Mutagenesis Advance Access originally published online on February 10, 2008
Mutagenesis 2008 23(2):93-99; doi:10.1093/mutage/gem048
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Antioxidant and anti-mutagenic effects of ebselen in yeast and in cultured mammalian V79 cells
1Laboratório de Genética Toxicológica, Universidade Luterana do Brasil (ULBRA), Canoas, RS, Brazil 2Departamento de Biofísica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
Ebselen has a wide spectrum of interesting therapeutic actions including antioxidant, cytoprotective, neuroprotective and anti-inflammatory activities. Since its antioxidant effect is very well known, this paper links the effects of ebselen in redox cellular status to its possible involvement in the maintenance of the integrity of genomic information by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defences and the mammalian V79 cell line. Using the alkaline comet assay, we showed that 5–10 µM ebselen does not induce DNA damage in V79 cells. Similarly, these same concentrations diminished the extent of the DNA damage induced by hydrogen peroxide (H2O2). The modified comet assay using DNA glycosylases (formamidopyrimidine-DNA glycosylase and endonuclease II) showed that after pre-treatment with ebselen followed by exposure to H2O2, oxidative damage as recognized by these enzymes was significantly lower. In the same way, ebselen showed strong activity against H2O2-induced oxidative damage in the anti-mutagenic assay using S.cerevisiae N123 strain and in the antioxidative assay by using S.cerevisiae strains lacking antioxidant defences. This antioxidant effect was more pronounced for the gpx3 
mutant, which indicated that ebselen acts by mimicking the GPx3 catalytic activity. The results confirm that ebselen is involved in antioxidant defence and that its antioxidant ability contributes to its anti-mutagenic and anti-genotoxic action.
 |
Introduction |
|---|
 Top Introduction Materials and methodsResultsDiscussionFundingReferences |
|---|
It has been shown that selenium, an essential trace element for mammals, prevents cancer in many animal model systems and enhances cancer chemopreventive efficacy in humans (1
). In spite of extensive literature describing the anti-mutagenic and anti-carcinogenic effects of selenium compounds, little is known about their mode of action. In addition, selenium compounds have a ‘Janus property’—products with a double face—due to their contrasting behaviour, which depends on the concentration used. At low doses, Se exerts beneficial effects, whereas at high doses, it is toxic and possibly carcinogenic. The threshold concentration for these opposing activities has not yet been established (2).
As the doses of the main known dietary sources of selenium, such as selenomethionine, selenocysteine and inorganic selenium, are limited by toxicity, synthetic derivatives have been developed. In the last few decades, there has been an increasing interest in organoselenium (OS) biochemistry because the pharmacology of synthetic OS compounds revealed molecules that could be used as antioxidants, enzyme inhibitors, neuroprotectors, anti-tumour and anti-infectious agents, cytokine inducers, and immunomodulators (2,3).
Ebselen (Figure 1), 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a cyclic OS, which exhibits interesting therapeutic potential against a number of disease states involving oxidative stress, such as neurological disorders, acute pancreatitis, noise-induced hearing loss and cardiotoxicity, due to antioxidant, cytoprotective, neuroprotective and anti-inflammatory activities (2,4–8). This molecule is a very well studied OS compound since it is a potent glutathione peroxidase (GPx) mimetic (2,5,8–16). Interestingly, the beneficial effects of ebselen have been demonstrated in clinical trials for the treatment of patients with delayed neurological deficits after aneurysmal subarachnoid haemorrhage and acute ischaemic stroke (6,7,17–20).
|
Since oxidative damage is an important cause of DNA damage and mutation and of carcinogenesis (21
), this study aimed at deepening the existing knowledge on the effects of ebselen on redox cellular status and on the integrity of genomic information. The antioxidant, mutagenic and anti-mutagenic effects of ebselen were investigated in the yeast Saccharomyces cerevisiae strains proficient and deficient in antioxidant defences, while the genotoxicity profile and the anti-genotoxic properties of ebselen were studied in a permanent lung fibroblast cell line derived from Chinese hamsters (V79 cells) using the comet assay. |
Materials and methods |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Chemicals
Ebselen (Chemical Abstracts Series registry number 60940-34-3), 4-nitroquinoline oxide, sodium azide, amino acids (L-histidine, L-threonine, L-methionine, L-tryptophan, L-leucine, L-lysine), L-canavanine, nitrogen bases (adenine and uracil) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM), foetal bovine serum (FBS), trypsin–ethylenediaminetetraacetic acid (EDTA), L-glutamine and antibiotics were purchased from Gibco BRL (Grand Island, NY, USA). Agarose and low-melting point agarose were obtained from Invitrogen (Carlsbad, CA, USA). Hydrogen peroxide (H2O2) at 30% was purchased from Aldrich (Milwaukee, WI, USA). Yeast extract, bacto-peptone and bacto-agar were obtained from Difco Laboratories (Detroit, MI, USA). Formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (EndoIII) were obtained from New England BioLabs (Beverly, MA, USA). All other reagents were of analytical grade. The tissue culture flasks (bottles and dishes) were supplied by Nunc (Wiesbaden, Germany).
Yeast strains and media
The relevant genotypes of S.cerevisiae strains used in this work are given in Table I. Haploid strains XV185-14c and N123 were used in the mutagenicity assay. The yeast strains defective in antioxidant defence, Yap1 transcription factor, superoxide dismutase and GPx3, as well as their isogenic wild-type strain, were used in the evaluation of antioxidant potential in yeast. Media, solutions and buffers were prepared according to Burke et al. (22). Complete YPD medium, containing 0.5% yeast extract, 2% bacto-peptone and 2% glucose was used for routine growth of yeast cells. For plates, the medium was solidified with 2% bacto-agar. The minimal medium (MM) contained 0.67% yeast nitrogen base without amino acids, 2% glucose and 2% bacto-agar supplemented with the appropriate amino acids. Synthetic complete (SC) medium was MM supplemented with 2 mg adenine, 2 mg arginine, 5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine, 2 mg uracil, 2 mg tryptophan and 24 mg threonine per 100 ml of MM. For XV-185-14c strain mutagenesis, the omission media lacking lysine (SC-lys), histidine (SC-his) or homoserine (SC-hom) were used. Synthetic medium without arginine, supplemented with 60 µg/ml canavanine, was used for the detection of forward mutation in N123 strain.
|
For cell treatment, ebselen stock solutions were prepared immediately prior to use by dissolution in DMSO. The subsequent dilutions were carried out in distilled water and DMSO concentration in the incubation mixture never exceeded 0.2%. All treatments were carried out in the dark.
Yeast growth
Stationary phase cultures were obtained by inoculation of an isolated colony into liquid YPD medium. After 48 hours incubation at 30°C with aeration by shaking, the cultures contained 1–2 x 108 cells/ml. Cells were harvested and washed twice with saline solution (NaCl 0.9%). Cell concentration and percentage of budding cells in each culture were determined in a Neubauer chamber by microscope counts.
Growth inhibition assay in yeast cells
An inoculation loop of cells from a stationary phase cell suspension was streaked from the center to the rim of a Petri dish with complete medium in one continuous stroke. A filter paper disk was placed in the center of the dish and increasing ebselen concentrations (10, 25, 50 and 100 µM) were applied onto the disk. Dishes were pre-incubated for 4 hours at 30°C. Afterwards, 5 µl 30% H2O2 was placed on a same filter paper disk and incubated for 2 days at 30°C. Impaired growth was measured as centimetre of growth inhibition from the border of the filter disk to the beginning of cell growth. Values ranged from 0 (complete growth to the filter disk) to 3 cm (absence of growth to the rim of the Petri dish).
Detection of ebselen-induced reverse and frameshift mutation in S.cerevisiae
The strain XV185-14c was used for this evaluation. A suspension of 2 x 108 cells/ml in stationary phase was incubated for 2 hours at 30°C with different ebselen concentrations in phosphate-buffered saline (PBS) solution, pH 7.4. After treatment, appropriate cell dilutions were plated onto SC medium to determine cell survival (3–5 days, 30°C) and in the appropriate omission media (7–10 days, 30°C) to evaluate mutation induction (LYS, HIS or HOM revertants). Whereas his1-7 is a non-suppressible missense allele and reversions result from mutation at the locus itself (23), lys1-1 is a suppressible ochre nonsense mutant allele (24) which can be reverted either by locus-specific or forward mutation in a suppressor gene (25). True reversions and forward (suppressor) mutations at the lys1-1 locus were differentiated according to Schuller and von Borstel (26), where the reduced adenine content of the SC-lys medium shows locus reversions as red and suppressor mutations as white colonies. The hom3-10 mutant allele of haploid strain XV185-14c was used for assaying putative frameshift mutagenesis. It is believed that hom3-10 contains a frameshift mutation due to its response to a range of diagnostic mutagens (25). Assays were repeated at least four times and plating was performed in triplicate for each dose.
Detection of ebselen-induced forward mutation in S.cerevisiae
N123 strain was used for the evaluation of mutagenicity as well as of the protective effect of the ebselen against H2O2-induced mutagenesis since this strain is very responsive to H2O2 because it shows low glutathione content (27). Forward mutation was measured with the canavanine resistance assay (CAN1 
can1). Wild-type yeast strains express the arginine transporter which also imports canavanine, toxic akin, lead up to cell death. Alterations in the CAN1 gene that impair Can1p functionality can increase cellular survival in the presence of canavanine. Cell suspensions in stationary growth phase (2 x 108 cells/ml) were incubated in PBS, for 1 hour at 30°C with different ebselen concentrations. After treatment, appropriate cell dilutions were plated onto SC plates to determine cell survival, and 100 µl aliquots of cell suspensions (2 x 108 cells/ml) were plated onto SC media supplemented with 60 µg/ml canavanine in order to determine forward mutation in CAN1 locus. Mutants were counted after 4–5 day incubation at 30°C.
For anti-mutagenesis evaluation, the cells were submitted to pre-treatment with non-cytotoxic ebselen concentration for 2 hours with shaking at 30°C. Cells were harvested, washed and H2O2 was added to a 4-mM final concentration. The mixture was further incubated at 30°C for another hour. After treatment, appropriate cell dilutions were plated onto SC plates to determine cell survival and were plated onto SC media supplemented with 60 µg/ml canavanine to determine forward mutagenesis. Plates were incubated in the dark at 30°C for 3–5 days before counting surviving and mutant colonies. Assays were repeated at least three times and plating was in triplicate for each dose.
V79 Cell culture and treatments
V79 cells (Chinese hamster lung fibroblasts) were grown as monolayers under standard conditions in DMEM supplemented with 10% heat-inactivated FBS, 0.2 mg/ml L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were maintained in tissue culture flasks at 37°C in a humidified atmosphere containing 5% CO2 and were harvested by treatment with 0.15% trypsin–0.08% EDTA in PBS. Cells were seeded (3 x 106 cells) in 5 ml of complete medium in a 25-cm2 flask and grown for 2 days to 70% confluence prior to the treatment with the test substance.
Cells were treated with ebselen (0, 5, 10 and 50 µM) in FBS-free medium for 2 hours at 37°C in a humidified atmosphere containing 5% CO2. To evaluate the anti-genotoxic potential of this OS molecule, cells were then washed with PBS at and submitted to the mutagen experimental protocol. To perform oxidative challenge, cells were exposed to 150 µM H2O2 for 1 hour under the same conditions. The culture flasks were protected from direct light during treatment with ebselen and H2O2.
Genotoxic evaluation using comet assay in V79 cells
The alkaline comet assay was performed as described by Singh (28) with minor modifications (29,30). After treatment, cells were washed with ice-cold PBS, trypsinized and re-suspended in complete medium. Then, 20 µl cell suspension (3 x 106 cells/ml) was mixed with 0.75% low-melting point agarose and immediately spread onto a glass microscope slide pre-coated with a layer of 1% normal melting point agarose. Agarose was allowed to set at 4°C for 5 min. Slides were incubated in ice-cold lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100 and 10% DMSO, pH 10.0) at 4°C for at least 1 hour to remove cell proteins, leaving DNA as ‘nucleoids’. In the modified comet assay, the slides were removed from the lysing solution and washed three times in enzyme buffer (40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 100 mM KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8.0), drained and incubated at 37°C in this buffer with 60 µl of FPG [for detection of oxidized purines, mainly 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG); 1 µg/ml solution; 100 mU per gel] for 30 min and EndoIII (for detection of oxidized pyrimidines; 1 µg/ml solution; 100 mU per gel) for 45 min. After lysis, slides were placed on a horizontal electrophoresis unit, covered fresh buffer (300 mM NaOH, 1 mM EDTA, pH 13.0) for 20 min at 4°C to allow DNA unwinding and the expression of alkali-labile sites. Electrophoresis was performed for 20 min at 25 V and 300 mA (0.90 V/cm). All the steps above were conducted under yellow light or in the dark in order to prevent additional DNA damage. Slides were then neutralized (0.4 M Tris, pH 7.5), washed in bi-distilled water and stained using a silver nitrate staining protocol as described by Nadin et al. (31). After drying at room temperature, gels were analysed using an optical microscope. One hundred cells (50 cells from each of the two replicate slides) were selected and analysed for the concentration of each test substance. When selecting cells, the edges and cells around air bubbles were avoided (32). Cells were visually scored according to tail length into five classes: (i) class 0: undamaged, without a tail; (ii) class 1: with a tail shorter than the diameter of the head (nucleus); (iii) class 2: with a tail length 1–2x the diameter of the head; (iv) class 3: with a tail longer than 2x the diameter of the head and (v) class 4: comets with no heads. A value [damage index (DI)] was assigned to each comet according to its class. For the visual score analysis, the slides were coded and scored blind.
International guidelines and recommendations for the comet assay consider that visual scoring of comets is a well-validated evaluation method (33). The DI is based on the length of migration and on the amount of DNA in the tail, and it is considered a sensitive DNA measurement. DI and damage frequency were emphasized in our analyses. The other parameters, as image length, were only used as complementary DNA damage parameters. DI ranged from 0 (completely undamaged: 100 cells x 0) to 400 (with maximum damage: 100 cells x 4) (29,30,32,34). Damage frequency (%) was calculated based on the number of cells with tails versus those without tails. The vehicle was used as negative control.
Statistical analysis
Data are presented as means ± standard deviation (34) and analysed using one-way analysis of variance with Dunnett's multiple comparison test. Differences between the control group and different doses of the ebselen were considered significant when P < 0.05.
|
Results |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Antioxidative assay in S.cerevisiae
The treatment with ebselen protects against H2O2 cytotoxicity in yeast lacking antioxidant defences and in wild-type strains, as shown in Figure 2. None of the applied ebselen concentrations alone had any significant cytotoxic effect on the yeast strains tested (survival of colony forming ability 90–95%, data not shown). Similarly, the ebselen concentrations did not inhibit growth. As expected, H2O2 had an important toxic effect in yeast, verified by the increased inhibition of growth ratio. The treatment of different mutant strains (sod1
, sod2, sod1sod2, gpx3, yap1 and wild type) with this OS compound markedly decreased the response to the toxic effect of the H2O2. This increase in the survival score does occur in a dose-related manner, and is identical for all strains used, independently of the type of lacking enzymatic defence. In this manner, at this concentration range, ebselen acts as an antioxidant in yeast.
|
Mutagenic and anti-mutagenic effects in S.cerevisiae
Results of mutagenicity tests are shown in Tables II and III. Ebselen was neither cytotoxic nor mutagenic, at the concentration range employed in yeast, since the survival rate did not decrease. Moreover, it did not significantly increase the revertants ratio at the his1, lys1 and hom3 loci in the strain XV185-14c (Table II). In the same way, in N123 strain, ebselen treatment did not induce forward mutation (Table III).
|
|
In this manner, we have chosen this concentration range to follow experiments to verify the protective effects of ebselen on the H2O2-induced forward mutation in N123 yeast strain. Table IV shows that ebselen treatment increases the survival of strain N123 during H2O2 treatment and simultaneously reduces, in a dose-dependent manner, the H2O2-induced forward mutagenesis. In this manner, our findings clearly show that ebselen exerts an anti-mutagenic effect against oxidative mutagenesis in yeast.
|
Effects in V79 cells
In order to evaluate the ebselen genotoxicity, we investigated whether this compound could induce DNA damage under these experimental conditions. The in vitro alkaline (pH >13) comet test is the most frequently used assay for routine screening of potential genotoxic agents (30
) and can be performed with a variety of cell types, including V79 cell lines. The alkaline version of the comet assay detects primary (repairable) DNA single- and double-strand breaks, alkali-labile sites cross-links and incomplete excision repair sites (30). Ebselen did not generate significant DNA damage at concentrations <50 µM (Table IV). However, it induced DNA damage at 50 µM (Table IV). We also investigated the antioxidant effect of this OS molecule against H2O2-induced DNA damage at concentrations from 5 to 50 µM. At 5 µM concentration, the treatment with this compound reduced the H2O2-induced DNA damage. At 10 and 50 µM, the protective effect is not observed and DI is increased, suggesting that at these concentrations it contributes to genotoxic effects.
In order to determine the nature of the antioxidant effect on V79 cells exerted by this interesting molecule, we carried out the modified comet assay. Table V presents the mean DNA damage of ebselen as DI after treatment with DNA repair enzymes EndoIII and FPG. Therefore, the results indicate that at 5 and 10 µM ebselen did not induce significant oxidative damage, as expected (Table V). H2O2 treatment increases the extent of oxidative DNA damage recognized by EndoIII and FPG in V79 cells, indicating the presence of oxidized pyrimidines and oxidized purines, respectively. When the cells were pre-treated with ebselen and then exposed to H2O2, the extension of oxidative damage recognized by these enzymes decreases significantly, indicating that at these concentrations ebselen has an anti-genotoxic effect on V79 cells due to its antioxidant effect (Figure 3). This effect was more pronounced in reduced FPG-sensitive sites induced by H2O2. This suggests that ebselen reveals the strongest inhibition of FPG-sensitive sites or EndoII-sensitive sites preventing oxidative damage in both treatments with these enzymes.
|
|
|
Discussion |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Ebselen—the most studied synthetic OS compound—has antioxidant properties that have attracted a great deal of attention since its discovery in 1984. In this study, we used S.cerevisiae to explore the antioxidant and anti-mutagenic effects of this molecule against oxidative mutagenesis since yeast seems to be a potentially useful eukaryotic model for studies on the molecular mechanisms underlying the effects of pharmacodynamic molecules (35
). Moreover, we evaluated the genotoxic profile of this OS in V79 fibroblasts, well characterized and commonly used in mutagenicity and cytotoxicity studies, as well as the protective effect of ebselen against DNA damage induced by oxidative challenge (36). H2O2 was chosen for oxidative challenge in yeast and V79 cells because it is genotoxic and capable of inducing oxidative DNA damage, including DNA strand breakage and base modification (21).
The yeast S.cerevisiae has been a useful model for studies of the eukaryotic response to oxidant challenge and for investigating the interplay between stress resistance and levels of damaged cell components, such as DNA. Although mutations are often induced at higher frequency in exponential as compared to stationary phase, in this work, we have used cells in stationary phase since these resemble cells of multicellular organisms in various aspects; for example, most energy comes from mitochondrial respiration, cells are in the G0 phase and, especially, damage accumulates over time. In stationary phase cells, damage cannot be diluted since cell division and new synthesis are not occurring (35).
Ebselen, at the concentration range employed, was neither cytotoxic nor mutagenic in yeast strains (Table III and Table IV), and the treatment was able to prevent oxidative mutagenesis induced by H2O2 (Table IV). In order to evaluate the role of the antioxidant effect of this OS molecule in its anti-mutagenic potential, a determination of the protection of ebselen against H2O2 cytotoxicity was performed using the growth inhibition assay in wild-type and isogenic strains lacking antioxidant defences. The results showed that after the treatment with ebselen, a decrease in the growth inhibition induced by the H2O2 took place, indicating the possibility of antioxidant protection (Figure 2). This antioxidant effect was more pronounced for the gpx3 mutant, which indicated that ebselen acts as scavenger by mimicking the catalytic activity of GPx3, as expected. It is also important to note the antioxidant response observed in the yap1 mutant, which is very sensitive to oxidative stress since Yap1 is a key regulator of oxidative stress tolerance in S.cerevisiae (37). Our results thus demonstrate a putative direct action of ebselen as reactive oxygen species (ROS) scavenger, probably by GPx-like action, rather than an induction of other antioxidant defences that would lead to an adaptive response in yeast.
Reinforcing the results obtained in yeast, the treatment with this OS molecule at low concentrations was not genotoxic to V79 cells. Moreover, it protects against H2O2-induced oxidative damage as verified by the decrease in the DI in the standard comet assay (Table III). The role of antioxidant action of ebselen in the anti-genotoxic effect on V79 cells was assessed using the modified comet assay. The standard alkaline method gives limited information on the type of DNA damage being measured as it is not possible to determine whether it is a consequence of direct effects of the damaging agent, or of indirect effects, such as oxidative damage. The sensitivity and specificity of the assay can be improved by incubating the lysed cells (nucleoids) with lesion-specific endonucleases, which recognize particular damaged bases and create additional breaks. In this work, we have used FPG, which is specific for oxidized purines, including 8-oxo-7,8-dihydroguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and other ring-opened purines, and also EndoIII, which recognizes oxidized pyrimidines, including thymine glycol and uracil glycol (38). As shown in Table V, ebselen reduces, more effectively, the FPG-sensitive sites by inhibiting the formation the oxidized purines.
The exposure of living cells to various oxidizing agents such as peroxides, singlet oxygen and ultraviolet and ionizing irradiation leads to hydroxylation of DNA constituents such as 2'-deoxyguanosine, which gives rise to 8-hydroxy-deoxyguanosine and its tautomer 8-oxodG at physiological pH (7,39–41). According to our findings, ebselen was able to decrease 8-oxodG levels in in vitro studies using rat liver DNA treated with peroxides (42). In addition, in a model of ischemic damage in rat brain, ebselen-treated animals had a reduction in cellular damage and a clear decrease in the levels of hydroxylated 2'-deoxyguanosine products (7). In rat liver, this OS molecule significantly inhibited the increase in the level of 8-oxodG generated by aflatoxin B1 treatment, which may contribute to its protective effect against the carcinogenicity of aflatoxin B1 (42).
The present study presents evidence that ebselen has a strong protective effect against H2O2-induced oxidative DNA damage in yeast and V79 cells. The reason for this protection probably resides in an additional antioxidant protection, mainly in virtue of its GPx-mimetic action. In this respect, ebselen has antioxidative properties as it can act as a Gpx mimic in reducing H2O2 and lipid hydroperoxides. Also, ebselen scavenges the highly reactive species peroxynitrite and inactivates free radical-generating enzymes such as lipoxygenase, cyclooxygenase and reduced nicotinamide adenine dinucleotide phosphate oxidase (43).
In in vitro models, ebselen protects against oxidative DNA damage induced by dopamine in the presence of copper ions (19). Similarly, it provides potent protection against ROS-induced cytotoxicity and DNA damage in HepG2 and HL-60 cell lines, through its antioxidant properties (44,45). Ebselen also protects against aflatoxin B1 cytotoxicity via its strong ability to scavenge intracellular ROS and to prevent oxidative damage (46). Furthermore, it can inhibit pre-neoplasic changes caused by aflatoxin B1, at least in part, due the protective effect against oxidative damage (42). In this sense, some previous studies confirm that OS compounds, as selenomethionine and selenocysteine, are neither toxic nor mutagenic in similar doses in yeast and have a protective effect against H2O2-induced mutagenesis by means of reinforcing antioxidant cell potential (47).
Similarly to other OS compounds, ebselen presents anti-genotoxic effect in V79 cell activity at low doses, in the range upon 5 µM. In agreement with literature data, this molecule reduces ROS formation and the DNA-damaging effect caused by H2O2 in HepG2 cells at 1–25 µM (44). Also, ebselen protects ECV-304 cells against oxysterols at 2 µM (48). The neuroprotective actions of ebselen are at 0.01–20 µM, with the best results obtained at 8 µM (49). In this manner, our results in V79 cells are consistent with those reported in the literature, which show that some OS compounds exhibit protective potential at lower concentrations.
However, ebselen is genotoxic to V79 cells in concentrations of up to 10 µM, as verified in the alkaline comet assay (Table IV). Indeed, it depletes glutathione in HepG2 cells in this concentration range, inducing apoptosis through rapid depletion of intracellular thiols (50). In view of this, ebselen has an interesting anti-proliferative potential in tumoural cells lines, as observed in the growth inhibition of MCF-7 cells and in the cell death induction in Sp2/O-Ag14 hybridoma cells and in the C6 glioma cell line (51–53). In addition, a recent in vivo study showed that ebselen presented a pro-oxidative effect on the livers of suckling rat pups (54). Our findings reinforce these cytotoxic effects above the threshold concentration and suggest the possibility that the DNA damage induced by ebselen can be involved, at least in part, in its anti-proliferative effect. Furthermore, DNA-damaging capacity is useful to trigger signalling pathways that lead to apoptosis, and this has been considered a promising characteristic in the search for new OS derivative compounds with anti-tumoural effect (55).
In summary, the present findings suggest that ebselen is capable of protecting against H2O2-induced cytotoxicity and DNA damage and mutation in the yeast S.cerevisiae as well as in V79 cultured cells. It is neither cytotoxic nor induces mutagenicity in yeast S.cerevisiae, and it is able to protect against growth inhibition induced by H2O2 in yeast strains defective in antioxidant defence. In V79 cells, ebselen is not genotoxic at concentrations of up to 10 µM, and it protects against oxidative DNA damage by its antioxidant activity, as observed in the modified comet assay. However, at concentrations above 10 µM in V79 cells, this molecule is genotoxic and induces DNA damage. Besides all these interesting properties, more studies concerning clinical trials are still necessary for its safe therapeutic application and for the development of more potent derivatives or those that present other pharmacological effects.
|
Funding |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq.
|
Acknowledgments |
|---|
Conflict of interest statement: None declared.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +55 51 34774000; Fax: +55 51 34779214; E-mail: jenifer.saffi{at}ulbra.br
|
References |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
-
1. Soriano-Garcia M. Organoselenium compounds as potential therapeutic and chemopreventive agents: a review. Curr. Med. Chem. (2004) 11:1657–1669.[Web of Science][Medline]
2. Nogueira CW, Zeni G, Rocha JB. Organoselenium and organotellurium compounds: toxicology and pharmacology. Chem. Rev. (2004) 104:6255–6285.[CrossRef][Web of Science][Medline]
3. Mugesh G, du Mont WW, Sies H. Chemistry of biologically important synthetic organoselenium compounds. Chem. Rev. (2001) 101:2125–2179.[CrossRef][Web of Science][Medline]
4. Bhabak KP, Mugesh G. Synthesis, characterization, and antioxidant activity of some ebselen analogues. Chemistry (2007) 13:4594–4601.[CrossRef][Web of Science][Medline]
5. Parnham M, Sies H. Ebselen: prospective therapy for cerebral ischaemia. Expert Opin. Investig. Drugs (2000) 9:607–619.[CrossRef][Web of Science][Medline]
6. Saito I, Asano T, Sano K, Takakura K, Abe H, Yoshimoto T, Kikuchi H, Ohta T, Ishibashi S. Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage. Neurosurgery (1998) 42:269–277. discussion, 277–278.[CrossRef][Web of Science][Medline]
7. Imai H, Graham DI, Masayasu H, Macrae IM. Antioxidant ebselen reduces oxidative damage in focal cerebral ischemia. Free Radic. Biol. Med. (2003) 34:56–63.[CrossRef][Web of Science][Medline]
8. Kil J, Pierce C, Tran H, Gu R, Lynch ED. Ebselen treatment reduces noise induced hearing loss via the mimicry and induction of glutathione peroxidase. Hear. Res. (2007) 226:44–51.[CrossRef][Web of Science][Medline]
9. Schewe T. Molecular actions of ebselen—an antiinflammatory antioxidant. Gen. Pharmacol. (1995) 26:1153–1169.[Web of Science][Medline]
10. Sies H. Ebselen, a selenoorganic compound as glutathione peroxidase mimic. Free Radic. Biol. Med. (1993) 14:313–323.[CrossRef][Web of Science][Medline]
11. Delanty N, Dichter MA. Antioxidant therapy in neurologic disease. Arch. Neurol. (2000) 57:1265–1270.
12. Zhao R, Holmgren A. A novel antioxidant mechanism of ebselen involving ebselen diselenide, a substrate of mammalian thioredoxin and thioredoxin reductase. J. Biol. Chem. (2002) 277:39456–39462.
13. Chang TC, Huang ML, Hsu WL, Hwang JM, Hsu LY. Synthesis and biological evaluation of ebselen and its acyclic derivatives. Chem. Pharm. Bull. (2003) 51:1413–1416.[CrossRef][Medline]
14. Morin D, Zini R, Ligeret H, Neckameyer W, Labidalle S, Tillement JP. Dual effect of ebselen on mitochondrial permeability transition. Biochem. Pharmacol. (2003) 65:1643–1651.[CrossRef][Web of Science][Medline]
15. Namura S, Nagata I, Takami S, Masayasu H, Kikuchi H. Ebselen reduces cytochrome c release from mitochondria and subsequent DNA fragmentation after transient focal cerebral ischemia in mice. Stroke (2001) 32:1906–1911.
16. Wojtowicz H, Kloc K, Maliszewska I, Mlochowski J, Pietka M, Piasecki E. Azaanalogues of ebselen as antimicrobial and antiviral agents: synthesis and properties. Farmaco (2004) 59:863–868.[CrossRef][Medline]
17. Yamaguchi T, Sano K, Takakura K, Saito I, Shinohara Y, Asano T, Yasuhara H. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group. Stroke (1998) 29:12–17.
18. Herin GA, Du S, Aizenman E. The neuroprotective agent ebselen modifies NMDA receptor function via the redox modulatory site. J. Neurochem. (2001) 78:1307–1314.[CrossRef][Web of Science][Medline]
19. Li Y, Cao Z. The neuroprotectant ebselen inhibits oxidative DNA damage induced by dopamine in the presence of copper ions. Neurosci. Lett. (2002) 330:69–73.[CrossRef][Web of Science][Medline]
20. Green AR, Ashwood T. Free radical trapping as a therapeutic approach to neuroprotection in stroke: experimental and clinical studies with NXY-059 and free radical scavengers. Curr. Drug Targets CNS Neurol. Disord. (2005) 4:109–118.[CrossRef][Medline]
21. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. (2006) 160:1–40.[CrossRef][Web of Science][Medline]
22. Burke D, Dawson D, Stearns T. Methods in Yeast Genetics (2000) CSH Laboratory Press, Plainview, NY, pp. 171–205.
23. Fogel S, Lax C, Hurst DD. Reversion at the HiS1 locus of yeast. Genetics (1978) 90:489–500.
24. Hawthorne DC. Identification of nonsense codons in yeast. J. Mol. Biol. (1969) 43:71–75.[CrossRef][Web of Science][Medline]
25. Von Borstel RC, Cain KT, Steinberg CM. Inheritance of spontaneous mutability in yeast. Genetics (1971) 69:17–27.
26. Schuller RC, Von Borstel RC. Spontaneous mutability in yeast. I. Stability of lysine reversion rates to variation of adenine concentration. Mutat. Res. (1974) 24:17–23.[CrossRef][Web of Science][Medline]
27. Rosa RM, Melecchi MI, da Costa Halmenschlager R, Abad FC, Simoni CR, Caramao EB, Henriques JA, Saffi J, de Paula Ramos AL. Antioxidant and antimutagenic properties of Hibiscus tiliaceus L. methanolic extract. J. Agric. Food Chem. (2006) 54:7324–7330.[CrossRef][Web of Science][Medline]
28. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. (1988) 175:184–191.[CrossRef][Web of Science][Medline]
29. Hartmann A, Speit G. The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay). Toxicol. Lett. (1997) 90:183–188.[CrossRef][Web of Science][Medline]
30. Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol. Biotechnol. (2004) 26:249–261.[CrossRef][Web of Science][Medline]
31. Nadin SB, Vargas-Roig LM, Ciocca DR. A silver staining method for single-cell gel assay. J. Histochem. Cytochem. (2001) 49:1183–1186.
32. Tice RR, Agurell E, Anderson D, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. (2000) 35:206–221.[CrossRef][Web of Science][Medline]
33. Burlinson B, Tice RR, Speit G, et al. Fourth International Workgroup on Genotoxicity testing: results of the in vivo comet assay workgroup. Mutat. Res. (2007) 627:31–35.[Web of Science][Medline]
34. Gallegos A, Berggren M, Gasdaska JR, Powis G. Mechanisms of the regulation of thioredoxin reductase activity in cancer cells by the chemopreventive agent selenium. Cancer Res. (1997) 57:4965–4970.
35. Mustacchi R, Hohmann S, Nielsen J. Yeast systems biology to unravel the network of life. Yeast (2006) 23:227–238.[CrossRef][Web of Science][Medline]
36. Bradley MO, Bhuyan B, Francis MC, Langenbach R, Peterson A, Huberman E. Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature. A report of the Gene-Tox Program. Mutat. Res. (1981) 87:81–142.[Web of Science][Medline]
37. Ikner A, Shiozaki K. Yeast signaling pathways in the oxidative stress response. Mutat. Res. (2005) 569:13–27.[Web of Science][Medline]
38. Dizdaroglu M. Base-excision repair of oxidative DNA damage by DNA glycosylases. Mutat. Res. (2005) 591:45–59.[Web of Science][Medline]
39. Culp SJ, Cho BP, Kadlubar FF, Evans FE. Structural and conformational analyses of 8-hydroxy-2'-deoxyguanosine. Chem. Res. Toxicol. (1989) 2:416–422.[CrossRef][Web of Science][Medline]
40. Cho BP, Kadlubar FF, Culp SJ, Evans FE. 15N nuclear magnetic resonance studies on the tautomerism of 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, and other C8-substituted guanine nucleosides. Chem. Res. Toxicol. (1990) 3:445–452.[CrossRef][Web of Science][Medline]
41. Hofer T, Moller L. Optimization of the workup procedure for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine with electrochemical detection. Chem. Res. Toxicol. (2002) 15:426–432.[CrossRef][Web of Science][Medline]
42. Yang CF, Liu J, Wasser S, Shen HM, Tan CE, Ong CN. Inhibition of ebselen on aflatoxin B(1)-induced hepatocarcinogenesis in Fischer 344 rats. Carcinogenesis (2000) 21:2237–2243.
43. Sakurai T, Kanayama M, Shibata T, Itoh K, Kobayashi A, Yamamoto M, Uchida K. Ebselen, a seleno-organic antioxidant, as an electrophile. Chem. Res. Toxicol. (2006) 19:1196–1204.[CrossRef][Web of Science][Medline]
44. Yang CF, Shen HM, Ong CN. Protective effect of ebselen against hydrogen peroxide-induced cytotoxicity and DNA damage in HepG2 cells. Biochem. Pharmacol. (1999) 57:273–279.[CrossRef][Web of Science][Medline]
45. Li J, Chen JJ, Zhang F, Zhang C. Ebselen protection against hydrogen peroxide-induced cytotoxicity and DNA damage in HL-60 cells. Acta Pharmacol. Sin. (2000) 21:455–459.[Web of Science][Medline]
46. Yang CF, Liu J, Shen HM, Ong CN. Protective effect of ebselen on aflatoxin B1-induced cytotoxicity in primary rat hepatocytes. Pharmacol. Toxicol. (2000) 86:156–161.[CrossRef][Web of Science][Medline]
47. Bronzetti G, Cini M, Caltavuturo L, Fiorio R, Croce CD. Antimutagenicity of sodium selenite in Chinese hamster V79 cells exposed to azoxymethane, methylmethansulphonate and hydrogen peroxide. Mutat. Res. (2003) 523–524:21–31.
48. Wu Q, Huang K. Protective effect of ebselen on cytotoxicity induced by cholestane-3 beta, 5 alpha, 6 beta-triol in ECV-304 cells. Biochim. Biophys. Acta (2006) 1761:350–359.[Medline]
49. Xu JH, Hu HT, Liu Y, Qian YH, Liu ZH, Tan QR, Zhang ZJ. Neuroprotective effects of ebselen are associated with the regulation of Bcl-2 and Bax proteins in cultured mouse cortical neurons. Neurosci. Lett. (2006) 399:210–214.[CrossRef][Web of Science][Medline]
50. Yang CF, Shen HM, Ong CN. Ebselen induces apoptosis in HepG(2) cells through rapid depletion of intracellular thiols. Arch. Biochem. Biophys. (2000) 374:142–152.[CrossRef][Web of Science][Medline]
51. Guerin PJ, Gauthier ER. Induction of cellular necrosis by the glutathione peroxidase mimetic ebselen. J. Cell. Biochem. (2003) 89:203–211.[CrossRef][Web of Science][Medline]
52. Engman L, Cotgreave I, Angulo M, Taylor CW, Paine-Murrieta GD, Powis G. Diaryl chalcogenides as selective inhibitors of thioredoxin reductase and potential antitumor agents. Anticancer Res. (1997) 17:4599–4605.[Web of Science][Medline]
53. Shi H, Liu S, Miyake M, Liu KJ. Ebselen induced C6 glioma cell death in oxygen and glucose deprivation. Chem. Res. Toxicol. (2006) 19:655–660.[CrossRef][Web of Science][Medline]
54. Farina M, Soares FA, Zeni G, Souza DO, Rocha JB. Additive pro-oxidative effects of methylmercury and ebselen in liver from suckling rat pups. Toxicol. Lett. (2004) 146:227–235.[CrossRef][Web of Science][Medline]
55. Zhou N, Xiao H, Li TK, Nur EKA, Liu LF. DNA damage-mediated apoptosis induced by selenium compounds. J. Biol. Chem. (2003) 278:29532–29537.
56. Hill KE, Zhou J, McMahan WJ, Motley AK, Burk RF. Neurological dysfunction occurs in mice with targeted deletion of the selenoprotein P gene. J. Nutr. (2004) 134:157–161.
Received on September 29, 2007; revised on November 9, 2007; accepted on November 21, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||