Arch Microbiol (2010) 192:221–228 DOI 10.1007/s00203-010-0551-2
ORIGINAL PAPER
EVect of vitamin A treatment on superoxide dismutase-deWcient yeast strains Rafael Roehrs · Daniela R. J. Freitas · Aoi Masuda · João A. P. Henriques · Temenouga N. Guecheva · Ana-Ligia L. P. Ramos · Jenifer SaY
Received: 20 November 2009 / Revised: 11 January 2010 / Accepted: 12 January 2010 / Published online: 4 February 2010 © Springer-Verlag 2010
Abstract Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, diVerent studies have been demonstrated the pro-oxidant eVects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A eVects on superoxide dismutase (SOD)deWcient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deWcient in CuZnSOD (sod1) and CuZnSOD/MnSOD (sod1sod2). We also observed the protective eVect of vitamin E against the Vit A-induced toxicity. Possible adaptation eVects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were
Communicated by Erko Stackebrandt. R. Roehrs · J. A. P. Henriques · T. N. Guecheva · A.-L. L. P. Ramos · J. SaY Departamento de Biofísica, IB/UFRGS, Porto Alegre, RS, Brazil T. N. Guecheva e-mail:
[email protected] D. R. J. Freitas · A. Masuda Centro de Biotecnologia do Estado do Rio Grande do Sul, Porto Alegre, RS, Brazil J. A. P. Henriques · J. SaY (&) Laboratório de Genética Toxicológica, Universidade Luterana do Brasil, Av. Farroupilha, 8001. Prédio 1, sala 122, Canoas, RS 92425-900, Brazil e-mail:
[email protected]
determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deWcient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not suYcient to prevent the Vit A-induced cell death in these mutants in exponential phase growth. Keywords Vitamin A · Reactive oxygen species (ROS) · Saccharomyces cerevisiae · Oxidative stress · Paraquat
Introduction Dietary sources of Vit A are provided either by retinol esters, which are present in foods of animal origin, or by plant carotenoids (De Flora et al. 1999). Carotenoids and Vit A share some protective mechanisms, such as scavenging of genotoxic ROS, modulation of signal transduction pathways, inhibition of cell transformation induced by physical and chemical agents and facilitation of intracellular communication inhibited by genotoxic compounds (De Flora et al. 1999). However, retinol and other derivatives were also observed to induce pro-oxidant eVects in diVerent cell types (Klamt et al. 2003; De Oliveira and Moreira 2008; Roehrs et al. 2009; De Oliveira et al. 2009). Murata and Kawanishi (2000) used the cytochrome c reduction method to show generation of superoxide anion from the autoxidation of retinoids and demonstrated that this process was signiWcantly correlated with 8-oxodG formation. The superoxide anion can damage DNA and proteins by directly attack or exert a toxic eVect indirectly, participating in Harber–Weiss reaction, forming the extremely reactive hydroxyl radical (Gralla and Kosma 1992; Flattery-O’Brien et al. 1997).
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In response to the destructive nature of ROS, aerobically growing organisms have evolved multiple defence mechanisms for prevention of cellular components damage. Primary biological defence mechanisms against oxidative damage include protective proteins able to remove ROS or to scavenge metal ions, while secondary defence mechanisms consist of enzymes that remove and repair the products of oxidatively damaged components (Moradas et al. 1996). To counteract the oxidative stress resulting from ROS, cells posses a range of non-enzymatic and enzymatic defence systems, including glutathione (GSH), thioredoxin, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (Halliwell and Gutteridge 2000). The single-cell eukaryote Saccharomyces cerevisiae is an ideal model organism to investigate whether chemicals exert antioxidant or pro-oxidant eVects in vivo. Its antioxidant defences are generally similar to those of higher organisms. Like most eukaryotes, it contains a cytosolic copper–zinc superoxide dismutase (CuZnSOD—Sod1) coded by the SOD1 gene; a mitochondrial manganese superoxide dismutase (MnSOD—Sod2) coded by the SOD2 nuclear gene and synthesized in the cytosol as a prepeptide which is cleaved after entering the mitochondria; a cytosolic catalase coded by the CTT1 gene; and a peroxisomal catalase coded by the CTA1 gene (reviewed by Jamieson 1998). It is known that yeast logarithmic phase growth (LOG) cells, using glucose as carbon source, are more sensitive to oxidative stress than stationary phase cells, due to glucose repression of many antioxidant defence mechanisms. Using microarrays, Gasch et al. (2000) showed that the induction of the antioxidant defences in yeast, i.e. cytosolic superoxide dismutase, cytosolic catalase and glutathione peroxidase, occurs during the diauxic shift. Bronzetti et al. (2001) showed that superoxide dismutase activity was highly induced by Vit A, treatment in LOG yeast cells, and thus can represent an important defence against Vit A-generated oxidative damage. Thus, the aim of this work was to evaluate the eVect of Vit A on cell survival, the enzymatic activity of catalase and glutathione peroxidase, as well as on the total glutathione content in exponentially growing superoxide dismutase-deWcient yeast strains.
Materials and methods Chemicals Vitamina A (Arovit®—retinyl palmitate), vitamin E (hydrosoluble form, Trolox®) and Paraquat (Gramoxone®) were purchased from Roche, Sigma and Bayer, respectively.
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Arch Microbiol (2010) 192:221–228 Table 1 S. cerevisiae strains used in this study Strains
Genotype
EG103 (WT)
MAT, leu20, his31, trp1-289, ura3-52
EG110 (sod2)
Like EG103 except sod2::TRP1
EG118 (sod1)
Like EG103 except sod1::URA3
EG133 (sod1sod2)
Like EG103 except sod2::TRP1, sod1::URA3
Yeast strains, media and growth conditions Saccharomyces cerevisiae strains used in this work were kindly provided by Dr. E. Gralla (University of California, Los Angeles, CA, USA). The relevant genotypes are listed in Table 1. Strains were routinely grown and stored on solid YPD medium (1% yeast extract, 2% glucose, 2% peptone and 2% agar) in appropriate conditions to avoid selection of petites or suppressors, as described by Gralla and Valentine (1991), Gralla and Kosma (1992). Treatment conditions Sensitivity assay of S. cerevisiae strains YPD-grown yeast cells from the early stationary phase were re-inoculated at an appropriate cell density in fresh synthetic media containing 5% glucose (glucose-repressing conditions) and supplemented with appropriate nutrients (Sherman et al. 1986), and grown for 4 h at 30°C to a density of 1–2 £ 107 cells/ml. These cells in exponential phase of growth (LOG cells) were harvested, washed and re-suspended in sterile saline (0.9% NaCl) to a Wnal concentration of 1–2 £ 106 cells/ml. To evaluate sensitivity to Vit A, cultures were exposed to concentrations of 10, 20 and 40 mg/ml (19.05, 38.1 and 76.2 mM, respectively) for 1 h at 30°C. Three diVerent treatment conditions were performed: Pre-treatment with Vit A: LOG cells were pre-treated with Vit A (10, 20 and 40 mg/ml) for 1 h and exposed for 1 h more at non-cytotoxic to the wild-type yeast strain concentrations of paraquat (0.1 and 0.5 mM); Post-treatment with Vit A: LOG cells were Wrst treated with non-cytotoxic concentrations of paraquat (0.1 and 0.5 mM) for 1 h and then exposed for 1 h to Vit A (10, 20 and 40 mg/ml); Co-treatment with Vit A: LOG cells were concomitantly treated with Vit A (10, 20 and 40 mg/ml) and paraquat (0.1 and 0.5 mM) or vitamin E (0.1 mM). Cells were appropriately diluted and plated on solid YPD (1% yeast extract, 2% peptone, 2% glucose and 2% agar). After 3 days, colony-forming units were counted. All biological tests were repeated at least twice, and plating
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was carried out in triplicate for each dose. Sensitivity is expressed as percentage of survival. Determination of glutathione content Yeast cells were grown as described for each treatment condition, and total glutathione was determined after 1-h exposure to Vit A, using 30 ml of LOG cell suspension in saline, according to Akerboom and Sies (1981). Protein content was determined by the method of Bradford using bovine serum albumin as standard (Bradford 1976). Enzyme activities Crude extracts from Vit A-treated yeast LOG cells were prepared by glass bead lysis: cells were suspended in lysis buVer (50 mM Tris 7.2, 150 mM NaCl, 5 mM EDTA and 0.2 M phenylmethylsulfonyl Xuoride) with an equal volume of acid-washed 0.5-mm glass beads and vortexed for 6–8 cycles of 30 s with cooling before each cycle. The mixture was then micro-centrifuged for 2 min at 10,000 rpm to remove cellular debris and glass beads. Catalase (EC 1.11.1.6) activity was determined spectrophotometrically by monitoring the disappearance of hydrogen peroxide at 240 nm (Longo et al. 1996). Glutathione peroxidase (EC 1.11.1.9) was assayed by measuring the oxidized glutathione using glutathione reductase as described by Flohe and Gunzler (1984). The reaction mixture (1.0 ml) containing 2.5 mM tert-butyl hydroperoxide, 10 mM glutathione, 50 mM potassium phosphate buVer (pH 7.0), 0.3 mM NADPH, 3.4 U/ml glutathione reductase and the enzyme was incubated at 25°C, and decrease of absorbance of the mixture at 340 nm was monitored. Protein concentration was determined by the Bradford assay (Bradford 1976).
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previous experiments based on the wild-type strain survival. The lowest dose used (10 mg/ml) was non-toxic in wild-type SOD strain, whereas the treatment with 20 and 40 mg/ml Vit A during 1 h decreased the survival to about 80 per cent (Table 2). Among the SOD-deWcient strains, sod1 and sod1sod2 presented accentuated sensitivity to the treatment (survival of 54.2 and 42.7%, respectively) at the higher dose used. The results in Table 2 show that WT cells treated with Vit A presented signiWcant elevation in the cellular antioxidant defences. On the other hand, CuZnSODdeWcient strain (sod1) presents lower levels of catalase, while sod2 and sod1sod2 strains show no detectable catalase activity. With regard to GPx levels, our results show that Vit A treatment induces GPx activity in all tested yeast strains, namely in wild-type cells. The total glutathione content also increased in wild-type cells, whereas it decreased signiWcantly in the double-mutant sod1sod2. Vit A-caused lethality in S. cerevisiae can be prevented by vitamin E In order to investigate the participation of ROS in the sensitivity presented to the Vit A treatment, the cells were concomitantly exposed to 0.1 mM vitamin E (a known antioxidant agent). Vit A was toxic at all tested doses in superoxide dismutase-deWcient strains (Fig. 1b–d) and at doses of 20 and 40 mg/ml in wild-type cells (Fig. 1a). The results show that vitamin E protects all yeast strains against toxic eVects of Vit A. However, vitamin E treatment alone decreased cell survival in sod1sod2 double mutant (Fig. 1d) reinforcing the importance of the dose used in the vitamin treatment. Vitamin A does not protect against paraquat-generated damage
Statistical analysis Statistics was performed using the one-way ANOVA on the mean of three independent experiments. Bonferroni’s multiples comparison test was carried out, accepting a probability of P < 0.05 as statistically signiWcant.
Results Vit A-treatment induces antioxidant protection in S. cerevisiae To determine the eVects of Vit A treatment on cellular antioxidant defences in SOD-deWcient cells, we measured the amount of total glutathione content and the activity of catalase and glutathione peroxidase in LOG cells. The concentration range of the Vit A treatment was determined in
A possible role of Vit A treatment in antioxidant or pro-oxidant eVects, as well as possible adaptation eVects induced by sub-lethal oxidative stress, was monitored by pre-, co- and post-treatment with the oxidative agent paraquat. LOG yeast cells were pre-treated for 1 h with Vit A (10, 20 and 40 mg/ml) and then exposed for 1 h to paraquat (0.1 and 0.5 mM). The results presented in Fig. 2 show that Vit A pre-treatment does not protect the cells against paraquatinduced oxidative stress with exception of pre-treatment with dose 10 mg/ml for 0.1 mM paraquat treatment in sod1sod2 mutant (Fig. 2d). Instead of this, an increased cell death was observed after Vit A pre-treatment in sod1 (Fig. 2b and sod1sod2 (Fig. 2d) mutant strains. The absence of MnSOD enzyme had no inXuence on the cell survival after Vit A pre-treatment (Fig. 2c). The WT strain also presented a decrease in survival after pre-treatment with 20 mg/ml Vit A (Fig. 2a), indicating the formation of
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Table 2 Survival and enzymatic activities in exponentially growing wild-type and sod mutant strains exposed to vitamin A Survival (%)
Catalase (U/mg protein)
GPx (nmol/min/mg protein)
Total glutathione (g/mg protein)
Saline
100.0 (§0.0)
0.021 (§0.003)
41.9 (§11.1)
5.8 (§2.3)
Vitamin A 10 mg/mL
101.3 (§1.9)
0.011 (§0.001)
42.5 (§4.6)
8.2 (§1.3)
Vitamin A 20 mg/mL
80.5 (§8.0)*
0.169 (§0.018)*
261.2 (§33.9)*
12.6 (§5.9)
Vitamin A 40 mg/ml
79.7 (§12.9)*
0.162 (§0.024)*
254.5 (§60.2)*
30.5 (§5.0)*
Saline
100.0 (§0.0)
0.015 (§0.001)
ND
11.6 (§1.7)
Vitamin A 10 mg/ml
78.9 (§8.3)*
0.004 (§0.002)*
ND
9.4 (§0.8)
Vitamin A 20 mg/ml
69.5 (§13.9)*
0.012 (§0.002)
130.1 (§47.2)
7.6 (§1.3)*
Vitamin A 40 mg/ml sod2 Saline
54.2 (§8.6)*
ND
17.9 (§2.7)
8.6 (§2.4)
100.0 (§0.0)
ND
49.8 (§12.8)
4.2 (§0.8)
Vitamin A 10 mg/ml
79.6 (§2.1)*
ND
43.5 (§1.0)
3.8 (§0.5)
Vitamin A 20 mg/ml
72.7 (§6.3)*
0.018 (§0.004)
73.9 (§5.9)
3.8 (§0.3)
Vitamin A 40 mg/ml
80.7 (§9.2)*
ND
65.3 (§10.0)
2.4 (§0.0)
Saline
100.0 (§0.0)
ND
ND
5.5 (§0.3)
Vitamin A 10 mg/ml
75.1 (§7.7)*
ND
21.7 (§6.7)
4.1 (§0.8)*
Vitamin A 20 mg/ml
58.8 (§10.5)*
ND
17.7 (§3.5)
1.4 (§0.4)*
Vitamin A 40 mg/ml
42.7 (§4.4)*
ND
28.2 (§12.5)
1.6 (§0.1)*
SOD
sod1
sod1sod2
ND not detectable * P < 0.05 compared to saline Fig. 1 Sensitivity of wild-type and sod mutant strains to vitamin A in the absence or presence of vitamin E. SigniWcantly diVerent at P < 0.05, a when compared to the negative control, b when compared to the vitamin A treatment only
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Fig. 2 Vitamin A pre-treatment: sensitivity of wild-type and sod mutant strains to previous incubation with vitamin A during 1 h followed by 1-h paraquat exposure. SigniWcantly diVerent at P < 0.05, a when
compared to the negative control, b when compared to the paraquat 0.1 mM treatment, c when compared to the paraquat 0.5 mM treatment
additional oxidative damage even in cells with intact antioxidant mechanisms. When LOG yeast cells were exposed to sub-lethal stress like heat shock at 40°C during 60 min, they acquired oxidative stress tolerance that could increase the survival in a subsequent treatment under lethal conditions (Pereira et al. 2001, 2003). In this respect, we treated LOG cells at low, sub-lethal concentrations of paraquat (0.1 and 0.5 mM) during 1 h and post-treated with Vit A (10, 20 and 40 mg/ml) for another hour. Figure 3 shows that sod1 (Fig. 3b) and sod2 (Fig. 3c) strains present an increased survival in Vit A treatment at 20 mg/ml, when pre-treated with 0.1 mM paraquat. This eVect is also evident in sod1sod2 mutant for doses of 20 and 40 mg/ml when pre-treated with 0.5 mM paraquat (Fig. 3d). Probably, the sub-lethal exposure doses of paraquat increased the level of antioxidant defences, thus protecting the cells from Vit A toxicity. To verify the possible anti- or pro-oxidant eVect of Vit A treatment, yeast LOG cells were treated concomitantly with
Vit A (10, 20 and 40 mg/ml) and paraquat (0.1 and 0.5 mM) during 1h, in glucose metabolism. Combined treatment with Vit A and paraquat (Fig. 4) presented tendency of decreased cell survival in all yeast strains, especially at the highest Vit A dose used. Interestingly, sod1 mutant presented diVerentiated response—increased cell survival after combined treatment with 0.1 mM paraquat (for doses 20 and 40 mg/ml Vit A) and strong cytotoxic eVect after combined treatment with 0.5 mM paraquat.
Discussion In this work, we investigated the eVects of Vit A on oxidative metabolism using superoxide dismutase-proWcient and dismutase-deWcient S. cerevisiae strains. The Vit A administration induces a higher toxicity in superoxide dismutasedeWcient cells, indicating that the increased cell death is associated with the accumulation of superoxide anion.
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Fig. 3 Vitamin A post-treatment: sensitivity of wild-type and sod mutant strains to previous incubation with paraquat during 1 h followed by 1-h vitamin A exposure. SigniWcantly diVerent at
P < 0.05, a when compared to the negative control, b when compared to the paraquat 0.1 mM treatment, c when compared to the paraquat 0.5 mM treatment
Superoxide dismutases (Sod) are antioxidant enzymes that disproportionate superoxide to O2 and H2O2, the latter being eliminated by catalase or glutathione peroxidase (Flattery-O’Brien et al. 1997). In S. cerevisiae, there are two types of superoxide dismutases, one mitochondrial (MnSOD—Sod2) and another cytosolic (CuZnSOD— Sod1) (Jamieson 1998). Sod1 represents at least 90% of the total SOD activity in yeast in the presence of high amounts of glucose, as Sod2 (mitochondrial) is largely glucoserepressed (Gralla and Kosma 1992). Catalase activity, which correlates with Sod2 (MnSOD) activity (Kujumdzieva-Savova et al. 1991), is also repressed by glucose and does not aVect the survival when cells are exposed to an oxidative stress in fermentative metabolism (Petrova et al. 2002). The correlation between catalase and superoxide dismutase activities was evidenced also in our study where we observed that CuZnSOD-deWcient strains have lower levels of catalase activity and that LOG cells of sod2 and sod1sod2 show no detectable catalase activity (Table 2). Our data demonstrated that Vit A treatment in LOG cells, under glucose metabolism, does not induce catalase activity
in SOD-lacking cells. Hydrogen peroxide can be detoxiWed either by catalase or by glutathione peroxidase that use GSH as a reductor to metabolize hydroperoxides and other peroxides (Grant et al. 1998). We found an increase in the total glutathione content in wild-type yeast cells whereas in superoxide dismutase-deWcient strains, the total glutathione content decreased. This decrease may be due to the consumption of GSH at increased GPx activity, considering that GSH is a substrate for GPx metabolism. The GSH oxidation to GSSG is involved in the transcriptional regulation of oxidative stress responsive genes (Pereira et al. 2003). Michels et al. (1994) reported that GPx has greater aYnity for H2O2 than catalase, suggesting that H2O2 is essentially degraded by GPx under normal conditions. The elevated GPx activity in SOD-deWcient strains in our study suggests the importance of this enzyme for the protection against Vit A-induced oxidative stress. Similarly, Manfredini et al. (2004) demonstrated that H2O2 resistance in sod1sod2 cells in stationary phase depends on GPx induction. Longo et al. (1996) found that both CuZnSOD and MnSOD participate in essential functions for cell survival, while catalase showed only a weak eVect and was associated with
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Fig. 4 Vitamin A co-treatment: sensitivity of wild-type and sod mutant strains to co-treatment with vitamin A and paraquat. SigniWcantly diVerent at P < 0.05, a when compared to the negative
control, b when compared to the paraquat 0.1 mM treatment, c when compared to the paraquat 0.5 mM treatment
MnSOD. Dal-Pizzol et al. (2001) showed that Vit A treatment increased catalase and GPx activities in Sertoli cells, whereas mannitol could inhibit this activity, suggesting that the eVects are due to ROS induction and not due to direct action of Vit A. In order to investigate the participation of ROS in the sensitivity presented in yeast strains treated with Vit A, the cells were concomitantly exposed to vitamin E, which is a wellknown antioxidant agent. The results presented in Fig. 1 showed that vitamin E was able to prevent the Vit Ainduced cell death, which is in agreement with a previous report (Klamt et al. 2003) showing that vitamin E can abolished the retinol-induced DNA damage in V79 cells using the comet assay. Murata and Kawanishi (2000) have also demonstrated that high doses of retinol induce DNA damage in HL-60 cells via superoxide radical generation. Oxidative cellular damage induced by retinol supplementation seems to be related to iron metabolism and to generation of highly reactive hydroxyl radicals (Dal-Pizzol et al. 2001). Contrary to the increased survival observed after concomitant treatment with Vit A and vitamin E (Fig. 1), the
combined treatment with Vit A and paraquat (Fig. 4) signiWcantly decreased survival, especially of the sod1 strains at the higher dose (0.5 mM) of paraquat. The critical action of Sod1 in removing free radicals is also evident by the extreme menadione sensitivity of sod1 cells under aerobic growth conditions (Gralla and Valentine 1991; Jamieson et al. 1994). On the other hand, the opposite eVect observed in the co-treatment with 0.1 mM paraquat and Vit A (i.e. signiWcantly increased survival of the combined treatment in relation to the corresponding paraquat treatment, Fig. 4b), suggested that Vit A can promote cell proliferation or apoptosis induction dependent on the cellular redox context. Increased proliferation after retinol treatment was reported in Sertoli cells (Zanotto-Filho et al. 2008). Proliferation induction is also observed in our study after 0.1 mM paraquat treatment in Vit A-pre-treated (at dose of 40 mg/ml) wild-type cells (Fig. 2a). From the survival results with Vit A and paraquat, we conclude that (a) CuZnSOD is the main enzyme that protects LOG cells against the eVects of Vit A; (b) MnSOD does not contribute for protection under our treatment con-
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ditions as survival levels of treated sod2 mutants do not change; and (c) in our conditions, Vit A treatment did not show protective eVects in yeast cells against paraquatinduced superoxide anion. Our results also demonstrate that Vit A-induced catalase and GPx activities in SOD-proWcient cells in LOG phase of growth, whereas only GPx was induced in SOD-deWcient mutants. This suggests that GPx is the main defence against Vit A-produced ROS in superoxide dismutase-deWcient S. cerevisiae cells but the elevation of GPx activity is not suYcient to prevent the induced cell death in these mutants. Moreover, the vitamin E co-treatment was able to abolish the reduced cell survival caused by Vit A treatment. Acknowledgments The authors would like to thank Dr. Edith Gralla (University of California, Los Angeles, CA, USA) for kindly providing yeast strains. This work was supported by the Brazilian Founding Agencies Conselho Nacional de Desenvolvimento CientíWco e Tecnológico (CNPq), Coordenação de Aperfeiçoamento e Formação de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).
References Akerboom TP, Sies H (1981) Assay of glutathione, glutathione disulphide and glutathione mixed disulphides in biological samples. Method Enzymol 77:373–382 Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. Annal Biochem 72:248–254 Bronzetti G, Cini M, Andreoli E, Caltavuturo L, Panunzio M, Croce CD (2001) Protective eVects of vitamins and selenium compounds in yeast. Mutat Res 496:105–115 Dal-Pizzol F, Klamt F, Benfato MS, Bernard EA, Moreira JCF (2001) Retinol supplementation induces oxidative stress and modulates antioxidant enzymes in rat sertoli cells. Free Rad Res 34:395–404 De Flora S, Bagnasco M, Vainio H (1999) Modulation of genotoxic and related eVects by carotenoids and vitamin A in experimental models: mechanistic issues. Mutagenesis 14:153–172 De Oliveira MR, Moreira JC (2008) Impaired redox state and respiratory chain enzyme activities in the cerebellum of vitamin A-treated rats. Toxicology 253:125–130 De Oliveira MR, Oliveira MW, HoV ML, Behr GA, da Rocha RF, Moreira JC (2009) Evaluation of redox and bioenergetics states in the liver of vitamin A-treated rats. Eur J Pharmacol 610:99–105 Flattery-O⬘Brien JA, Grant CM, Dawes IW (1997) Stationary-phase regulation of the Saccharomyces cerevisiae SOD2 gene is dependent on additive eVects of HAP2/3/4/5- and STRE- binding elements. Mol Microbiol 23:303–312 Flohe L, Gunzler WA (1984) Assays of glutathione peroxidase. Method Enzymol 105:114–121 Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Gisela S, Botstein D, Brown PO (2000) Genomic expression programs in
123
Arch Microbiol (2010) 192:221–228 the response of yeast cells to environmental changes. Mol Biol Cell 11:4241–4257 Gralla EB, Kosma DJ (1992) Molecular genetics of superoxide dismutases in yeasts and related fungi. Adv Gent 30:251–319 Gralla EB, Valentine JS (1991) Null mutants of S. cerevisiae Cu, Zn superoxide dismutase: characterization and spontaneous rates. J Bacteriol 173:5918–5920 Grant CM, Perrone G, Dawes IW (1998) Glutathione and catalase provide overlapping defenses for protection against hydrogen Peroxide in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 253:893–898 Halliwell B, Gutteridge JMC (2000) Free radical in biology and medicine, 3rd edn. Claredon, Oxford Jamieson DJ (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14:1511–1527 Jamieson DJ, Rivers SL, Stephen DWS (1994) Analysis of Saccharomyces cerevisiae proteins induced by peroxide and superoxide stress. Microbiology 140:3277–3283 Klamt F, Dal-Pizzol F, Roehrs R, Oliveira RM, Dalmolin R, Henriques JAP, Andrades HHR, Ramos ALLP, SaY J, Moreira JCF (2003) Genotoxicity, recombinogenicity and cellular preneoplasic transformation induced by Vitamin A supplementation. Mutat Res 539:117–125 Kujumdzieva-Savova V, Savov A, Georgieva EI (1991) Role of superoxide dismutase in the oxidation of n-alkanes by yeast. Free Rad Biol Med 11:263–268 Longo VD, Gralla EB, Valentine JS (1996) Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. J Biol Chem 271:12275–12280 Manfredini V, Roehrs R, Peralba MCR, Henriques JAP, SaY J, Ramos ALL, Benfato MS (2004) Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1sod2 double mutants against oxidative damage. Braz J Med Biol Res 37:159–165 Michels C, Raes M, Toussaint O, Remacle J (1994) Importance of Se-Glutathione peroxidase, catalase and Cu/Zn-SOD for cell survival against oxidative stress. Free Rad Biol Med 17:235–248 Moradas FP, Costa V, Mager W (1996) The molecular defences against reactive oxygen species in yeast. Mol Microbiol 19:651–658 Murata M, Kawanishi S (2000) Oxidative DNA damages by vitamin A and its derivative via superoxide generation. J Biol Chem 275:2003–2008 Pereira MD, Herdeiro RS, Fernandes PN, Eleutherio ECA, Panek AD (2003) Targets of oxidative stress in yeast sod mutants. Biochim Biophis Acta 1620:245–251 Petrova VY, Rasheva TV, Kujumdzieva AV (2002) Catalase enzyme in mitochondria of Saccharomyces cerevisiae. Eletronic J Biotechnol [online] 5:1 [cited 20 November 2009]. Available from: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S071734582002000100010&lng=es&nrm=iso. ISSN 0717-3458. doi: 10.4067/S0717-34582002000100010 Roehrs M, Valentini J, Bulcão R, Moreira JC, Biesalski H, Limberger RP, Grune T, Garcia SC (2009) The plasma retinol levels as prooxidant/oxidant agents in haemodialysis patients. Nephrol Dial Transplant 24:2212–2218 Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spirng Harbor, NY Zanotto-Filho A, Schröder R, Moreira JC (2008) Xanthine oxidasedependent ROS production mediates vitamin A pro-oxidant eVects in cultured Sertoli cells. Free Radic Res 42:593–601
