous

tubules structure, with normal aspect of the spermatogonia (white arrow), spermatocyte (black arrow) and spermatids (arrowheads)

(H&E, 400� ), (B) (PhSe)2 group shows normal seminiferous tubules structure (H&E, 400� ), (C) CdCl2 group shows the

spermatogonia (white arrow), spermatocyte (black arrow) and spermatids (arrowheads) cells with intense cytoplasmic eosinophilia

and the absence of elongating spermatids (arrowheads) (H&E, 400� ), (D) CdCl2 group shows intense cytoplasmic eosinophilia in

the Leydig cells (white arrow) and the presence of erythrocytes (*) in the interstitial space (H&E, 400� ), (E) CdCl2+(PhSe)2 group

shows the seminiferous tubules with histological characteristics more preserved than those of the CdCl2 group, with moderate

cytoplasmic eosinophilia of spermatogonia (white arrow), spermatocyte (black arrow), spermatids (arrowheads) and Leydig cells (*)

(H&E, 400� ).

R. Brandao et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 324–333330

In agreement with the above reported point, we foundthat CdCl2 exposure caused an increase in lipidperoxidation, a depletion of NPSH and ascorbic acidlevels (non-enzymatic antioxidant defenses) and aninhibition of ALA-D activity in testes of mice. It isknown that the oxidative effect of cadmium is indirectand based mainly on the depletion of SH-group-containing compounds [41]. Evidence has been gatheredabout the important role played by glutathione (GSH),the major NPSH in tissues, in the detoxification andprotection from oxidative injuries. GSH acts as a redoxbuffer to prevent oxidative damage due to its reducing

and nucleophilic properties, it behaves as a free-radicalscavenger and also helps in regenerating other antiox-idants [42]. Therefore, the depletion of NPSH levels inmice exposed to CdCl2 demonstrated in this study couldcontribute to oxidative stress in testes of mice. Inaddition, GSH is required to recycle ascorbic acid fromdehydroascorbic acid (DHA) (oxidized form of ascorbicacid) [43]. Thus, the depletion on NPSH levels may berelated to the reduction in ascorbic acid levels found inmice exposed to CdCl2.

d-ALA-D is a sulfhydryl enzyme very sensitive tosituations in which oxidative stress plays a role [44]. In

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the current study, testes of mice exposed to CdCl2showed oxidative stress characterized by an increase inlipid peroxidation and the depletion of NPSH andascorbic acid levels, which could justify d-ALA-Dinhibition. The inhibition of d-ALA-D activity couldbe also linked to the oxidation of –SH groups located atthe active center of this enzyme [45]. One can alsohypothesize that cadmium causes a Zn2+ displacementleading to enzyme inhibition since the mammalian d-ALA-D is a metalloenzyme that requires Zn2+ formaximal catalytic activity [46].

Although (PhSe)2 treatment was effective in amelior-ating the depletion of NPSH and ascorbic acid levels aswell as the inhibition of ALA-D activity caused byCdCl2, lipid peroxidation remained unaltered. Oneexplanation for this fact is that the increase of NPSHand ascorbic acid levels was not enough to counteractlipid peroxidation in (PhSe)2- and CdCl2-exposed mice.In fact, the levels of non-enzymatic antioxidant defensesin mice treated with (PhSe)2 could indicate an adaptivemechanism to overwhelm oxidative stress.

The results demonstrate also that CdCl2 exposure tomice caused an increase both in the activities of ASTand ALT, markers of hepatic damage, and in LDHactivity. In accordance, an increase in plasmaticactivities of AST and ALT in animals exposed to CdCl2has been reported [47]. (PhSe)2 administration waseffective in protecting against the increase in AST,ALT and LDH activities caused by CdCl2 exposure tomice.

Organic forms of selenium have been suggested aspossible antioxidant agents [21]. In fact, studies haveshown that (PhSe)2 protects against cadmium-inducedtoxicity through its antioxidant action [10,47]. More-over, the formation of a complex between (PhSe)2 andcadmium has been also reported as a possible mechan-ism of (PhSe)2 action [10,47]. In the present study weclearly demonstrate that (PhSe)2 reduced damage causedby CdCl2 in testes of mice and the involvement of non-enzymatic antioxidant defenses (ascorbic acid andNPSH) in (PhSe)2 protective effect. In addition,selenium is a structural component of several enzymeswith physiological antioxidant properties, includingglutathione peroxidase (GPx) [21]. Therefore, thepotential contribution of increased GPx activity in theprotective effects of (PhSe)2 cannot be excluded. Thisidea is supported by elevated concentrations of glu-tathione, the major NPSH, found in the present study.Glutathione is the substrate of GPx, which is acomponent of the enzymatic antioxidant defenses. Inaccordance, (PhSe)2 has been reported to possessglutathione peroxidase-like activity in vitro [21].

Importantly, (PhSe)2 did not alter markers of hepatic(AST and ALT activities), renal (creatinine levels) andgeneral tissue damage (LDH activity), suggesting thatthe oral administration of (PhSe)2 was non-toxic, at

least, at the dose tested in this experimental protocol. Itis noteworthy to point out that the oral route ofadministration shows numerous advantages when com-pared to others (subcutaneous, intraperitoneal, intrave-nous, etc).

In conclusion, oral therapy with (PhSe)2 reducedacute toxicity caused by CdCl2 in testes of mice.Ascorbic acid and NPSH, non-enzymatic antioxidantdefenses, are involved in the protective effect of (PhSe)2against testicular damage caused by CdCl2 in mice.Further researches have to be conducted to provideinformation for the possible use of (PhSe)2 in humans inthe future.

Acknowledgements

The financial support by FAPERGS, CAPES andCNPq is gratefully acknowledged.

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