pharmacology and toxicology of diphenyl diselenide in ...2departamento de farmacologia, instituto de...

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Braz J Med Biol Res 40(10) 2007

Biological effects of diphenyl diselenide

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Pharmacology and toxicology ofdiphenyl diselenide in severalbiological models

1Departamento de Biofísica, Instituto de Biociências,2Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde,Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil3Laboratório de Genética Toxicológica, Universidade Luterana do Brasil,Canoas, RS, Brasil4Instituto de Química, Universidade Federal de Santa Maria, Santa Maria,RS, Brasil

R.M. Rosa1, R. Roesler2,A.L. Braga4, J. Saffi1,3

and J.A.P. Henriques1,3

Abstract

The pharmacology of synthetic organoselenium compounds indicatesthat they can be used as antioxidants, enzyme inhibitors, neuroprotec-tors, anti-tumor and anti-infectious agents, and immunomodulators.In this review, we focus on the effects of diphenyl diselenide (DPDS)in various biological model organisms. DPDS possesses antioxidantactivity, confirmed in several in vitro and in vivo systems, and thus hasa protective effect against hepatic, renal and gastric injuries, in addi-tion to its neuroprotective activity. The activity of the compound onthe central nervous system has been studied since DPDS has lipophiliccharacteristics, increasing adenylyl cyclase activity and inhibitingglutamate and MK-801 binding to rat synaptic membranes. Systemicadministration facilitates the formation of long-term object recogni-tion memory in mice and has a protective effect against brain ischemiaand on reserpine-induced orofacial dyskinesia in rats. On the otherhand, DPDS may be toxic, mainly because of its interaction with thiolgroups. In the yeast Saccharomyces cerevisiae, the molecule acts as apro-oxidant by depleting free glutathione. Administration to miceduring cadmium intoxication has the opposite effect, reducing oxida-tive stress in various tissues. DPDS is a potent inhibitor of δ-aminolev-ulinate dehydratase and chronic exposure to high doses of this com-pound has central effects on mouse brain, as well as liver and renaltoxicity. Genotoxicity of this compound has been assessed in bacteria,haploid and diploid yeast and in a tumor cell line.

CorrespondenceJ.A.P. Henriques

Agronomia, UFRGS

Av. Bento Gonçalves, 9500

Ed. 43422, Laboratório 210

91501-970 Porto Alegre, RS

Brasil

Fax: +55-51-3316-7309

E-mail: [email protected]

Research supported by CNPq,

CAPES, and GENOTOX (Genotoxicity

Laboratory, Instituto Royal,

Brazil). R.M. Rosa is the

recipient of a doctoral grant

from CAPES.

Received January 18, 2007

Accepted May 21, 2007

Key words• Diphenyl diselenide• Organoselenium• Antioxidants• Neuroprotection• Saccharomyces cerevisiae• Mutagenesis

Introduction

The element selenium was discovered in1818 by the Swedish chemist Berzelius andwas named after the Greek goddess of the

moon, Selene. First reports were related toits toxicity. Although evidence was obtainedas early as 1842 for the toxicity of selenium,the first authentic written record of seleniumpoisoning was reported in 1856 by Madison,

Brazilian Journal of Medical and Biological Research (2007) 40: 1287-1304ISSN 0100-879X Review

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an army surgeon stationed at Fort Randall,then in the Nebraska territory (1). He de-scribed a fatal disease among horses grazingcertain areas near the fort. In 1929, investi-gations started to study toxic grains at theSouth Dakota Experimental Station and in1933 the toxic principle in these grains wassuggested to be selenium, which was con-firmed a year later. The element was re-ported to be a potent carcinogen in 1943 (2,3). Selenium was thus considered to be anabsolute poison until Schwarz and Foltz iden-tified it as a micronutrient for bacteria, mam-mals and birds (1). After years of empiricalstudies on selenium deficiency syndromesin animal models, selenium biochemistryemerged in 1973, when the bacterial en-zymes formate dehydrogenase and glycinereductase were reported to contain selenium.At the same time, this atom was found in theactive site of the antioxidant enzyme gluta-thione peroxidase (GPX) in mammals (1).The number of selenoproteins identified hasgrown substantially in recent years and mo-lecular mechanisms and regulatory pathwaysof seleno amino acid incorporation have beenelucidated (2).

The biological importance of seleniumand its inorganic forms led to the develop-ment of pharmacologically active organose-lenium (OS) compounds with low toxicity,since the selenium atom was not delivered tothe intracellular selenium pool. Althoughthe first synthetic OS compound, diethyldiselenide, was prepared by Lowig as earlyas in 1836, the highly malodorous nature ofselenium compounds, difficulties in purifi-cation, and the instability of many of thederivatives hampered further developments.The discovery of several useful new reac-tions and a variety of novel structures withunusual properties led to further OS devel-opment. Interest in the use of these mol-ecules in biochemistry started with the find-ings that they are much less toxic than inor-

ganic selenium species (1).Diphenyl diselenide (DPDS; Figure 1)

(4) is a simple and stable OS compound. Itis an electrophilic reagent used in the syn-thesis of a variety of pharmacologically ac-tive OS compounds (4). Recently, the bio-logical activities of DPDS have been studiedand this compound has become a good can-didate for therapeutic purposes. In this re-view we provide a comprehensive coverageof the toxicology and pharmacology of thismolecule.

Toxicology

Although the molecular mechanisms un-derlying selenium toxicity are still poorlyunderstood, Painter (see Ref. 1) proposedthat it could be related to the oxidation ofthiols of biological importance. The interac-tion of inorganic selenium with thiols wasdemonstrated experimentally by Tsen andTappel (5), who showed that selenite cata-lytically increased the oxidation of glutathi-one. Subsequently, the effectiveness of OScompounds as catalytic oxidants of thiolswas reported (5-9). Inorganic and organicforms of selenium can also increase thioloxidation by accelerating electron transferfrom thiols to oxidized acceptors such ascytochrome c and methylene blue (8).

The mechanism of oxidation of thiolgroups in low- and high-molecular weightmolecules by selenium varies with the chem-ical structure of the compounds and caninvolve the formation of selenoxides, sele-notrisulfides and selenylsulfide (10,11). OScompounds such as selenocystine and a va-riety of diselenides can react with thiols suchas cysteine, dithiothreitol (DTT) and reducedglutathione (GSH) to produce selenocys-teine and selenols, respectively, and disul-fides. The interaction between DPDS andthiol groups has been investigated in severalbiological models, such as the enzyme

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δ-aminolevulinate dehydratase (ALA-D)from various sources, strains of Saccharo-myces cerevisiae defective in GSH biosyn-thesis, and in in vitro systems.

Interaction with δδδδδ-aminolevulinatedehydratase

ALA-D, or porphobilinogen synthase, isan enzyme sensitive to sulfydryl (SH)-block-ing agents that participates in the secondstep of heme, chlorophyll and corrin biosyn-thesis (11,12). It catalyzes the condensationof two molecules of 5-aminolevulinic acidto porphobilinogen, thus playing an essen-tial role in animals and photosynthetic or-ganisms (11,12). ALA-D inhibition inhibitsheme biosynthesis, resulting in accumula-tion of aminolevulinic acid, which affectsaerobic metabolism, and has some pro-oxi-dant activity (13). Inhibition of heme bio-synthesis may disturb the production of cy-tochrome P450, fundamental to metabolism.

The investigation of the effects of DPDStreatment on ALA-D activity is of particularsignificance from an academic and toxico-logical point of view because of the impor-tance of reduced thiol groups for ALA-Dactivity and the oxidant properties of or-ganic and inorganic selenium compounds.Studies have indicated whether this enzymeis a potential target for DPDS and whether itcan be considered a marker for OS exposure,as was found for lead intoxication.

The first study was carried out in vitrousing ALA-D purified from liver, kidneyand brain of adult rats. DPDS, as well asother diselenide bond-containing com-pounds, did indeed have an inhibitory poten-tial on ALA-D, even without pre-incuba-tion. However, after pre-incubation of theenzyme preparation with DPDS, inhibitionwas significantly stronger. The effect wasreversed in a concentration-dependent man-ner by DTT treatment, which protects SHgroups of the enzyme, and also by GSH, the

Figure 1. Chemical structure ofdiphenyl diselenide, some di-phenyl diselenide derivativesand ebselen.

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main cellular thiol (14). Moreover, the in-hibitory action of propyl-2-methoxy-2-phen-yl selenide and propyl-2,2-diphenyl disele-nide (Figure 1) on ALA-D activity seems tobe due to their decomposition to DPDS (14).Other OS compounds such as phenylselenoacetylene inhibit ALA-D in vitro viaconversion to DPDS (15).

The mechanism underlying the inhibito-ry effect of DPDS is related to oxidation ofessential cysteinyl residues located at theactive site of the enzyme. Since oxyradicalscavengers do not interfere with the inhibi-tory effect of the tested compounds, DPDSapparently acts by oxidizing cysteinyl resi-dues in a similar way to that described forlow-molecular weight thiols (14). Based onthis evidence, Maciel and co-workers (16)showed that DPDS inhibits ALA-D fromliver, kidney and brain of mice in vivo. Theeffects were tissue-specific and dependenton exposure protocol (single dose or chronictreatment). The acute treatment of mice in-hibited the activity of ALA-D from liver andbrain but not kidney. After chronic expo-sure, brain and liver ALA-D were inhibitedin a dose-dependent manner, with the effectsbeing greater in liver than in brain. Oneinteresting finding is that the treatment spe-cifically affects enzyme activity without re-ducing the total content of non-protein SHgroups. In contrast to in vitro results (8),DTT could not completely reactivate theinhibited enzyme, suggesting that the oxida-tion caused by organoselenium might havealtered the enzyme conformation in such away that DTT could no longer reactivate it(14,16).

DPDS inhibits mammalian ALA-D butnot the plant enzyme. This is presumablyrelated to differences in the primary struc-ture of these enzymes, i.e., in the quantityand spatial proximity of cysteinyl residueswithin the protein. The mammalian enzymepossesses two distinct classes of zinc-bind-

ing sites, one containing a single cysteineresidue and the other four cysteine residues,whereas the plant enzyme has no cysteineresidues in these regions. This led Rocha andco-workers (17,18) to propose a mechanismfor the differential action of DPDS. In thefirst step, enzyme reaction with DPDS givesan unstable intermediate of the enzyme-Cys-S-SePh and selenophenol type, probably dueto a thiol nucleophilic attack in the seleniumatom of DPDS. Subsequently, the other cys-teinyl residue, due to its close spatial prox-imity to the more reactive residue, attacksthe sulfurselenium bond of the intermediate,producing the oxidized enzyme (inactive),and regenerates a second molecule of sele-nophenol. Support for this mechanism hasbeen obtained using low-molecular weightthiol-containing molecules. The selenophe-nol molecules formed after reaction with thethiol group are oxidized back to DPDS byatmospheric O2. The oxidation of selenophe-nol helps to explain the previous observationthat the inhibitory effect on rat ALA-D de-creases considerably in an anaerobic atmos-phere (17,18).

In agreement with these observations,enzyme inhibition in human erythrocytescan be avoided by mercaptan DTT and theactivity of inhibited ALA-D can be restoredby treatment with this compound. However,the inhibitory effect was not reversed byGSH; even 3 mM GSH gave only a modestincrease in ALA-D activity that had beeninhibited by 40 and 100 µM DPDS (19).These results clearly reinforce the sugges-tion that DPDS inhibits ALA-D in humanblood by interacting with cysteinyl residuesthat are important for enzyme activity. Theimportance of thiol groups for ALA-D ca-talysis is well known (20). In this case, DTTwas more protective than GSH; only themore hydrophobic SH-reducing agent is ableto reactivate SH groups localized in thedeeper regions of the enzyme. Alternatively,

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the oxidation caused by DPDS in ALA-Dmay modify enzyme conformation in such away that GSH is unable to reactivate theenzyme. In this manner, in vitro DPDS inac-tivates human erythrocyte ALA-D by inter-acting with the SH groups essential to theenzyme (19).

As pointed out above, OS compoundscan interact directly with low-molecularweight thiols, oxidizing them to disulfidesthat in turn oxidize cysteinyl residues inproteins. ALA-D is an SH-containing en-zyme that is extremely sensitive to oxidizingagents and thus represents an excellent toolto study this type of interaction. DPDS, inoxidizing essential SH groups from ALA-D,is converted into a selenol and the resultsobtained with mammalian ALA-D and DPDSsuggest that proteins containing cysteinylresidues in close spatial proximity in theirthree-dimensional structure will react morepromptly with DPDS. Recently, it waspointed out that this OS compound does notinhibit mouse hepatic ALA-D by an oxida-tive stress-mediated mechanism, since thelevels of thiobarbituric reactive substances(TBARS), a marker of oxidative damage,were not increased (20). The cellular toxic-ity of this compound may thus be related, atleast in part, to the oxidation of vicinal SHgroups of target proteins. In addition, ALA-D has been demonstrated to be a potentialmolecular target for selenium and OS intoxi-cation.

Pro-oxidant activity in yeast

Since DPDS has high thiol peroxidaseand GPX mimetic activities, we investigatedits antioxidant properties using several invitro assays and mutant strains of Saccharo-myces cerevisiae deficient in superoxide dis-mutase, GSH biosynthesis and the yAP-1transcription factor. Unexpectedly, DPDSper se had no cytotoxic effects on S. cerevi-

siae. However, it sensitized yeast cells tooxidative mutagens, hydrogen peroxide,paraquat, tert-butyl-hydroperoxide, and so-dium nitrite, suggesting that it has an indi-rect pro-oxidant effect (21). In spite of thislack of antioxidant defense, the response ofall strains of S. cerevisiae tested was identi-cal, indicating the existence of a general, butnot necessarily nonspecific, mechanism ofindirect cytotoxicity. The GSH constitutesthe common and main defense system againstreactive oxygen species (ROS). This mole-cule also contributes to detoxification ofelectrophilic compounds, sequestration oftoxic metals and modulation of the cellularredox and thiol-disulfide status of proteins(22).

Measurement of GSH in yeast cellstreated with the OS molecule confirmed thatthis drug depleted cellular GSH without in-creasing GSH oxidation rate, explaining thepro-oxidant effect. Accordingly, the DPDSpro-oxidant activity could be lowered by N-acetylcysteine pre-treatment, increasing GSHlevels in yeast (21).

We have frequently employed a set ofmutant strains involved in GSH biosynthesisin our investigations. The yeast gsh1 mutantis unable to synthesize the first dipeptideintermediate of GSH and, lacking endoge-nous GSH synthesis, requires exogenous glu-tathione to survive and grow in minimalmedia. When a second mutation, lwg1-1, isintroduced into the gsh1 mutant, the result-ing double mutant does not require any ex-ogenous glutathione since the lwg1 missensemutation, affecting the PRO2 gene and en-coding an altered γ-glutamyl phosphate re-ductase, can synthesize a small amount of γ-glutamyl-cysteinyl dipeptide, thus allowingsynthesis of a minimal amount of GSH. Thedifferential response to DPDS of the gsh1mutant of low GSH content and of the doublemutant gsh1 lwg1-1 confirms the involve-ment of GSH in protection against this OS in

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a dose-dependent manner (21).The thiol moiety of GSH also has unique

redox and strong nucleophilic properties,permitting its conjugation with electrophiliccompounds, xenobiotics in particular (23).DPDS, an electrophilic molecule, is able toform an as yet undescribed GSH adduct,most probably by conjugating via nucleo-philic attack to the thiol moiety of GSH; thisreaction apparently occurs spontaneously invitro and is predicted to result in a reducedstate of one or both selenium moieties inDPDS. This reaction is more likely than areduction of the diselenide bond by GSH,which is not favored since the redox poten-tial of GSH is much higher than that of thediselenide bond (21,23). This phenomenonwould explain the depletion of intracellularfree GSH by DPDS that results in the ob-served pro-oxidant properties of the drug,i.e., sensitization of DPDS-pre-treated yeastcells, regardless of their genetic endowmentwith anti-oxidant defense mechanisms, to anoxidative mutagen challenge.

Maciel and co-workers (16) observed thatDTT, GSH, and other thiols could be oxi-

dized by DPDS, but that oxidation of DTT isfavored over oxidation of GSH and cysteine.In the interaction between ALA-D andDPDS, oxidation is the main mechanism bywhich DPDS indirectly acts as a pro-oxidantby enzyme inhibition. In yeast we could notshow accumulation of extracellular oxidizedglutathione (GSSG) after DPDS treatment,as would be expected after direct reductionof the diselenide bond. We suggest, there-fore, that the DPDS-induced pro-oxidant ef-fect in yeast is a direct result of GSH deple-tion via the production of DPDS-GSH ad-ducts.

Genetic toxicology

Our group has also investigated the po-tential genotoxicity of DPDS. This wasdeemed important since DPDS is used as anintermediate and reagent in organic synthe-ses, which increases the risk of human expo-sure in the workplace. This information isalso very important to evaluate the safety ofpossible future pharmacological applicationsof the compound. The genotoxicity of DPDShas been assayed in Salmonella typhimu-rium and in the simple eukaryote S. cerevi-siae, whose mutagenesis and DNA repairmechanisms were assessed using haploidstrains, whereas recombinogenesis was testedin a diploid strain (4).

We suggested also that the mutageniceffect of DPDS is an indirect effect mediatedby free radicals. DPDS was able to stronglyenhance the oxidative mutagenesis inducedby hydrogen peroxide in S. typhimuriumTA102, which has a proven ability to detectmutagens that generate ROS and cause oxi-dative DNA damage. A modulatory (en-hancer) role in the effect of oxidative mu-tagens is suggested for DPDS (Figure 2).Moreover, it is known that DPDS acts as apro-oxidant in yeast strains, causing GSHdepletion without increasing the rate of GSH

YeastBacteria

Frameshift mutation

DPDS-induced DNA intercalation

Glutathione depletion

Increased ROS

SSB/DSB

SeSe

SeSe

Figure 2. A possible model of the genotoxic mechanism of action of diphenyl diselenide(DPDS). ROS = reactive oxygen species; SSB = single-strand breaks; DSB = double-strand breaks.

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oxidation (22). Thus, as expected, N-acetyl-cysteine, a GSH biosynthesis precursor, abol-ished the mutagenicity of DPDS in yeast,probably by conjugation with DPDS, block-ing its DNA damaging potential.

To gain insight into the mechanisms ofthe genotoxic action of DPDS, we have stud-ied the response of S. cerevisiae mutantsdefective in DNA repair to treatment withthis OS compound. Mutants defective inexcision-resynthesis repair (rad3-e5, rad1∆),in error-prone repair (rad6∆) and in recom-binational repair (rad52-1) were used in thisinvestigation. In this respect, the single mu-tants rad3-e5 and rad1∆, blocked in exci-sion repair, and rad6∆, defective in the mu-tagenic repair pathway, showed practicallythe same sensitivity as the wild-type strain.The hypersensitivity of the rad52-1 mutantstrain suggests that recombinational repair iscritical for the processing of potentially le-thal genetic lesions resulting from DPDS-induced DNA damage. Thus, it appears thatthe OS compound probably induces single(SSB)- and/or double-strand breaks (DSB)in DNA (4).

The cytotoxic responses of the singlemutant rad52-1 and double mutant rad3-e5rad52-1 to DPDS are identical, which is aconsequence of the absence of the RAD52pathway. In S. cerevisiae, members of theRAD52 epitasis group are indispensable forsuccessful meiotic and mitotic recombina-tion and survival after treatment with ioniz-ing radiations, alkylating agents, some oxi-dative mutagens, and cross-linking agents(24). These results suggest that the DSBrepair system is the main pathway requiredto overcome the induced lesions, whereasthe excision repair and mutagenic systemsare not primarily involved.

In order to examine the contribution ofthe base excision DNA repair pathway (BER)to DPDS-induced DNA damage, severalsingle, double, triple and quadruple mutants

were evaluated regarding survival of theirmutation rate following exposure to DPDS.Figure 3 shows that the recombination-defi-cient rad52 mutant strain presents highsensitivity to DPDS, whereas the other mu-tants showed the same sensitivity as thewild-type strain. The quadruple mutant ntg1ntg2 apn1 rad52 had the same sensitivity asthe single mutant rad52, supporting the par-ticipation of the recombination pathway indefense against genotoxic lesions generatedby DPDS.

The results shown in Table 1 suggest thatDPDS treatment increases the mutation ratein BER-deficient strains. The increase in thefrequency of forward mutation induced byDPDS in the quadruple mutant blocked inboth BER and nucleotide excision repair(NER) (ntg1 ntg2 apn1 rad1) suggests thatan error-prone pathway repairs (some of) theDNA lesions. However, the quadruple mu-tant for BER and translesion synthesis (ntg1ntg2 apn1 rev3), after DPDS exposure, showsinduction of forward mutation. In this re-spect, the results of mutagenesis, also sup-ported by the resistance of the single mutantrev3∆, show that other translesion enzymes,like Rad30p (24), contribute to the cell’sDPDS tolerance. Furthermore, the increasein the frequency of forward mutation in-duced by this molecule in the quadruplemutant for BER and recombinational repair(ntg1 ntg2 apn1 rad52) suggest an interac-tion of different DNA repair pathways re-lated to the response to DPDS.

On this basis, our findings suggest thatthe N-glycosylases and AP-lyases of theBER pathway, which primarily involves therepair of small, helix non-distorting baselesions, abasic sites, and oxidative DNAdamage, have a crucial role in preventing theoxidative mutagenesis induced by this mol-ecule. However, the recombinational repairsystem is the main pathway required to over-come the lesions generated by the oxidative

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Wild-type BY4741Wild-type SJR751ntg1∆ntg1∆ ntg2∆ntg2∆apn1∆rad1∆rev3∆ntg1∆ ntg2∆ apn1∆ntg1∆ ntg2∆ apn1∆ rad1∆ntg1∆ ntg2∆ apn1∆ rev3∆

DPDS (µM)

rad52∆

ntg1∆ ntg2∆ apn1∆ rad52∆

Table 1. Induction of forward mutation of the CAN1 locus (Canr mutants per 108 survivors) in haploid strains of Saccharomyces cerevisiaedefective in DNA repair pathways, after diphenyl diselenide (DPDS) treatment during exponential growth in liquid-rich medium containing DPDS atdesired concentrations.

Relevant genotype/ Mutation rate (Canr mutants per 108 survivors)DPDS exposure

0 0.01 mM 0.1 mM 1 mM 10 mM

Wild-type BY4741 0.22 ± 0.09 0.32 ± 0.10 0.39 ± 0.11 0.16 ± 0.05 0.28 ± 0.09rad1∆ 0.26 ± 0.12 0.34 ± 0.11 0.20 ± 0.05 0.29 ± 0.10 0.31 ± 0.06rev3∆ 0.38 ± 0.02 0.41 ± 0.05 0.41 ± 0.08 0.45 ± 0.10 0.48 ± 0.10rad52∆ 0.31 ± 0.08 0.53 ± 0.07* 0.85 ± 0.06* 1.12 ± 0.36* 1.52 ± 0.10*Wild-type SJR751 3.8 ± 0.70 4.1 ± 1.10 2.9 ± 0.80 5.0 ± 2.10 4.8 ± 1.90ntg1∆ 9.5 ± 1.34 8.0 ± 1.77 14.3 ± 2.85 24.3 ± 5.47* 31.5 ± 9.44*ntg2∆ 11.2 ± 0.90 12.5 ± 0.70 10.9 ± 2.10 18.4 ± 1.80 26.9 ± 0.55*ntg1∆ ntg2∆ 18.8 ± 2.44 21.5 ± 3.00 14.9 ± 1.20 52.4 ± 6.44* 72.0 ± 1.00*ntg1∆ ntg2∆ apn1∆ 12.3 ± 0.50 18.8 ± 2.30 15.4 ± 2.47 32.9 ± 5.00* 55.8 ± 9.00*ntg1∆ ntg2∆ apn1∆ rad1∆ 259.0 ± 21.30 312.2 ± 18.47* 405.8 ± 21.00* 667.1 ± 50.45* 730.0 ± 81.00*ntg1∆ ntg2∆ apn1∆ rad52∆ 85.0 ± 14.70 159.8 ± 9.20* 232.9 ± 21.30* 389.8 ± 62.00* 400.7 ± 32.10*ntg1∆ ntg2∆ apn1∆ rev3∆ 168.4 ± 12.80 258.1 ± 22.00* 325.7 ± 6.47* 554.1 ± 18.00* 861.3 ± 14.80*

Data are reported as means ± SD for 4 independent experiments carried out in triplicate. The strains rad1∆, rev3∆, and rad52∆ are derivatives ofthe wild-type BY4741, whereas the other mutants are isogenic to strain SJR751.*P < 0.05 compared to DPDS non-treated cells (ANOVA followed by the Tukey test).

Figure 3. Survival of Saccharomycescerevisiae strains deficient in severalDNA repair pathways after treatmentwith different doses of diphenyl disele-nide (DPDS) during exponential growthin liquid-rich medium.

Sur

viva

l (%

)

100

10

10 2 4 6 8 10

DPDS (µM)

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damage - DNA DSB and closely locatedSSB - or by the SSB repair process that can,in turn, result in DSB - and rescue the sur-vival after DPDS exposure.

Although oxidative damage can be theprime mechanism explaining the genotoxic-ity of this molecule in yeast, a possibleintercalant effect may be also involved in thegenotoxic effect. DPDS cytotoxicity may belinked to its ability to intercalate into DNA.As observed in the Ames test, DPDS clearlycaused frame shift mutations only in expo-nentially growing cells of S. cerevisiae. Sincechemical compounds with planar topologiesand electrophilicity may intercalate betweenDNA bases, the mutagenic response is ob-served mainly as frame shift mutagenicity inmicrobial systems. DPDS is an electrophilicmolecule with an aromatic planar structure,which induces frame shift mutation onlyduring cellular growth, when the access toDNA is facilitated; we believe that this com-pound is a weak intercalator mutagen. Frameshift mutations induced by DNA-intercalat-ing drugs have been correlated with DNAstrand breaks induced by inhibition of DNAtopoisomerases. Since DNA topoisomeraseII inhibition induces breaks which are re-paired almost exclusively by Rad52-depend-ent homologous recombination in yeast, thehigh sensitivity of the rad52 mutant couldinvolve, at least in part, the interference ofDPDS with topoisomerase II activity (24).Thus, the putative intercalant effect of DPDSmay involve strand break generation by theaction of topoisomerases.

In view of its genotoxic properties inmicroorganisms, we investigated whetherDPDS might be inducing DNA damage inMCF7 cells, a mammary adenocarcinomacell line, using the in vitro alkaline cometassay. The alkaline version of the cometassay detects primary (repairable) DNA SSBand DSB and alkali-labile sites. DPDS didnot generate significant DNA damage in a

Figure 4. Genotoxicity of diphenyl diselenide (DPDS)treatment for 3 h in serum-free medium to the MCF7cell line evaluated by the comet assay. Data are re-ported as means ± SD for 6 independent experiments.*P < 0.05 compared to control (ANOVA followed by theTukey test). Positive control: 40 µM methylmethanesul-fonate. Damage index is an arbitrary score calculatedfrom cells in different damage classes, which are clas-sified in the visual score by the measurement of DNAmigration length and on the amount of DNA in the tail.

Figure 5. Pro-oxidant effect of treatment with diphenyldiselenide (DPDS) for 3 h in serum-free medium on thegenotoxicity of 150 µM H2O2 to the MCF7 cell lineevaluated by the comet assay. Data are reported asmeans ± SD for 6 independent experiments. *P < 0.05compared to cells treated only with hydrogen peroxide(one-way ANOVA followed by the Tukey test). Damageindex is an arbitrary score calculated from cells indifferent damage classes, which are classified in thevisual score by the measurement of DNA migrationlength and on the amount of DNA in the tail.

dose-response manner (Figure 4). In addi-tion, it can be seen that pre-treatment withDPDS potentiated the DNA damage inducedby hydrogen peroxide, supporting the viewof a clear pro-oxidant ability (Figure 5). This

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finding supports the genotoxic effects of DPDSon mammalian tumor cells and suggests itspotential in antiproliferative therapy.

Recently, we evaluated the cytotoxic, pro-oxidant, genotoxic, and mutagenic proper-ties of this molecule in V79 Chinese lungfibroblast cells (25). When cells were treatedwith increasing concentrations of DPDS, itscytotoxic activity, as determined using fourcell viability endpoints, occurred at doses upto 50 µM. The MTT reduction was stimulat-ed, which may indicate ROS generation.Accordingly, the treatment of cells for 3 hwith cytotoxic doses of DPDS increasedTBARS levels and sensitized cells to oxida-tive challenge, indicating a pro-oxidant ef-fect. The measurement of total, reduced, andoxidized GSH showed that DPDS can leadto intracellular GSH depletion, with no in-crease in the oxidation rate in a dose- andtime-dependent manner. At the higher doses,DPDS generated DNA strand breaks, as ob-served using the comet assay. The treatmentalso induced an increase in the number ofbinucleated cells in the micronucleus test,showing the mutagenic risk of this moleculeat high concentrations. Finally, pre-incuba-tion with N-acetylcysteine, which restoredGSH to normal levels, annulled the pro-oxidant and genotoxic effects of DPDS.These findings show that DPDS-inducedoxidative stress and toxicity are closely re-lated to intracellular level of reduced GSH,in agreement with our previous findings inyeast and bacteria.

Reproductive toxicology

The reproductive and teratogenic effectsof DPDS have been evaluated. Favero et al.(26,27) showed that a single subcutaneousinjection of 50 and 100 mg/kg DPDS led toan increased incidence of skeletal variationsor anomalies in pregnant Wistar rats, but didnot cause externally visible malformations

in rat fetuses and did not affect the progeny.DPDS administered either intraperitoneally(acute treatment) or subcutaneously (sub-chronic treatment) to adult rats also had noreproductive toxicology (27,28). However,recent studies on the effects on embryo-fetaldevelopment in dams that were treated sub-cutaneously with DPDS at 6 to 15 days ofpregnancy have shown evidence of toxicity.A decrease in maternal body weight gain, areduction in fetal weight and crown-rumplength, and signs of delayed ossification inthe skull, sternebrae and limbs were ob-served. Exposure of dams to DPDS alsoresulted in altered placental morphology thatmay have contributed to an adverse repro-ductive outcome. The teratogenic and po-tential reproductive toxicologic aspects ofDPDS and the mechanisms involved remainunclear.

Neurotoxicology

Exposure for 2 months to high doses ofDPDS (250 µmol/kg, subcutaneous admin-istration) induced a 3-fold increase of thetotal selenium content of the brain, showingthat DPDS crosses the blood-brain barrierand brain selenium levels increase in miceafter acute and chronic exposure (29,30).This supports the hypothesis that the brain isa potential target for the toxicity of highlylipophilic OS compounds and possibly fortheir pharmacological and therapeutic ac-tions. The mechanisms underlying the neu-rotoxicity of OS compounds and their de-rivatives have been investigated. When neu-rotoxicologic effects are considered, the tox-icity of OS compounds depends on theirchemical form, experimental model, age,physiological state, nutrition and dietary in-teractions, and the route of administration.

In terms of toxicity, the OS compoundebselen (Figure 1) (molecular weight = 274.2)was more toxic than DPDS (molecular weight

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= 310.0) in rats acutely treated by the intra-peritoneal route (with an LD50 of 1200 µmol/kg for DPDS and of 400 µmol/kg for ebselen)(31,32). In addition, DPDS was more toxicin mice than rats when administered by theintraperitoneal route (LD50 of 210 and 1200µmol/kg, respectively) and had no acute toxiceffects when administered by the subcutane-ous route (32).

OS compounds have convulsing activ-ity. Intraperitoneal, but not subcutaneous,administration of DPDS provoked seizuresin mice but not in rats. In addition, GABA-ergic allosteric modulators such as diaze-pam, phenobarbital and muscimol, and acompetitive muscarinic antagonist of theacetylcholine receptor, atropine, were ableto consistently abolish or reduce the seizureepisodes, suggesting that the modulation ofone or more neuronal systems can accountfor the convulsive effect (33,34).

DPDS also affected several neuronal pro-cesses. It inhibited the binding of (3H)-gluta-mate, (3H)-MK-801, and unstable (3H)-guan-ylyl-imidodiphate (GMP-PNP) to rat synap-tic membrane preparations after both in vitroand ex vivo exposure; these are importantindicators of glutamatergic system function-ality (18). An inhibitory effect on (3H)-glu-tamate and (3H)-MK-801 binding indicatedthat the toxicologic properties of DPDS maybe related, at least in part, to the inhibition ofthe physiological excitatory neurotransmit-ter system. Experimental results from acuteexposure (25 µmol/kg) showed that DPDSdrastically reduced (3H)-GMP-PNP bindingto G-proteins and the response of adenylatecyclase activity to GMP-PNP, and also in-creased basal adenylate cyclase activity, sug-gesting a direct effect on this enzyme. Thisstudy also revealed reduction of (3H)-gluta-mate uptake by brain synaptosomes (18). Inhuman platelets this molecule caused a sig-nificant inhibition of Na+-independent glu-tamate binding to the membrane. In the same

study, it inhibited (3H)-glutamate uptake (18).Calcium channels, mediating Ca2+ in-

flux, play a central role in neurotransmissionand chemical agents that potentially inter-fere with Ca2+ homeostasis are potential toxicagents. DPDS reduced 45Ca influx into iso-lated nerve endings of rat synaptosomes whena non-depolarizing condition was used orwhen 4-aminopyridine was used as a depo-larizing agent (35). However, the same ef-fects were not observed on excitotoxicityinduced by glutamate in an isolated chickretina model (36).

In summary, the neurotoxic effects ofDPDS could result from its action at differ-ent levels of neural signal transduction path-ways, certainly involving other neurotrans-mitters besides the glutamatergic system.

Pharmacological effects

The pharmacology of synthetic OS com-pounds that have been subjected to morethan just a biological screening was criti-cally evaluated in a review by Parnham andGraf (37, and see Refs. 1,6). During the pastdecade, a lot of effort has been directedtoward the development of stable OS com-pounds that could be used as antioxidants,enzyme inhibitors, anti-tumor and anti-in-fectious agents, cytokine inducers, and im-munomodulators (1). In addition, many com-pounds have been studied. In this section,we explore the potential pharmacologicalapplications of DPDS.

Antioxidant effects

The essential dietary trace element sele-nium plays a crucial role in the catalyticcenter of GPX, an enzyme that catalyzes thereduction of various hydroperoxides by GSHand scavenges ROS, thus protecting cellsagainst oxidative stress (3). However, GPXhas some shortcomings, e.g., instability, poor

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availability and high molecular weight. Thesedrawbacks limit its therapeutic applicationand research has, therefore, centered on de-veloping synthetic drugs with superior per-formance (6,38-40).

Research in recent years has providedsolid evidence for the role of ROS in humandisease and this knowledge has led to newideas for the therapy of a variety of diseases.The concept that selenium-containing mol-ecules may be better nucleophiles than clas-sical antioxidants has led to the design ofsynthetic OS drugs. Intensive research in OSbiochemistry during the last two decades hasyielded a variety of compounds with poten-tial antioxidant activity, including ebselenanalogues, benzoselenazolinones, selena-mide and related derivatives, diaryl disele-nides, various tellurides and ditellurides, andthe semi-synthetic enzyme selenosubtilisin(1,3,41). The promising compound ebselenis an example of such a molecule; it hasantioxidant properties and is known as themost important glutathione peroxidase mi-metic agent. Unlike inorganic selenium andselenomethionine, it is not toxic to mammalssince it contains bound selenium and thusdoes not contribute to the cellular seleniumpool (7,39,40).

The properties of some synthetic disele-nides suggest that the cleavage and reduc-tion of their diselenide bond produces theselenol group, and therefore the diselenideswere successful in mimicking GPX (1). Di-selenides are good candidates as antioxidantagents because they have some chemicaland biochemical characteristics in commonwith ebselen (18). Of particular importance,DPDS has been shown to be even moreactive as a GPX mimetic and less toxic torodents than ebselen (31,32).

The in vitro catalytic activity of DPDS inhydrogen peroxide decomposition was stud-ied by the method that uses benzenethiol asan alternative to GSH. The initial rates of

hydrogen peroxide reduction by thiol in thepresence of various catalysts were deter-mined in methanol medium by monitoringthe UV absorption at 305 nm due to theformation of DPDS. In this system, DPDSexhibits good initial rates of hydrogen per-oxide reduction (0.55 against 0.15 µM/minin the absence of catalysts). Indeed, DPDSexhibits GPX-like activity and low-GSHoxidase activity; in the presence of a 10-foldexcess of GSH, the diselenide bond in DPDSis reduced to form selenol as the predomi-nant species, which reacts very rapidly with0.5 equivalents of t-butyl-hydroperoxide(23). The reduction of the diselenide bond inDPDS by GSH may not be an easy reaction,since the redox potential of GSH (Eo = -240mV) is much higher than that of the bond (Eo= -380 mV) (1,18,23).

DPDS, as well as ebselen, reduced thespontaneous and quinolinic acid- and so-dium nitroprusside-induced TBARS in ratbrain homogenates to basal rates, demon-strating its antioxidant activity (41,42). Theantioxidant potential, thiol peroxidase activ-ity and rate of DTT and GSH oxidation havebeen evaluated in rats and mice. DPDS alsoefficiently reduced lipid peroxidation inmouse and rat brain, with similar resultsapart from species differences. It also exhib-ited high-thiol peroxidase activity and dem-onstrated better antioxidant potential thanthe other diselenide substitutes tested (41,42).

DPDS has been recently shown to beeffective in restoring acute cadmium-inducedoxidative damage in mouse testes, and moreeffective than the chelating compounds suci-mer and 2,3-dimercapto-1-propanesulfonicacid (43-46). Lipid peroxidation has longbeen considered to be the primary mechan-ism for cadmium toxicity despite its inabil-ity to generate free radicals under physi-ological conditions. Thus, it is believed thatan antioxidant should be one of the impor-tant components of an effective treatment

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for cadmium poisoning. Santos and co-work-ers (44) clearly showed the ability of DPDStherapy to restore enhancement of TBARSlevels caused by sub-chronic cadmium ex-posure in liver and brain of mice. This indi-cates that the use of DPDS as an antioxidantmight be useful in the treatment of cadmiumpoisoning since it has the capacity to allevi-ate many of the harmful effects of cadmium.Unexpectedly, DPDS treatment potentiatedthe testicular damage caused by cadmium ina model using sub-chronic cadmium expo-sure. This finding again indicated the pro-oxidant capacity of DPDS, and the two-faceproperties of OS compounds.

DPDS also protects against inhibition ofliver ALA-D during paracetamol intoxica-tion by its antioxidant GPX mimetic activity(18). Recently, an in vitro evaluation of theantioxidant effect of DPDS against sodiumnitroprusside-induced lipid peroxidation wascarried out. At 2 µM, this OS molecule sig-nificantly protected human platelets and re-stored GPX activity. These phenomena seemto be related to its thiol peroxidase-like ac-tivity (47). Hence DPDS is a promising anti-oxidant molecule.

However, although the peroxidase-likeactivity of diselenides may account for theirantioxidant properties, the thiol-diselenideexchange catalyzed by chalcogenides maycontribute to their toxicologic properties byoxidizing relevant thiol-containing metabo-lites and proteins without consuming toxicsubstances like peroxides. Selenides can re-act with -SH groups, forming selenosulfideor -SeH and metabolites.

In contrast, using assays such as 1-chloro-2,4-dinitrobenzene complex formation andUV absorption, our group has recently shownthat DPDS can spontaneously react withGSH, forming stable adducts. This propertyconfers not an antioxidant but a pro-oxidantability, a common paradox in the biologicaleffects of OS. Thus, the main reactions be-

tween DPDS and thiols are oxidation andconjugation. These results have been ob-tained in vivo using ALA-D inhibition andthe investigation of pro-oxidant activity in S.cerevisiae, as previously mentioned (21).

Liver and kidney

The hepatic toxicity of DPDS was thefirst of its biological properties to be studied.The acute toxicity of DPDS in the maleSwiss mouse was enhanced by pre-treat-ment with phenobarbital, which induces thecytochrome P-450 system. In this study,DPDS decreased hepatic GSH content by50% after 1 h and OS metabolites werefound in urine but not in bile or feces (48).DPDS was more toxic to mice than ratswhen administered by the intraperitonealroute and had no toxic effects when admin-istered subcutaneously (31). However, athigher doses DPDS did not alter hepaticenzyme activity and did not change urea orcreatinine parameters, suggesting no renaltoxicity (31). Several research groups havedemonstrated that liver damage is a thera-peutic target of OS compounds, as well asthe various clinical conditions in which oxi-dative stress plays a main role (18).

Pre-treatment with DPDS preventedhepatotoxicity and cellular damage in the ratliver exposed to 2-nitropropane and sug-gested that the administration of 100 µmol/kg could be effective in mitigating the haz-ards of 2-nitropropane exposure through itsantioxidant properties, increasing superox-ide dismutase and catalase activities (49,50).It has also been reported to act as a hepato-protector in diabetic rats (51). In addition,DPDS protected against acute renal damage,as demonstrated by a decrease in urea levelsin rats exposed to 2-nitropropan. The mech-anism involved is the increase of catalaseand superoxide dismutase activity and againit is correlated with the antioxidant potential

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of DPDS (50). These studies provide practi-cal indications of the benefits of DPDS ad-ministration in protecting humans against avariety of hazardous environmental toxicantsthat induce liver and kidney damage.

Gastric mucosa

The major side effect of all non-steroidalanti-inflammatory drugs (NSAIDS) is dam-age to the gastric mucosa. In this respect, it isof considerable importance that OS compoundshave no such irritant or damaging effect. DPDShas been recently reported to prevent ethanol-and indomethacin-induced ulcers, as well asto inhibit gastric acid secretion in pylorus-ligated rats. It also protects against mucosaldamage by inhibiting the oxidation of the SHgroups of H+-K+ ATPase. This effect on theenzyme responsible for acid secretion cancounteract the undesirable side effects causedby NSAIDS. Thus, as anti-inflammatory drugs,OS compounds are versatile with regard togastric ulcers because not only do they blockthe inflammatory cascade, which can impairgastric endogenous protective factors againstHCl, but also inhibit HCl secretion. Since thetherapeutic approach to a variety of diseases ischanging from a single target to a multitargetone, it would be very useful to evaluate thepossible synergism of the association of OScompounds with other NSAIDS with respectto the anti-inflammatory response and to theantagonism of gastric ulcer development (18).

Anti-inflammatory activity

The role of ROS in inflammatory disor-ders has been the subject of intensive inves-tigation. Considerable effort has been ex-pended in the search for low-toxicity scav-engers and inhibitors of ROS. One of theapproaches has been to investigate syntheticOS compounds that may be therapeuticallybeneficial for the treatment of inflammatory

diseases.DPDS displayed the most promising pro-

file against carragenin-induced paw edema;p-methoxyphenyl diselenide and p-chloro-diphenyl diselenide (Figure 1) were less ef-ficient, p-methyldiphenyl diselenide (Figure1) being the weakest. DPDS has also beendemonstrated to be an anti-nociceptive com-pound, having an effective action in experi-mental models such as tail-flick, formalin,acetic acid-induced abdominal writhing, andcapsaicin. Interestingly, the anti-nociceptiveand anti-inflammatory potency of DPDS washigher than that of ebselen and was notrelated to opioid mechanisms (34,52). In-deed, the mechanisms underlying the anal-gesic action may be related to serotonergicpathways, but still remain unclear.

Neuroprotection

New drugs that preserve neuronal integ-rity, acting after an ischemic insult, are un-der investigation, including inducible nitricoxide synthase inhibitors, antioxidants, gluta-mate antagonists, and calcium channel block-ers. Data from clinical trials have consis-tently demonstrated that the OS compoundebselen reduced brain damage in patientswith delayed neurological deficits after an-eurysmal subarachnoid hemorrhage and im-proved the outcome of acute ischemic stroke,suggesting that ebselen may be a promisingneuroprotective agent (18,40).

In this context, reports on the neuropro-tective effects of other GPX mimetics haveappeared in the literature in recent years.DPDS has shown a neuroprotective role inan in vitro ischemia model using oxygen-glucose deprivation in rat hippocampal slices.It simultaneously prevented two effects inthis model: cell death and increase in induc-ible nitric oxide synthase immunocontent.These effects were dose-dependent and werestronger than those of ebselen (53).

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The effects of systemic administration ofDPDS on memory formation were studiedusing the novel object recognition task inmice. Novel object recognition is a type ofnon-aversive, non-spatial memory that re-quires glutamate receptor activation in thehippocampus. The results show that, in acutepre-training, systemic administration ofDPDS facilitated novel object recognition inmice without affecting other behavioral pa-rameters such as training performance, short-term memory or total time spent exploringboth objects. In addition, ebselen was shownto induce memory-impairing effects in aninhibitory avoidance task in rats when in-fused post-training into the dorsal hippo-campus (54). However, DPDS facilitatednovel object recognition in mice (55).

In neurodegenerative diseases, the cyto-skeleton is abnormally assembled and im-pairment of neurotransmission occurs. Cu-mulative evidence suggests that neurode-generative diseases and psychiatric illnessare associated with cytoskeleton and loss ofsympathetic connectivity and of the abilityto transmit incoming axonal information tothe somatodendritic domain. DPDS pre-vented the inhibitory effects of the neuro-toxicant methylmercury on the in vitro phos-phorylation of intermediate filament cyto-skeletal proteins in slices of cerebral cortexof rats, but did not prevent the hyperphos-phorylation induced by diphenyl ditelluride(56,57).

Tardive dyskinesia is a motor side effectof long-term treatment with typical neuro-leptics; it involves involuntary movementsof the face, mouth and tongue, but differentparts of the body may be affected. An animalmodel for tardive dyskinesia is obtained bythe acute administration of reserpine to rats;this produces persistent oral dyskinesia. Thepathophysiology of the syndrome is unclear,but the neurodegeneration is linked to oxida-tive stress. Burger and co-workers (58,59)

used this model to study the effect of DPDSon behavioral and neurochemical param-eters after acute reserpine administration toold rats (15 months). The results showed thatthe antioxidant properties of DPDS, as wellas its anti-inflammatory properties, had onlya modest effect in this animal model (58).This OS molecule also reverses haloperidol-induced orofacial dyskinesia, another modelfor studying this extrapyramidal syndrome,possibly by its antioxidant properties (59).

Recently, using DPDS as a prototypecompound, the diselenide-structural analog3’3-ditrifluoromethyldiphenyl diselenide(DFDD, Figure 4) was developed. DFDDhas been shown to attenuate apomorphine-induced stereotypy at a dose which did notaffect other behavioral parameters, clearlyindicating an antipsychotic potential (60). Inaddition, this derivative is less toxic thanDPDS and does not induce seizures in mice(30). As is the case for DPDS, DFDD de-creased lipoperoxidation in mouse and ratbrain (41).

Concluding remarks

Characteristically, all OS compounds (in-cluding DPDS) show interesting biologicaleffects in different models and species (Table2), but they also exhibit paradoxical phar-macological properties that cannot be easilyexplained by the current biochemical meth-odologies. DPDS has antioxidative proper-ties both in vitro mammalian cell culturesand in murine models, despite the inhibitionof ALA-D. On the other hand, DPDS showspro-oxidative properties in yeast cells, whichcould be explained by the depletion of cellu-lar glutathione. Another paradoxical effectof DPDS, observed in murine models, is itsmechanism of action on the central nervoussystem. Although DPDS antagonizes theNMDA glutamatergic receptor, which im-pairs the formation of memory, this com-

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Table 2. Pharmacology and toxicology of diphenyl diselenide in several biological models.

Pharmacology Toxicology

Effect Model References Effect Model References

Antioxidative effects Chemical systems: glutathione 1,18,23 Thiol group Chemical systems 8peroxidase and thiol peroxidase oxidationactivities; protective against sodiumnitroprusside-induced lipidperoxidation

Murine models: quinolinic acid- 18,41-46 Murine models 14,16and sodium nitroprusside-induced Inhibition of ALA-D 17-19TBARS in rat brain homogenates in several organsand against oxidative stress induced and tissues inby cadmium chloride mammals

Neuroprotective agent In vitro ischemia model using 53 GSH Chemical systems: 16,21oxygen-glucose deprivation in rat depletion CDNB complexation andhippocampal slices GSH-DPDS interaction

Protection against methylmercury 56,57 Yeast strains (wild-type 21effects on the in vitro phosphorylation and antioxidant defenseof intermediate filament cytoskeletal defective strains)proteins in slices of rat cerebral cortex

Hepatoprotective effect Hepatotoxicity induced by 49,50 V79 cell culture 252-nitropropane in rats

Gastric mucosal Ethanol and indomethacin-induced 18 Neurotoxic Impairment of glutamatergic 18,33,34protection ulcers in rats effects transmission in mice and rats

Pylorus-ligated rats 18 Genotoxic and Ames test 4mutagenic

Cognitive enhancer Novel object recognition task in mice 55 Mutagenesis and 4recombinogenesis in yeast

Anti-inflammatory Carragenin-induced paw edema in 34,52 Comet assay and micro- 25mice nucleus evaluation in

V79 cells

Antinociceptive properties Tail-flick test in mice 34,52 Comet assay in MCF7 cells Presentstudy

Acetic acid-induced abdominal 34,52 Teratogenic Murine models 26-28writhing in mice evidence

Formalin-induced abdominal writhing 34,52in mice

Capsaicin effects in mice 34,52

Effect in animal models Acute administration of 58of tardive dyskinesia reserpine to old rats

Haloperidol-induced orofacial 59dyskinesia in rats

TBARS = thiobarbituric acid reactive substances; ALA-D = δ-aminolevulinate dehydratase; GSH = glutathione; CDNB = 1-chloro-2,4-dinitroben-zene; DPDS = diphenyl diselenide.

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pound is able to stimulate memory forma-tion in mice. Unfortunately, the biochemicalprinciples that drive this phenomenon arelargely unknown and more empirical dataare necessary to conclude how DPDS act onthe nervous system.

Taking into account all the biologicaleffects of DPDS and the other OS com-pounds reviewed here, more experimentsare needed to elucidate how they act oneukaryotic cells. Once a clearer pictureemerges, it might be possible to synthesizenew OS substances specifically applicable

to clinically important metabolic routes.

Acknowledgments

The authors thank Dr. Christine Gaylarde(Universidade Federal do Rio Grande doSul, Porto Alegre, RS, Brazil), Dr. MartinBrendel (Universidade Estadual de SantaCruz, Ilhéus, BA, Brazil) and Dr. CristinaPungartnick (Universidade Estadual de SantaCruz, Ilhéus, BA, Brazil) for critically read-ing the manuscript.

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