Toxicology in Vitro 27 (2013) 59–70

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Toxicology in Vitro

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Mitochondrial electron transfer chain complexes inhibition by differentorganochalcogens

Robson L. Puntel a,⇑, Daniel H. Roos b, Rodrigo Lopes Seeger b, João B.T. Rocha b,⇑a Universidade Federal do Pampa – Campus Uruguaiana BR-472 Km 7, Uruguaiana 97500-970, RS, Brazilb Departamento de Química, Centro de Ciências 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 a b s t r a c t

Article history:Received 12 June 2012Accepted 13 October 2012Available online 25 October 2012

Keywords:Mitochondrial dysfunctionOrganochalcogens toxicityThiol oxidation

0887-2333 � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.tiv.2012.10.011

⇑ Corresponding authors. Tel.: +55 3413 4321 (R.L.Rocha).

E-mail addresses: robson_puntel@yahoo.com.br (Ryahoo.com.br (D.H. Roos), rodrigo.see.ger@hotmail.cyahoo.com.br (J.B.T. Rocha).

Open access under the El

Mitochondrial dysfunction plays a pivotal role in the cell toxicology and death decision. The aim of thepresent study was to investigate the effect of three organocompounds (ebselen [Ebs], diphenyl diselenide[(PhSe)2] and diphenyl ditelluride [(PhTe)2]) on mitochondrial complexes (I, II, I–III, II–III and IV) activityfrom rat liver and kidney to determine their potential role as molecular targets of organochalcogens. Allstudied organochalcogens caused a statistically significant inhibition of the mitochondrial complex Iactivity. Ebs and (PhTe)2 caused a statistically significant inhibition of the mitochondrial complex II activ-ity in both hepatic and renal membranes. Hepatic mitochondrial complex II activity was practicallyunchanged by (PhSe)2, whereas it significantly inhibited renal complex II activity. Mitochondrial complexIV activity was practically unchanged by the organochalcogens. Furthermore, organochalcogens inhibitedthe mitochondrial respiration supported by complex I or complex II substrates. The inhibitory effect ofEbs, (PhSe)2 and (PhTe)2 on mitochondrial complex I was prevented by NADH, but it was not preventedby catalase (CAT) and/or superoxide dismutase (SOD). Additionally, the organochalcogens-induced inhi-bition of complex I and II was completely reversed by reduced glutathione (GSH). In conclusion, Ebs,(PhSe)2 and (PhTe)2 were more effective inhibitors of renal and hepatic mitochondrial complex I thancomplex II, whereas complexes III and IV were little modified by these compounds. Taking into accountthe presented results, we suggest that organochalcogen-induced mitochondrial complexes I and II inhi-bition can be mediated by their thiol oxidation activity, i.e., Ebs, (PhSe)2 and (PhTe)2 can oxidize criticalthiol groups from mitochondrial complexes I and II. So, mitochondrial dysfunction can be considered animportant factor in the toxicity of Ebs, (PhSe)2 and (PhTe)2.

� 2012 Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction

Living organisms use a series of integral membrane proteincomplexes for energy conversion and ATP synthesis (Hatefi,1985). In addition to their crucial role in energy production andmetabolic pathways, the mitochondrial complexes also play keyroles in integrating cell death stimuli and executing the apoptoticprogram (Navarro and Boveris, 2007). Accordingly, several humandiseases, such as Alzheimer’s disease, Friedreich’s ataxia, familialamyotrophic lateral sclerosis, and Huntington’s disease, are associ-ated with mitochondrial electron transport chain inhibition, en-ergy metabolism impairment and oxidative stress (Beal, 1998;Nicholls and Budd, 2000). Additionally, biochemical studies indi-cate a decline of electron transport and in some bioenergetic activ-

Puntel), +55 3220 8140 (J.B.T.

.L. Puntel), danielvarzeano@om (R.L. Seeger), jbtrocha@

sevier OA license.

ities of mitochondria during aging and ischemia–reperfusion(Cadenas and Davies, 2000; Caspersen et al., 2005; Cortopassiand Wong, 1999; Hagen et al., 1998; Hauptmann et al., 2006;Navarro and Boveris, 2007; Nicholls, 2002; Saris and Eriksson,1995; Sastre et al., 2003). Thus, mitochondrial dysfunction can beassociated with different degenerative cellular processes.

Organoselenium and organotellurium compounds have beenextensively studied because of their potential antioxidant capacity(Arteel and Sies, 2001; Barbosa et al., 2006, 2008; de Bem et al.,2009; de Freitas et al., 2009; Hort et al., 2011; Moretto et al., 2007;Nogueira and Rocha, 2011; Parnham and Graf, 1991; Prauchneret al., 2011; Prigol et al., 2008; Puntel et al., 2007). In line with this,literature data have indicated that these compounds can provideprotective effect against lipid peroxidation induced by a variety ofpro-oxidants agents (Barbosa et al., 2006, 2008; Moretto et al.,2007; Nogueira and Rocha, 2010, 2011; Parnham and Graf, 1991;Puntel et al., 2007; Rossato et al., 2002). The antioxidant activity ofthese organochalcogens has been ascribed either to their glutathi-one peroxidase-like activity (Maiorino et al., 1988; Santos et al.,2005; Sies, 1993, 1995) or to the fact that they can be substrates

60 R.L. Puntel et al. / Toxicology in Vitro 27 (2013) 59–70

of mammalian thioredoxin reductase (de Freitas and Rocha, 2011;Sausen de Freitas et al., 2010; Zhao and Holmgren, 2002; Zhaoet al., 2002). Thus, in order to exert antioxidant properties, theselenium containing compounds have to be metabolized toselenol/selenolate intermediates, a reaction which can be accom-plished via reduction of the Se moiety by different types of thiols(Nogueira and Rocha, 2011; Wendel et al., 1984) (Scheme 1). Fororganotellurium compounds, it has been postulated that the antiox-idant activity is linked to changes in the oxidation state of the Teatom (Te(II) M Te(IV)) (Engman et al., 1995; Leonard et al., 1996;You et al., 2003).

Thus, the thiol-peroxidase or thioredoxin-thiol-peroxidase-likeactivity of organochalcogens (Nogueira and Rocha, 2011; Sausende Freitas et al., 2010; Zhao and Holmgren, 2002; Zhao et al.,2002) can be of biological and therapeutic significance via artificialmodulation of the cellular levels of peroxides. However, the exces-sive oxidation of thiols, including those in mitochondrial mem-branes, by organochalcogens without a concomitant reduction ofperoxides may be toxic to living cells (thiol-oxidation activity)(Nogueira and Rocha, 2011; Puntel et al., 2010) (Scheme 1). Ineffect, mitochondrial dysfunction caused by thiol oxidation is clo-sely related to the apoptotic cell death (Morin et al., 2003; Zhaoet al., 2006). Accordingly, the organochalcogens should be consid-ered as putative candidates for apoptotic cell death inducer viamitochondrial dysfunction, which may explain, at least in part,their pharmacological/toxicological action (Ardais et al., 2010;Nogueira and Rocha, 2010; Santos et al., 2009a,b). In line with this,recently our group showed that both Ebselen (Ebs) and diphenyldiselenide [(PhSe)2] induced mitochondrial dysfunction via inter-action with critical mitochondria thiols (Puntel et al., 2010).

Considering that mitochondrial complexes play a central role incellular metabolism and in the regulation of apoptotic cell death,we sought to determine whether these mitochondrial complexescould be considered molecular targets for the thiol-oxidation activ-ity of Ebs, (PhSe)2 or diphenyl ditelluride [(PhTe)2]. Specifically, our

Scheme 1. Thiol peroxidation and thiol oxidation cycle of dichalcogenides. In thetoxic pathway the formation of the selenol/tellurol is associated with a futileoxidation of low-molecular- (RSH) or protein-thiol groups (PSH) causing depletionof GSH levels and/or protein loss of function. In the therapeutic pathwayorganochalcogens decompose peroxides either as a substrate for TrxR or as amimic of GPx via the formation of the selenol/selenolate or tellurol/tellurate (PhXH/PhX-) intermediate (X can be Se or Te).

main objective in this study was to determine whether Ebs, (PhSe)2

and (PhTe)2 could cause mitochondrial complex(es) I, II, I–III, II–IIIand IV inhibition using mitochondrial membranes prepared fromrat liver and kidney. We also determined whether organochalco-gens could cause mitochondrial respiration inhibition using intactmitochondria in order to better understand their toxicological siteof action at the molecular level.

2. Materials and methods

2.1. Chemicals

Chemicals, including NADH, mannitol, rotenone, succinic acid,malonate, potassium cyanide (KCN), sucrose, HEPES and cyto-chrome c were obtained from Sigma Chemical Company (St Louis,MO, USA). All other reagents were commercial products of thehighest purity grade available.

2.2. Animals

Adult male Wistar rats (250–350 g) from our own breeding col-ony were used in this study. The animals were housed in plasticcages with water and food ad libitum, at 22–23 �C, 56% humidity,and 12 h light cycle. The diet of the rats containing (in g/100 g):52 carbohydrate, 20 crude protein, 5 fat, 6 crude fiber, 5 mineralsand 11 moisture. Diet contained 0.1 mg/kg of Se and 30 IU/kg ofvitamin E (for complete mineral and vitamin contents, see refer-ence (Puntel et al., 2010)). The protocol was approved by the Insti-tutional Animal Care and Use Committee of Federal University ofde Santa Maria (42/2010) and conducted in accordance with theGuide for the Care and Use of Laboratory Animals.

2.3. Isolation of rat liver and kidney mitochondria and mitochondrialmembranes preparation

Liver and kidney mitochondria were isolated in a solution con-taining 0.23 M mannitol, 0.07 M sucrose, 15 mM HEPES (pH 7.2) ata ratio of 1 g of tissue/9 mL of homogenization medium in a Potterhomogenizer with a Teflon pestle. The homogenate was centri-fuged at 700g for 10 min, and the supernatant centrifuged at8000g for 10 min to yield a mitochondria pellet that was washedonce in the same buffer. Mitochondrial protein concentrationwas adjusted to 20 mg/mL (Peterson, 1977) and the samples wereimmediately frozen and kept at �80 �C. Mitochondria were dis-rupted and homogenized by twice freezing and thawing and bypassage through 15/10 tuberculin needles to produce the mito-chondrial membrane preparation according to (Navarro et al.,2002) which were used to the mitochondrial complexes activityassay. In order to study the organochalcogens effect on mitochon-drial respiration (oxygen consumption measurements) intact mito-chondria were used.

2.4. Mitochondrial complexes activities assay

The activities of complexes I, I–III, II, II–III, and IV were deter-mined spectrophotometrically at 30 �C with mitochondrial mem-branes (0.5 mg/mL) suspended in 100 mM phosphate buffer (pH7.4) as previously described (Navarro et al., 2002, 2004) with minormodifications. The mitochondrial membranes were pre-incubatedin phosphate buffer in the presence of different organochalcogensconcentration (Ebs 0–50 lM; [(PhSe)2] 0–100 lM; [(PhTe)2] 0–100 lM, vehicle (DMSO), or the respective classical inhibitor (posi-tive controls) for 10 min. After pre-incubation, the reaction wasstarted with the addition of the corresponding substrate, exceptfor the experiments using reduced glutathione (GSH), as described

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below. The activities of mitochondrial complexes were carried outindependently at least 3–5 times (each series of experiment wasperformed in duplicate) by use of different biological samples(samples obtained from different animals).

For NADH:ubiquinone oxidoreductase (complex I) activity as-say, mitochondrial membranes (0.5 mg/mL) in 100 mM phosphatebuffer, were incubated with different organocompounds or rote-none (100 lM) for 10 min. The reaction was started after 10 minby adding NADH to a final concentration of 100 lM. The enzymaticactivity was determined, either in the absence or presence ofsuperoxide dismutase (SOD; 100 UI/mL) and/or catalase (CAT;100 UI/mL), following the decrease in absorbance at 340 nm during180 s. In order to study the efficacy of GSH to reverse the organoch-alcogens-induced complex I inhibition, the mitochondrial mem-branes were pre-incubated in phosphate buffer in the presence oforganochalcogens (Ebs 25 lM; [(PhSe)2] 50 lM; [(PhTe)2] 50 lMfor 10 min in the absence of GSH. Thereafter the membranes werewashed in phosphate buffer and centrifuged at 12,000g for 10 minat 4 �C to remove the organochalcogens. Then, the membraneswere incubated 5 min with GSH (500 lM; to allow the potentialGSH reversion of the organochalcogens-induced inhibition). After-ward the mitochondrial complex I activity was assayed as de-scribed above by determining NADH oxidation.

For NADH–cytochrome c (complexes I–III) activity assay, wecarried out the experiments using two different conditions. In thecondition 1, mitochondrial membranes were pre-incubated with200 lM NADH (as substrate), 1 mM KCN and with differentorganocompounds or 100 lM rotenone for 10 min (pre-incubationwith organocompounds in the presence of NADH). The reactionwas started after addition of 100 lM cytochrome c3 (oxidizedcytochrome). In the condition 2, mitochondrial membranes werepre-incubated with 100 lM cytochrome c3 (oxidized cytochrome),1 mM KCN and with different organocompounds or rotenone pre-incubated for 10 min (pre-incubation with organocompounds inthe absence of NADH). The reaction was then started by adding200 lM NADH to the reaction mixture. In both conditions, theenzymatic activity was determined at 550 nm (e = 19 mM�1 cm�1)during 120 s.

For succinate:ubiquinone oxidoreductase (complex II) activityassay, we carried out the experiments in two different conditions.In the condition 1, mitochondrial membranes in 100 mM phos-phate buffer were incubated with succinate 5 mM, differentorganocompounds or malonate 8 mM. In the condition 2,mitochondrial membranes were pre-incubated with differentorganocompounds or malonate 8 mM (pre-incubation in theabsence of succinate). After 10 min of pre-incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 1 mg/mL;condition 1) or MTT and succinate (5 mM; condition 2) were addedto reaction medium. The reaction was stopped 3 min after MTT byadding 1 mL of ethanol and the absorbance was determined at570 nm. In order to study the efficacy of GSH to reverse the organ-ochalcogens-induced complex II inhibition, the mitochondrialmembranes were pre-incubated in phosphate buffer in the pres-ence of organochalcogens (Ebs 25 lM; [(PhSe)2] 50 lM; [(PhTe)2]50 lM for 10 min (according condition 1) in the absence of GSH.After, the membranes were washed in phosphate buffer to removethe organochalcogens. Afterward, centrifugations at 12,000g for10 min at 4 �C, mitochondrial membranes were resuspended inphosphate buffer. Subsequently, mitochondrial membranes wereincubated with GSH (500 lM during 5 min and the mitochondrialcomplex II activity was assayed according to condition 1 describedabove (by adding MTT). Mitochondrial complex II activity was as-sessed by the conversion of the MTT dye to formazan. This assay isbased on the reduction of MTT to formazan by mitochondrialsuccinate dehydrogenase (SDH). Because selenol/telurol mightreduce MTT per se, we inactivated the succinate dehydrogenase

by heat (10 min at 100 �C) in order to discount the potential non-enzymatic reduction of MTT.

For succinate–cytochrome c reductase (complexes II–III) activ-ity assay, mitochondrial membranes (0.5 mg/mL) were supple-mented with succinate 5 mM as substrate and with 1 mM KCN,and incubated for 10 min with different organocompounds (incu-bation with organocompounds in the presence of succinate). Thereaction was started by adding 100 lM cytochrome c3 (oxidizedcytochrome). The enzymatic activity was determined at 550 nm(e = 19 mM�1 cm�1) during 120 s.

For cytochrome oxidase (complex IV) activity assay, mitochon-drial membranes (0.5 mg/mL) were incubated in 100 mMphosphate buffer for 10 min with different organochalcogens or10 mM KCN. The reaction was started after reduced cytochromec2 100 lM addition, and monitored during 180 s. The rate of cyto-chrome c2 oxidation was calculated as first-order reaction constantk per milligram protein.

2.5. Oxygen consumption measurements

Oxygen consumption was measured in an oxymeter fitted witha water-jacket Clark-type electrode (Oxytherm – Hansatech Instru-ments Ltd.). The intact isolated liver mitochondria (approximately0.5 mg/mL) were pre incubated during 10 min in the standard res-piration buffer (100 mM sucrose, 65 mM KCl, 10 mM K+-HEPESbuffer (pH 7.2), 50 lM EGTA, 400 lM MgCl2) either in the absenceor presence of organochalcogens (Ebs 25 lM; (PhSe)2 50 lM;(PhTe)2 50 lM) in order to mimic the same conditions used tomeasured mitochondrial complexes activity. The oxygen consump-tion measurements were determined in the presence of complex I(pyruvate/glutamate 2.5 mM each) or complex II (succinate 5 mM)substrates.

2.6. Synthesis of organochalcogens

(PhSe)2 was synthesized using the method previously described(Paulmier, 1986), (PhTe)2 according to Petragnani (Petragnani,1994) and Ebs as described by Engman (Engman, 1989). Solutionsof organochalcogens were prepared freshly in dimethylsulfoxide(DMSO) and the final concentration of DMSO in each tube was 3%.

2.7. Statistical analysis

Data were analyzed by one-way ANOVA followed by Duncansmultiple range test when appropriate. The data of Figs. 3(A andB) and 4(A–C) and the effect of the respective classical inhibitors(vs. control) were analyzed by Student’s t Test. Linear regressionanalysis was performed in order to evaluate the concentrationdependence effect of organochalcogens on mitochondrial com-plexes. A p < 0.05 value was considered statistically significant.

3. Results

3.1. Effect of organochalcogens in mitochondrial complex I activity

Statistical analysis indicated that Ebs, (PhSe)2 and (PhTe)2 sig-nificantly inhibited complex I activity from liver and kidney mito-chondria (Fig. 1A and B, respectively). The inhibitory effect wasconcentration dependent in liver membranes, as revealed by thelinear regression analysis (p < 0.05 for all studied organochalcogens).Ebs-induced complex I inhibition was statistically significant from5 lM onwards, while both (PhSe)2 and (PhTe)2 caused mitochon-drial complex I inhibition from 10 lM onwards (Fig. 1A and B).The IC50 (lM) values for inhibition by organochalcogens ofmitochondrial complex I activity are showed in Table 1. Rotenone

Fig. 1. Effect of organochalcogens on mitochondrial complex I activity. Mitochondria membranes were incubated (0.5 mg/mL) for 10 min at 30 �C in a medium containing100 mM phosphate buffer, pH 7.4, in the presence of indicated concentrations of organochalcogens (Ebs 0–50 M; [(PhSe)2] 0–100 lM; [(PhTe)2] 0–100 lM. Complex I activityis expressed as % of control (without organochalcogens addition). (A) Liver mitochondria membranes; (B) Kidney mitochondrial membranes. —N— (PhSe)2, —d— (PhTe)2,—j— Ebs, — + — Rotenone (100 lM). The control values (without organochalcogens) were: liver 100.31 ± 4.27; kidney 100.74 ± 6.51. Data are expressed as means ± SEM from5 independent experiments carried out in duplicate. ⁄p < 0.05 from respective control by Duncan’s multiple range test. #p < 0.05 from control by t test.

Table 1IC50 (lM) values for inhibition by organochalcogens of mitochondrial complex(es) activity.

Complex(es) Organochalcogen Tissue

Liver Kidney

Complex I Ebs 14.50 ± 2.03 12.41 ± 1.56(PhSe)2 31.09 ± 1.97 2.92 ± 0.67(PhTe)2 44.71 ± 2.38 3.88 ± 0.95

Complexes I–III Ebs 3.03 ± 0.51 26.37 ± 3.17(PhSe)2 ND ND(PhTe)2 ND ND

Condition 1 Condition 2 Condition 1 Condition 2Complex II Ebs 18.50 ± 1.43 28.50 ± 1.94 0.92 ± 0.07 16.70 ± 1.07

(PhSe)2 ND ND 2.12 ± 0.30 ND(PhTe)2 2.70 ± 0.19 16.01 ± 2.07 0.81 ± 0.03 11.70 ± 0.88

Complexes II–III Ebs 31.01 ± 1.44 2.61 ± 0.42(PhSe)2 ND 20.08 ± 1.70(PhTe)2 13.19 ± 1.60 3.93 ± 0.19

Complex IV Ebs ND ND(PhSe)2 27.43 ± 2.05 ND(PhTe)2 ND ND

ND: Not determined.

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(100 lM), a classical complex I inhibitor, caused a significant inhi-bition of the mitochondrial complex I activity (Fig. 1A–B).

3.2. Effect of organochalcogens in mitochondrial complexes I–IIIactivity

Fig. 2 shows that Ebs significantly inhibited the complexes I–IIIactivity from liver mitochondrial membranes from 10 lM on-wards, with maximal effect at 50 lM (Fig. 2A). The inhibitory effectof Ebs on renal mitochondrial complexes I–III activity was statisti-cally evident only at 50 lM (Fig. 2B). (PhSe)2 and (PhTe)2 did notchange the mitochondrial complexes I–III activity from liver(Fig. 2A) or kidney (Fig. 2B). The IC50 (lM) values for inhibitionby organochalcogens of mitochondrial complexes I–III activityare showed in Table 1.

In order to better understand the inhibitory effect of differentorganochalcogens in mitochondrial complexes I–III activity, wecarried out experiments using two different conditions. In brief,in the condition 1 the membranes were pre-incubated with theorganocompounds (at different concentrations) in the presence ofNADH and the reaction was started with cytochrome c3. In the

condition 2, the mitochondrial membranes were pre-incubatedwith different concentrations of oganocompounds and cytochomec3, and the reaction was started by NADH.

Under condition 1, Ebs (5 lM) significantly inhibited complexesI–III activity from liver (Fig. 3A), without affecting renal complexesI–III activity (Fig. 3C). (PhSe)2 and (PhTe)2 did not inhibit the mito-chondrial complexes I–III activity from liver or kidney (Fig. 3A andB, respectively). However, under condition 2 Ebs, (PhSe)2 and(PhTe)2 (5 lM) significantly inhibited complexes I–III activity fromliver (Fig. 3A) and kidney membranes (Fig. 3B). Rotenone (100 lM)caused a significant inhibition of the mitochondrial complexes I–IIIactivity that varied from 30% to 70% (Fig. 3A–B).

Since inhibition of respiratory chain complex I could be at leastin part mediated by reactive oxygen species (including H2O2 andsuperoxide), we have investigated the potential protection of anti-oxidant enzymes on Complex I inhibition by Ebs, (PhSe)2 and(PhTe)2. Results depicted in Fig. 4 indicate that complex I inhibitionby Ebs, (PhSe)2 and (PhTe)2 was not modified by the addition ofSOD (Fig. 4A), CAT (Fig. 4B) or SOD + CAT (Fig. 4C).

In order to test the hypothesis that organochalcogens-inducedcomplex I inhibition is mediated by oxidation of thiol groups, we

Fig. 2. Effect of organochalcogens on mitochondrial complexes I–III activity. The mitochondria membranes were incubated (0.5 mg/mL) for 10 min at 30 �C in a mediumcontaining 100 mM phosphate buffer, NADH (200 lM), pH 7.4, in the presence of indicated concentrations of organochalcogens. Complexes I–III activity is expressed as % ofcontrol (without organochalcogens addition). (A) Liver mitochondria membranes; (B) kidney mitochondrial membranes. —N— (PhSe)2, —d— (PhTe)2, —j— Ebs. The controlvalues (without organochalcogens) were: liver 99.94 ± 2.21; kidney 101.03 ± 3.12. Data are expressed as means ± SEM from five independent experiments carried out induplicate. ⁄p < 0.05 from respective control by Duncan’s multiple range test.

Fig. 3. Effect of organochalcogens on mitochondrial complexes I–II activity under different assay conditions. The mitochondria membranes were incubated (0.5 mg/mL) for10 min at 30 �C in a medium containing 100 mM phosphate buffer, pH 7.4, in the presence of indicated concentrations of organochalcogens with (white bars) or without (graybars) NADH (200 lM). Complexes I–III activity is expressed as % of control (without organochalcogens addition). (A) Liver mitochondria membranes; (B) kidney mitochondrialmembranes. Control (absence of organocompounds); (PhSe)2 (5 lM); (PhTe)2 (5 lM); Ebs (5 lM); Rotenone (100 lM). Data are expressed as means ± SEM from 5independent experiments carried out in duplicate. ⁄p < 0.05 from respective control by t test. #p < 0.05 condition 1 vs condition 2 by t test.

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investigated the efficacy of GSH to reverse the organochalcogens-induced inhibition of complex I. Fig. 5 shows that GSH (500 lM)completely reversed the organochalcogens-induced complex Iinhibition in hepatic (Fig. 5A) and in renal (Fig. 5B) membranes.

3.3. Effect of organochalcogens in mitochondrial complex II activity

In order to check the inhibitory effect of different organochalcogensin mitochondria complex II activity, we carried out experiments attwo different conditions. In brief, in condition 1 the membraneswere incubated with the organocompounds (at different concen-trations) in the presence of succinate 5 mM for 10 min. The reac-tion was stopped 3 min after MTT by addition of ethanol. Incondition 2, the mitochondrial membranes were incubated withvarious concentrations of organocompounds in the absence of suc-cinate for 10 min. Succinate (5 mM) and MTT were then added andthe reaction stopped after 3 min by the addition of ethanol.

Statistical analysis indicates that Ebs and (PhTe)2 significantlyinhibited both hepatic and renal complex II activity in both condi-

tions (Fig. 6). In contrast, (PhSe)2 did not change the mitochondrialcomplex II activity from liver (Fig. 6A and B), but inhibited renalcomplex II activity under condition 1 (Fig. 6C), without inhibitingit under experimental condition 2 (Fig. 6D). The IC50 (lM) valuesfor inhibition by organochalcogens of mitochondrial complex IIactivity, in both conditions, are showed in Table 1. Malonate(8 mM) caused a significant inhibition of the mitochondrial com-plex II activity that varied from 40% to 70% inhibition (seeFig. 6A–D). GSH (500 lM) completely reversed the organochalcogens-induced complex II inhibition both in hepatic (Fig. 7A) and renal(Fig. 7B) membranes.

3.4. Effect of organochalcogens in mitochondrial complexes II–IIIactivity

Ebs and (PhTe)2 inhibited the mitochondrial complexes II–IIIactivity from liver (Fig. 8A) and kidney (Fig. 8B). (PhSe)2 did notinhibit hepatic complexes II–III activity (Fig. 8A), but significantlyinhibited renal complexes II–III activity (Fig. 8B). The IC50 (lM)

Fig. 4. Effect of SOD and/or CAT on organochalcogens-induced hepatic mitochondrial complex I inhibition. Hepatic mitochondria membranes were incubated (0.5 mg/mL) for10 min at 30 �C in a medium containing 100 mM phosphate buffer, pH 7.4, in the presence of organochalcogens (Ebs 25 lM; [(PhSe)2] 50 lM; [(PhTe)2] 50 lM either in theabsence or presence of SOD (100 UI/mL) (A); CAT (100 UI/mL) (B); SOD + CAT (100 UI/mL each) (C). Complex I activity is expressed as % of control (without organochalcogensaddition). Data are expressed as means ± SEM from five independent experiments carried out in duplicate. Comparisons among the experiments with vs. without antioxidantenzymes were done by t test. ⁄p < 0.05 from respective control by Duncan’s multiple range test.

Fig. 5. Reversion by GSH of Ebs-, (PhSe)2-, or PhTe)2-induced mitochondrial complex I inhibition. Mitochondria membranes were incubated (0.5 mg/mL) for 10 min at 30 �C ina medium containing 100 mM phosphate buffer, pH 7.4, in the presence of organochalcogens (Ebs 25 lM; [(PhSe)2] 50 lM; [(PhTe)2] 50 lM. The activity of mitochondrialcomplex I was determined after washing by centrifugation (12,000g for 10 min at 4 �C) the membranes to remove the organochalcogens and, sequentially, after incubating for5 min in the presence of GSH (500 lM). (A) Liver mitochondria membranes; (B) kidney mitochondrial membranes. Complex I activity is expressed as % of control (withoutorganochalcogens addition). Data are expressed as means ± SEM from 3–5 independent experiments carried out in duplicate. ⁄p < 0.05 from respective control by Duncan’smultiple range test. #p < 0.05 without vs with GSH by t test.

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Fig. 6. Effect of organochalcogens on mitochondrial complex II activity. The mitochondria membranes were incubated (0.5 mg/mL) for 10 min at 30 �C in a mediumcontaining 100 mM phosphate buffer, pH 7.4, in the presence of indicated concentrations organochalcogens of under two different conditions: Condition 1 (with succinate5 mM); Condition 2 (without succinate). Values of complex II activity are expressed as % of control (without organochalcogens addition). (A) Liver mitochondrial membranesunder condition 1 (Control value 98.50 ± 5.08); (B) liver mitochondrial membranes under condition 2 (Control value 100.09 ± 3.21); (C) kidney mitochondrial membranesunder condition 1 (Control value 102.55 ± 6.98); (D) Kidney mitochondrial membranes under condition 2 (Control value 101.95 ± 5.66). —N— (PhSe)2, —d— (PhTe)2, —j—Ebs, — + — Malonate (8 mM). Data are expressed as means ± SEM from 5 independent experiments carried out in duplicate. ⁄p < 0.05 from respective control by Duncan’smultiple range test. #p < 0.05 from control by t test.

R.L. Puntel et al. / Toxicology in Vitro 27 (2013) 59–70 65

values for inhibition by organochalcogens of mitochondrial com-plexes II–III activity are showed in Table 1.

3.5. Effect of organochalcogens in mitochondrial complex IV activity

Statistical analysis revealed that Ebs did not modify the hepatic(Fig. 9A) or renal (Fig. 9B) complex IV activity. (PhSe)2 slightlyinhibited complex IV activity from liver and kidney (Fig. 9A andB), whereas (PhTe)2 did not change the renal complex IV activity(Fig. 9B); but it inhibited hepatic complex IV activity at 50 lM(Fig. 9A). The IC50 (lM) value for inhibition by (PhSe)2 of mitochon-drial complex IV activity is showed in Table 1. Potassium cyanide(KCN, 10 mM) caused a significant inhibition of the mitochondrialcomplex IV activity that varied from 93% to 100% inhibition (Fig. 9Aand B).

3.6. Effects of organochalcogens in oxygen consumption in intactmitochondria

Ebs (25 lM), (PhSe)2 (50 lM), and (PhTe)2 (50 lM) inhibitedoxygen consumption in intact liver mitochondria supported either

by pyruvate/glutamate (complex I substrates; Fig. 10A) orsuccinate (complex II substrate; Fig. 10B) as substrates. For mito-chondrial oxygen consumption, the inhibitory potency order wasEbs � (PhTe)2 > (PhSe)2, independent of the substrate used. In fact,Ebs and (PhTe)2 completely inhibits oxygen consumption, whereas(PhSe)2 was less active. Taking into account, the results obtainedwith intact mitochondria are in accordance with our findings usingmitochondrial membranes (isolated complexes assay).

4. Discussion

The results presented here indicate that the hepatic and renaltoxicity of organochalcogens can be, at least in part, mediated bymitochondrial dysfunction via inhibition of different mithochondri-al complexes, which can explain our previous results (Puntel et al.,2010). Here we found that mitochondrial complex I was the keycomplex targeted by the organocompounds (almost 100% inhib-ited), followed by the complex II, whereas the inhibition of complexIII and complex IV was negligible. Furthermore, both hepatic andrenal mitochondrial preparations seem to be similarly inhibitedby organochalcogens. In fact, despite of different susceptibility of

Fig. 7. Reversion by GSH of Ebs-, (PhSe)2-, or PhTe)2-induced mitochondrial complex II inhibition. Mitochondria membranes were incubated (0.5 mg/mL) for 10 min at 30 �Cin a medium containing 100 mM phosphate buffer, pH 7.4, in the presence of organochalcogens (Ebs 25 lM; [(PhSe)2] 50 lM; [(PhTe)2] 50 lM. The activity of mitochondrialcomplex II was determined after washing the membranes to remove the organochalcogens and after incubating washed membranes for 5 min in the presence of GSH(500 lM). (A) Liver mitochondria membranes; (B) kidney mitochondrial membranes. Complex II activity is expressed as % of control (without organochalcogens addition).Data are expressed as means ± SEM from 3–4 independent experiments carried out in duplicate. ⁄p < 0.05 from respective control by Duncan’s multiple range test. #p < 0.05without vs with GSH by t test.

Fig. 8. Effect of organochalcogens on mitochondrial complexes II–III activity. The mitochondria membranes were incubated (0.5 mg/mL) for 10 min at 30 �C in a mediumcontaining 100 mM phosphate buffer, pH 7.4, in the presence of indicated concentrations of organochalcogens. Complexes II–III activity is expressed as % of control (withoutorganochalcogens addition). (A) Liver mitochondria membranes; (B) kidney mitochondrial membranes. —N— (PhSe)2, —d— (PhTe)2, —j— Ebs. The control values (withoutorganochalcogens) were: liver 99.56 ± 2.50; kidney 101.16 ± 1.12. Data are expressed as means ± SEM from 5 independent experiments carried out in duplicate. ⁄p < 0.05 fromrespective control by Duncan’s multiple range test.

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liver and kidney tissues after in vitro or in vivo exposure toorganochalcogens (Nogueira and Rocha, 2010, 2011), isolatedhepatic and renal mitochondria tended to respond similarly to theinhibitory properties of organochalcogens. Thus we suggest thatthe differences in the tissues susceptibility when exposed to organ-ochalcogens (Nogueira and Rocha, 2010, 2011) can be associatedwith other factors, such as differential distribution and metabolismof organochalcogens in these tissues.

Moreover, here we have used mitochondrial membranes in or-der to study the direct effect of organochalcogens on the com-plexes activity to avoid indirect effects of organochalcogen oncomplexes via modification of mitochondrial functionality (extentof polarization, presence of additional membrane barriers, etc., fordetails see (Puntel et al., 2010)). We know that the amounts oractivities of specific complexes and enzymes can be useful to test

specific hypotheses but should generally be held in reserve andnot used as the primary assay for mitochondrial dysfunction(Brand and Nicholls, 2011). Hence, oxygen consumption data usingintact mitochondria (Fig. 10) validated our findings with mitochon-drial membranes and suggested that the inhibition of mitochon-drial complexes is involved in the reduction of oxygenconsumption by intact mitochondria.

Although there are no compelling data in the literature reportingthe intracellular concentrations of these chalcogenides after acuteor chronic treatment, we have previously observed that exposureto toxic doses of (PhSe)2 increased selenium deposition in liver tolevels as high as 100 lM (Maciel et al., 2003). Thus, it is reasonableto suggest that after exposure to high doses, mitochondria may playan important role in the toxicity of organochalcogens. Accordingly,(PhSe)2 have been reported to cause cytotoxicity to a neuronal cell

Fig. 9. Effect of organochalcogens on mitochondrial complex IV activity. The mitochondria membranes were incubated (0.5 mg/mL) for 10 min at 30 �C in a mediumcontaining 100 mM phosphate buffer, pH 7.4, in the presence of indicated concentrations of organochalcogens. Complex IV activity is expressed as % of control (withoutorganochalcogens addition). (A) Liver mitochondria membranes; (B) kidney mitochondrial membranes. —N— (PhSe)2, —d— (PhTe)2, —j— Ebs, —�— KCN (10 mM). Thecontrol values (without organochalcogens) were: liver 101.05 ± 3.05; kidney 99.37 ± 2.03. Data are expressed as means ± SEM from 5 independent experiments carried out induplicate. ⁄p < 0.05 from respective control by Duncan’s multiple range test. #p < 0.05 from control by t test.

Fig. 10. Effect of organochalcogens on Hepatic Mitochondrial oxygen consumption. Isolated rat liver mitochondria (0.5 mg/mL) were pre incubated 10 min in the absence orpresence of different orcanochalcogens studied at indicated concentrations and the oxygen consumption was measured as described in the Materials and methods usingPyruvate/glutamate (2.5 mM each) (A) or succinate (5 mM) (B) as substrates. The arrows indicate sequential additions of: Mit, 0.5 mg/mL mitochondria; Py/Glu, 2.5 mMpyruvate and 2.5 mM glutamate; Succ, 5 mM succinate; ADP/Pi, 0.3 mM ADP and 0.6 mM inorganic phosphate; Olig, 2 lM oligomycin; and 2,4 DNP, 10 lM dinitrophenol.—e— Control, —N— (PhSe)2, —d— (PhTe)2, —j— Ebs. Data (oxygen content in the medium) are expressed as means ± SEM from 4 independent experiments carried out induplicate. The numbers in the Figures represent the respiratory rate (expressed as rate medium of 4 independent experiments performed in duplicates).

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line from 10 lM onwards, by induction of apoptosis via ERK1/2pathway (Posser et al., 2011). However, literature data about thecytotoxicity of these compounds are scarce. In fact, Ebs (at 50–75 lM) was toxic to human hepatoma cells (HepG2) and inducedapoptosis via disruption of mitochondrial physiology that wasdependent on cellular thiol depletion (Yang et al., 2000). The corre-lation of these concentrations with the concentration of organoch-alcogens used here in isolated mitochondria is difficult to be done.However, since authors have exposed HepG2 cells only briefly tothese relatively high concentration of Ebs (50–75 lM), we can sup-pose that mitochondria exposure to lM concentrations of organ-ochalcogens is plausible to occur in vitro and after in vivoexposure to high doses of these agents. However, literature hasnot explored the concentrations of organochalcogens that couldbe toxic to primary cells and there is no study about their effectson mitochondria respiration using intact cells and/or tissue slices.Thus, it is important to emphasize that this is the first report con-

cerning the inhibitory effect of studied organochalcogens on mito-chondrial complexes activity.

Taken together, our results indicate that Ebs, (PhSe)2, and(PhTe)2 inhibit the activity of mitochondrial complexes I and IIfrom liver and kidney. This inhibition probably involves the inter-action of these compounds with essential cysteinyl residues ofmitochondrial complexes (represented by PSH in Scheme 1), asindicated by our data using GSH (see Figs. 5 and 7). In fact, themitochondrial complexes (complexes I–IV) are well known to beoxidatively modified in physiological and non-physiological condi-tions, which can culminate with their inhibition (Beltran et al.,2000; Clementi et al., 1998; Le-Quoc et al., 1981; Navarro andBoveris, 2007; Navarro et al., 2002, 2004, 2005; Ohnishi et al.,1998). In line with this, Ebs, (PhSe)2, and (PhTe)2 were reportedto inhibit d-ALA-D (Barbosa et al., 1998; Folmer et al., 2005; Macielet al., 2000; Rocha et al., 2012), Na+K+/ATPase (Borges et al., 2005),and LDH (Lugokenski et al., 2011) by binding to sulfhydryl groups

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of these enzymes. Thus, we can hypothesize that the organochom-pounds studied here inhibited the mitochondrial complexes viatheir thiol oxidation activity (Scheme 1).

Our assumption is further supported by the results presented inFig. 3(A–B) where we have used different assay conditions. In fact,we found that incubation of mitochondrial membranes with thecomplex I substrate (NADH) partially prevents the inhibitory effectof chalcogens on complex I activity. Our hypothesis is further sup-ported by previous data from our laboratory showing that (PhSe)2-induced LDH inhibition was attenuated or abolished by NADH(Lugokenski et al., 2011). These data indicate that NADH can mod-ulate enzyme conformation preventing the critical thiols from theattack by organochalcogens. Based on the presented results, wesuggest that organochalcogen-induced mitochondrial complex Iinhibition is linked to their interaction with critical thiol groupspresent in the active site of the NADH:ubiquinone oxidoreductase(Lin et al., 2002).

As mentioned above, the complex I inhibition by organochalco-gens was more pronounced than complex II. We suggest that, de-spite of succinate dehydrogenase (SDH) being described topossess sulfhydryl group essential for catalytic activity, located inthe substrate site (Le-Quoc et al., 1981), the organochalcogens-induced mitochondrial complex II inhibition could be due to theirinteraction with other thiols critical to enzyme activity, than thatlocated in the active site of the SDH (Lin et al., 2002). Our dataare further supported by previous data showing that complex IIis less prone to inactivation than complex I (Cadenas and Davies,2000; Orrenius et al., 2007; Zhang et al., 1990). Thus, based onthe presented results (Figs. 5 and 7) we suggest that both com-plexes I and II were directly affected by the organochalogens, beingthe thiols groups the molecular site of action for the organochalc-ogens. Our hypothesis is further supported by the data showingthat organochalcogens induced complex I inhibition was not med-iated by ROS formation (Figs. 4A–C).

However, as seen in Figs. 6 and 8, (PhSe)2 has differential effecton complex II in liver and kidney. At the present moment, these re-sults are not completely understood, but they can be related to dif-ferences in the molecular composition of mitochondria obtainedfrom different tissues (Benard et al., 2006). Thus, we speculate thatthe liver and kidney could present different contents and isoformsof complex II enzymes, which resulted in different inhibition by(PhSe)2. Our assumption is based on previous data showing that,at least, two different isoforms of complex II have been reportedin the literature (Tomitsuka et al., 2003a,b; Tomitsuka et al., 2009).

In addition to complexes I and II, the activities of the mitochon-drial complexes III and IV (both from rat liver and kidney) were prac-tically not targeted by organocompounds. In fact, mitochondrialcomplex III was minimally inhibited by the treatment with studiedcompounds, whereas complex IV was nearly unchanged. Thus,organochalcogens possibly did not inhibit mitochondrial complexesIII and IV due to steric hindrance of their sulfhydryl groups to theorganochalcogens (Lin et al., 2002). Our findings are supported byprevious report showing that thiol groups from complex IV are lessprone to oxidation than that from complex I (Orrenius et al., 2007).

As pointed previously, damage to the mitochondrial electrontransport chain has been suggested to be an important factor inthe pathogenesis of a range of diseases where the oxidative stressseems to be involved (Beltran et al., 2000; Clementi et al., 1998;Dahm et al., 2006; Fattal et al., 2006; Hurd et al., 2007; Le-Quocet al., 1981; Madrigal et al., 2001; Navarro and Boveris, 2007;Navarro et al., 2002, 2004, 2005; Ohnishi et al., 1998; Tayloret al., 2003). In addition, mitochondrial dysfunction (as evidencedby decline in respiratory chain activity) is closely linked to bothage and ischemia–reperfusion-associated mitochondrial changes,that culminates, in some cases, to apoptotic cell death (Cadenasand Davies, 2000; Caspersen et al., 2005; Hauptmann et al.,

2006; Navarro and Boveris, 2007; Nicholls, 2002; Sastre et al.,2003). Thus, based on the presented results and in the previouslypublished data (Puntel et al., 2010) it is reasonable to suggest thatmitochondrial dysfunction could be a central process in the hepa-totoxicity of organochalcogens after in vivo exposure. Kidney couldalso be targeted by high doses of organochalcogens; however, thedeposition of these compounds in kidney is less accentuated thanin liver (Maciel et al., 2003).

In conclusion, here we clearly demonstrate that Ebs, (PhSe)2,and (PhTe)2 – induced mitochondrial complexes inhibition, andthat their effects virtually did not vary among the hepatic and renalmitochondria. The mitochondrial complex I was the most suscep-tible to organochalcogens-induced inhibition, followed by complexII. Based on our data, we believe that inhibitory effect of organoch-alcogens could be attributed to oxidation of essential thiols in theenzyme complexes. Taking this into account, we suggest that mito-chondrial complex I and II could be considered important molecu-lar targets of organochalcogens after exposure to high dosages ofthese compounds.

Acknowledgments

This work was supported by grants from UNIPAMPA (UniversidadeFederal do Pampa), UFSM (Universidade Federal de Santa Maria),CNPq/FAPERGS/DECIT/SCTIE-MS/PRONEM #11/2029-1, CAPES(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior),FINEP (Rede Instituto Brasileiro de Neurociência (IBN-Net) #01.06.0842-00), FAPERGS-PRONEX and INCT-EN (Instituto Nacion-al de Ciência e Tecnologia em Excitotoxicidade e Neuroproteção).

References

Ardais, A.P., Viola, G.G., Costa, M.S., Nunes, F., Behr, G.A., Klamt, F., Moreira, J.C.,Souza, D.O., Rocha, J.B., Porciuncula, L.O., 2010. Acute treatment with diphenyldiselenide inhibits glutamate uptake into rat hippocampal slices and modifiesglutamate transporters, SNAP-25, and GFAP immunocontent. Toxicol. Sci. 113,434–443.

Arteel, G.E., Sies, H., 2001. The biochemistry of selenium and the glutathionesystem. Environ. Toxicol. Pharmacol. 10, 153–158.

Barbosa, N.B., Rocha, J.B., Soares, J.C., Wondracek, D.C., Goncalves, J.F., Schetinger,M.R., Nogueira, C.W., 2008. Dietary diphenyl diselenide reduces the STZ-induced toxicity. Food Chem. Toxicol. 46, 186–194.

Barbosa, N.B., Rocha, J.B., Wondracek, D.C., Perottoni, J., Zeni, G., Nogueira, C.W.,2006. Diphenyl diselenide reduces temporarily hyperglycemia: possiblerelationship with oxidative stress. Chem. Biol. Interact. 163, 230–238.

Barbosa, N.B., Rocha, J.B., Zeni, G., Emanuelli, T., Beque, M.C., Braga, A.L., 1998. Effectof organic forms of selenium on delta-aminolevulinate dehydratase from liver,kidney, and brain of adult rats. Toxicol. Appl. Pharmacol. 149, 243–253.

Beal, M.F., 1998. Mitochondrial dysfunction in neurodegenerative diseases.Biochim. Biophys. Acta 1366, 211–223.

Beltran, B., Mathur, A., Duchen, M.R., Erusalimsky, J.D., Moncada, S., 2000. The effectof nitric oxide on cell respiration: a key to understanding its role in cell survivalor death. Proc. Natl. Acad. Sci. USA 97, 14602–14607.

Benard, G., Faustin, B., Passerieux, E., Galinier, A., Rocher, C., Bellance, N., Delage, J.P.,Casteilla, L., Letellier, T., Rossignol, R., 2006. Physiological diversity ofmitochondrial oxidative phosphorylation. Am. J. Physiol. Cell Physiol. 291,C1172–1182.

Borges, V.C., Rocha, J.B., Nogueira, C.W., 2005. Effect of diphenyl diselenide, diphenylditelluride and ebselen on cerebral Na(+), K(+)-ATPase activity in rats.Toxicology 215, 191–197.

Brand, M.D., Nicholls, D.G., 2011. Assessing mitochondrial dysfunction in cells.Biochem. J. 435, 297–312.

Cadenas, E., Davies, K.J., 2000. Mitochondrial free radical generation, oxidativestress, and aging. Free Radical Biol. Med. 29, 222–230.

Caspersen, C., Wang, N., Yao, J., Sosunov, A., Chen, X., Lustbader, J.W., Xu, H.W.,Stern, D., McKhann, G., Yan, S.D., 2005. Mitochondrial Abeta: a potential focalpoint for neuronal metabolic dysfunction in Alzheimer’s disease. FASEB J. 19,2040–2041.

Clementi, E., Brown, G.C., Feelisch, M., Moncada, S., 1998. Persistent inhibition ofcell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrialcomplex I and protective action of glutathione. Proc. Natl. Acad. Sci. USA 95,7631–7636.

Cortopassi, G.A., Wong, A., 1999. Mitochondria in organismal aging anddegeneration. Biochim. Biophys. Acta 1410, 183–193.

Dahm, C.C., Moore, K., Murphy, M.P., 2006. Persistent S-nitrosation of complex I andother mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide

R.L. Puntel et al. / Toxicology in Vitro 27 (2013) 59–70 69

or peroxynitrite: implications for the interaction of nitric oxide withmitochondria. J. Biol. Chem. 281, 10056–10065.

de Bem, A.F., Portella Rde, L., Colpo, E., Duarte, M.M., Frediane, A., Taube, P.S.,Nogueira, C.W., Farina, M., da Silva, E.L., Teixeira Rocha, J.B., 2009.Diphenyl diselenide decreases serum levels of total cholesterol and tissueoxidative stress in cholesterol-fed rabbits. Basic Clin. Pharmacol. Toxicol.105, 17–23.

de Freitas, A.S., Funck, V.R., Rotta Mdos, S., Bohrer, D., Morschbacher, V., Puntel, R.L.,Nogueira, C.W., Farina, M., Aschner, M., Rocha, J.B., 2009. Diphenyl diselenide, asimple organoselenium compound, decreases methylmercury-induced cerebral,hepatic and renal oxidative stress and mercury deposition in adult mice. BrainRes. Bull. 79, 77–84.

de Freitas, A.S., Rocha, J.B., 2011. Diphenyl diselenide and analogs are substrates ofcerebral rat thioredoxin reductase: a pathway for their neuroprotective effects.Neurosci. Lett. 503, 1–5.

Engman, L., Persson, J., Vessman, K., Ekstrom, M., Berglund, M., Andersson, C.M.,1995. Organotellurium compounds as efficient retarders of lipid peroxidation inmethanol. Free Radical Biol. Med. 19, 441–452.

Engman, L.J., 1989. Expedient synthesis of Ebselen and related-compounds. J. Org.Chem. 54, 2964–2966.

Fattal, O., Budur, K., Vaughan, A.J., Franco, K., 2006. Review of the literature on majormental disorders in adult patients with mitochondrial diseases. Psychosomatics47, 1–7.

Folmer, V., Bolzan, R.C., Farina, M., Zeni, G., Nogueira, C.W., Emanuelli, T., Rocha, J.B.,2005. Mechanism of delta-aminolevulinate dehydratase inhibition by phenylselenoacetylene involves its conversion to diphenyl diselenide. Toxicology 206,403–411.

Hagen, T.M., Wehr, C.M., Ames, B.N., 1998. Mitochondrial decay in aging. Reversalthrough supplementation of acetyl-L-carnitine and N-tert-butyl-alpha-phenyl-nitrone. Ann. N.Y. Acad. Sci. 854, 214–223.

Hatefi, Y., 1985. The mitochondrial electron transport and oxidativephosphorylation system. Annu. Rev. Biochem. 54, 1015–1069.

Hauptmann, S., Keil, U., Scherping, I., Bonert, A., Eckert, A., Muller, W.E., 2006.Mitochondrial dysfunction in sporadic and genetic Alzheimer’s disease. Exp.Gerontol. 41, 668–673.

Hort, M.A., Straliotto, M.R., Netto, P.M., da Rocha, J.B., de Bem, A.F., Ribeiro-do-Valle,R.M., 2011. Diphenyl diselenide effectively reduces atherosclerotic lesions inLDLr �/� mice by attenuation of oxidative stress and inflammation. J.Cardiovasc. Pharmacol. 58, 91–101.

Hurd, T.R., Prime, T.A., Harbour, M.E., Lilley, K.S., Murphy, M.P., 2007. Detection ofreactive oxygen species-sensitive thiol proteins by redox difference gelelectrophoresis: implications for mitochondrial redox signaling. J. Biol. Chem.282, 22040–22051.

Le-Quoc, K., Le-Quoc, D., Gaudemer, Y., 1981. Evidence for the existence of twoclasses of sulfhydryl groups essential for membrane-bound succinatedehydrogenase activity. Biochemistry 20, 1705–1710.

Leonard, K.A., Zhou, F., Detty, M.R., 1996. Chalcogen(IV)–chalcogen(II) redoxcycles.1. Halogenation of organic substrates with dihaloselenium(IV) and -tellurium(IV) derivatives. Dehalogenation of vicinal dibromides with diaryltellurides. Organomtallics 15, 4285–4292.

Lin, T.K., Hughes, G., Muratovska, A., Blaikie, F.H., Brookes, P.S., Darley-Usmar, V.,Smith, R.A., Murphy, M.P., 2002. Specific modification of mitochondrial proteinthiols in response to oxidative stress: a proteomics approach. J. Biol. Chem. 277,17048–17056.

Lugokenski, T.H., Muller, L.G., Taube, P.S., Rocha, J.B., Pereira, M.E., 2011. Inhibitoryeffect of ebselen on lactate dehydrogenase activity from mammals: acomparative study with diphenyl diselenide and diphenyl ditelluride. DrugChem. Toxicol. 34, 66–76.

Maciel, E.N., Bolzan, R.C., Braga, A.L., Rocha, J.B., 2000. Diphenyl diselenide anddiphenyl ditelluride differentially affect delta-aminolevulinate dehydratasefrom liver, kidney, and brain of mice. J. Biochem. Mol. Toxicol. 14, 310–319.

Maciel, E.N., Flores, E.M., Rocha, J.B., Folmer, V., 2003. Comparative deposition ofdiphenyl diselenide in liver, kidney, and brain of mice. Bull. Environ. Contam.Toxicol. 70, 470–476.

Madrigal, J.L., Olivenza, R., Moro, M.A., Lizasoain, I., Lorenzo, P., Rodrigo, J., Leza, J.C.,2001. Glutathione depletion, lipid peroxidation and mitochondrial dysfunctionare induced by chronic stress in rat brain. Neuropsychopharmacology 24, 420–429.

Maiorino, M., Roveri, A., Coassin, M., Ursini, F., 1988. Kinetic mechanism andsubstrate specificity of glutathione peroxidase activity of ebselen (PZ51).Biochem. Pharmacol. 37, 2267–2271.

Moretto, M.B., Thomazi, A.P., Godinho, G., Roessler, T.M., Nogueira, C.W., Souza, D.O.,Wofchuk, S., Rocha, J.B., 2007. Ebselen and diorganylchalcogenides decreasein vitro glutamate uptake by rat brain slices: prevention by DTT and GSH.Toxicol. In Vitro 21, 639–645.

Morin, D., Zini, R., Ligeret, H., Neckameyer, W., Labidalle, S., Tillement, J.P., 2003.Dual effect of ebselen on mitochondrial permeability transition. Biochem.Pharmacol. 65, 1643–1651.

Navarro, A., Boveris, A., 2007. The mitochondrial energy transduction system andthe aging process. Am. J. Physiol. Cell Physiol. 292, C670–686.

Navarro, A., Gomez, C., Lopez-Cepero, J.M., Boveris, A., 2004. Beneficial effects ofmoderate exercise on mice aging: survival, behavior, oxidative stress, andmitochondrial electron transfer. Am. J. Physiol. Regul. Integr. Comp. Physiol.286, R505–R511.

Navarro, A., Gomez, C., Sanchez-Pino, M.J., Gonzalez, H., Bandez, M.J., Boveris, A.D.,Boveris, A., 2005. Vitamin E at high doses improves survival, neurological

performance, and brain mitochondrial function in aging male mice. Am. J.Physiol. Regul. Integr. Comp. Physiol. 289, R1392–1399.

Navarro, A., Sanchez Del Pino, M.J., Gomez, C., Peralta, J.L., Boveris, A., 2002.Behavioral dysfunction, brain oxidative stress, and impaired mitochondrialelectron transfer in aging mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282,R985–992.

Nicholls, D.G., 2002. Mitochondrial function and dysfunction in the cell: itsrelevance to aging and aging-related disease. Int. J. Biochem. Cell Biol. 34,1372–1381.

Nicholls, D.G., Budd, S.L., 2000. Mitochondria and neuronal survival. Physiol. Rev.80, 315–360.

Nogueira, C.W., Rocha, J.B., 2011. Toxicology and pharmacology of selenium:emphasis on synthetic organoselenium compounds. Arch. Toxicol. 85, 1313–1359.

Nogueira, C.W., Rocha, J.B.T., 2010. Diphenyl Diselenide a janus-faced molecule. J.Braz. Chem. Soc. 21, 2055–2071.

Ohnishi, T., Sled, V.D., Yano, T., Yagi, T., Burbaev, D.S., Vinogradov, A.D., 1998.Structure-function studies of iron-sulfur clusters and semiquinones in theNADH-Q oxidoreductase segment of the respiratory chain. Biochim. Biophys.Acta 1365, 301–308.

Orrenius, S., Gogvadze, V., Zhivotovsky, B., 2007. Mitochondrial oxidative stress:implications for cell death. Annu. Rev. Pharmacol. Toxicol. 47, 143–183.

Parnham, M.J., Graf, E., 1991. Pharmacology of synthetic organic seleniumcompounds. Prog. Drug Res. 36, 9–47.

Paulmier, C., 1986. Selenium Reagents and Intermediates in Organic Synthesis.Pergamon Press, New York, p. 463.

Peterson, G.L., 1977. A simplification of the protein assay method of Lowry et al.which is more generally applicable. Anal. Biochem. 83, 346–356.

Petragnani, N., 1994. Tellurium in Organic Synthesis. Academic Press, San Diego, p.248.

Posser, T., de Paula, M.T., Franco, J.L., Leal, R.B., da Rocha, J.B., 2011. Diphenyldiselenide induces apoptotic cell death and modulates ERK1/2 phosphorylationin human neuroblastoma SH-SY5Y cells. Arch. Toxicol. 85, 645–651.

Prauchner, C.A., Prestes Ade, S., da Rocha, J.B., 2011. Effects of diphenyl diselenide onoxidative stress induced by sepsis in rats. Pathol. Res. Pract. 207, 554–558.

Prigol, M., Wilhelm, E.A., Schneider, C.C., Nogueira, C.W., 2008. Protective effect ofunsymmetrical dichalcogenide, a novel antioxidant agent, in vitro and an in vivomodel of brain oxidative damage. Chem. Biol. Interact. 176, 129–136.

Puntel, R.L., Roos, D.H., Folmer, V., Nogueira, C.W., Galina, A., Aschner, M., Rocha, J.B.,2010. Mitochondrial dysfunction induced by different organochalcogens ismediated by thiol oxidation and is not dependent of the classical mitochondrialpermeability transition pore opening. Toxicol. Sci. 117, 133–143.

Puntel, R.L., Roos, D.H., Paixao, M.W., Braga, A.L., Zeni, G., Nogueira, C.W., Rocha, J.B.,2007. Oxalate modulates thiobarbituric acid reactive species (TBARS)production in supernatants of homogenates from rat brain, liver and kidney:effect of diphenyl diselenide and diphenyl ditelluride. Chem. Biol. Interact. 165,87–98.

Rocha, J.B.T., Saraiva, R.A., Garcia, S.C., Gravina, F.S., Nogueira, C.W., 2012.Aminolevulinate dehydratase (d-ALA-D) as marker protein of intoxicationwith metals and other pro-oxidant situations. Toxicol. Res. 1, 85–102.

Rossato, J.I., Ketzer, L.A., Centuriao, F.B., Silva, S.J., Ludtke, D.S., Zeni, G., Braga, A.L.,Rubin, M.A., Rocha, J.B., 2002. Antioxidant properties of new chalcogenidesagainst lipid peroxidation in rat brain. Neurochem. Res. 27, 297–303.

Santos, D.B., Schiar, V.P., Paixao, M.W., Meinerz, D.F., Nogueira, C.W., Aschner, M.,Rocha, J.B., Barbosa, N.B., 2009a. Hemolytic and genotoxic evaluation oforganochalcogens in human blood cells in vitro. Toxicol. In Vitro 23, 1195–1204.

Santos, D.B., Schiar, V.P., Ribeiro, M.C., Schwab, R.S., Meinerz, D.F., Allebrandt, J.,Rocha, J.B., Nogueira, C.W., Aschner, M., Barbosa, N.B., 2009b. Genotoxicity oforganoselenium compounds in human leukocytes in vitro. Mutat. Res. 676, 21–26.

Santos, F.W., Zeni, G., Rocha, J.B., do Nascimento, P.C., Marques, M.S., Nogueira, C.W.,2005. Efficacy of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and diphenyldiselenide on cadmium induced testicular damage in mice. Food Chem. Toxicol.43, 1723–1730.

Saris, N.E., Eriksson, K.O., 1995. Mitochondrial dysfunction in ischaemia-reperfusion. Acta Anaesthesiol. Scand. Suppl. 107, 171–176.

Sastre, J., Pallardo, F.V., Vina, J., 2003. The role of mitochondrial oxidative stress inaging. Free Radical Biol. Med. 35, 1–8.

Sausen de Freitas, A., de Souza Prestes, A., Wagner, C., Haigert Sudati, J., Alves, D.,Oliveira Porciuncula, L., Kade, I.J., Teixeira Rocha, J.B., 2010. Reduction ofdiphenyl diselenide and analogs by mammalian thioredoxin reductase isindependent of their gluthathione peroxidase-like activity: a possible novelpathway for their antioxidant activity. Molecules 15, 7699–7714.

Sies, H., 1993. Ebselen, a selenoorganic compound as glutathione peroxidase mimic.Free Radical Biol. Med. 14, 313–323.

Sies, H., 1995. Ebselen. Methods Enzymol. 252, 341–342.Taylor, E.R., Hurrell, F., Shannon, R.J., Lin, T.K., Hirst, J., Murphy, M.P., 2003.

Reversible glutathionylation of complex I increases mitochondrial superoxideformation. J. Biol. Chem. 278, 19603–19610.

Tomitsuka, E., Goto, Y., Taniwaki, M., Kita, K., 2003a. Direct evidence for expressionof type II flavoprotein subunit in human complex II (succinate-ubiquinonereductase). Biochem. Biophys. Res. Commun. 311, 774–779.

Tomitsuka, E., Hirawake, H., Goto, Y., Taniwaki, M., Harada, S., Kita, K., 2003b. Directevidence for two distinct forms of the flavoprotein subunit of humanmitochondrial complex II (succinate-ubiquinone reductase). J. Biochem. 134,191–195.

70 R.L. Puntel et al. / Toxicology in Vitro 27 (2013) 59–70

Tomitsuka, E., Kita, K., Esumi, H., 2009. Regulation of succinate-ubiquinonereductase and fumarate reductase activities in human complex II byphosphorylation of its flavoprotein subunit. Proc. Jpn. Acad. Ser. B: Phys. Biol.Sci. 85, 258–265.

Wendel, A., Fausel, M., Safayhi, H., Tiegs, G., Otter, R., 1984. A novel biologicallyactive seleno-organic compound-II. Activity of PZ 51 in relation to glutathioneperoxidase. Biochem. Pharmacol. 33, 3241–3245.

Yang, C.F., Shen, H.M., Ong, C.N., 2000. Intracellular thiol depletion causesmitochondrial permeability transition in ebselen-induced apoptosis. Arch.Biochem. Biophys. 380, 319–330.

You, Y., Ahsan, K., Detty, M.R., 2003. Mechanistic studies of the tellurium(II)/tellurium(IV) redox cycle in thiol peroxidase-like reactions of diorgano-tellurides in methanol. J. Am. Chem. Soc. 125, 4918–4927.

Zhang, Y., Marcillat, O., Giulivi, C., Ernster, L., Davies, K.J., 1990. The oxidativeinactivation of mitochondrial electron transport chain components and ATPase.J. Biol. Chem. 265, 16330–16336.

Zhao, R., Domann, F.E., Zhong, W., 2006. Apoptosis induced by selenomethionineand methioninase is superoxide mediated and p53 dependent in humanprostate cancer cells. Mol. Cancer Ther. 5, 3275–3284.

Zhao, R., Holmgren, A., 2002. A novel antioxidant mechanism of ebselen involvingebselen diselenide, a substrate of mammalian thioredoxin and thioredoxinreductase. J. Biol. Chem. 277, 39456–39462.

Zhao, R., Masayasu, H., Holmgren, A., 2002. Ebselen: a substrate for humanthioredoxin reductase strongly stimulating its hydroperoxide reductaseactivity and a superfast thioredoxin oxidant. Proc. Natl. Acad. Sci. USA 99,8579–8584.