Effect of Myricetin, Pyrogallol, and Phloroglucinol on Yeast Resistance to Oxidative Stress
The health beneficial effects of dietary polyphenols have been attributed to their intrinsic antioxidant activity, which depends on the structure of the compound and number of hydroxyl groups. In this study, the protective effects of pyrogallol, phloroglucinol, and myricetin on the yeast Saccharomyces cerevisiae were investigated. Pyrogallol and myricetin, which have a pyrogallol structure in the B ring, increased H2O2 resistance associated with a reduction in intracellular oxidation and protein carbonylation, whereas phloroglucinol did not exert protective effects. The acquisition of oxidative stress resistance in cells pretreated with pyrogallol and myricetin was not associated with an induction of endogenous antioxidant defences as assessed by the analysis of superoxide dismutase and catalase activities. However, myricetin, which provided greater stress resistance, prevented H2O2-induced glutathione oxidation. Moreover, myricetin increased the chronological lifespan of yeast lacking the mitochondrial superoxide dismutase (Sod2p), which exhibited a premature aging phenotype and oxidative stress sensitivity. These findings show that the presence of hydroxyl groups in the ortho position of the B ring in pyrogallol and myricetin contributes to the antioxidant protection afforded by these compounds. In addition, myricetin may alleviate aging-induced oxidative stress, particularly when redox homeostasis is compromised due to downregulation of endogenous defences present in mitochondria.
Oxidative stress is a hallmark of human disorders such as cancer and age-associated diseases . It results from an unbalance between the levels of reactive oxygen species (ROS) or reactive nitrogen species (RNS) and cellular antioxidant defenses. The toxicity of high levels of ROS and RNS is associated with the accumulation of damaged molecules, including proteins, lipids, and nucleic acids . Under normal physiological conditions, ROS are kept at low levels by antioxidant defenses such as superoxide dismutases (SOD) that catalyze the dismutation of superoxide radicals into hydrogen peroxide, catalases, or peroxidases that reduce H2O2 into water, as well as nonenzymatic defenses, including glutathione that plays critical roles in redox homeostasis and cellular detoxification . In addition, antioxidants obtained in the diet, such as vitamins C and E and phenolic compounds, play essential role in cellular protection .
Phenolic compounds are natural antioxidants present in the human diet through the consumption of fruits, vegetables, and drinks such as juice, tea, coffee, and wine [4, 5]. Structurally, these compounds are characterized by having one or more hydroxyl groups attached to at least one aromatic ring . The number and position of hydroxyl groups are important features that affect the antioxidant activity of phenolic compounds . These compounds possess antiproliferative, proapoptotic, and anti-inflammatory properties and they have been associated with the prevention of cancer and cardiovascular, neurodegenerative, and metabolic disorders [7, 8]. The protective effects of these compounds have been attributed not only to their intrinsic antioxidant activity but also to the modulation of cell signaling pathways, including mitogen-activated protein kinase cascades, which regulate oxidative stress responses [9–11].
The budding yeast Saccharomyces cerevisiae has been used as a eukaryotic model organism to characterize the molecular mechanisms underlying oxidative stress resistance and to evaluate the antioxidant potential of dietary extracts and phenolic compounds . We have previously reported that quercetin, the most common flavonol in the diet, increases yeast oxidative stress resistance  and exerts its protective effects against oxidative stress by inducing the biosynthesis of trehalose, a stress protectant disaccharide, and the activation of the cell wall integrity pathway . Other studies have shown that resveratrol and catechin increase oxidative stress resistance in yeast by mechanisms associated with the activation of catalase , whereas delphinidin 3-glucoside and petunidin 3-glucoside protect yeast through activation of the stress response regulators Msn2p and Msn4p . Moreover, the sirtuin Hst3p has been implicated in oxidative stress protection afforded by a polyphenol-enriched cocoa powder .
Pyrogallol and phloroglucinol are simple phenols that contain three hydroxyl groups in the ortho- and metaposition, respectively, of a benzene ring (Figure 1(a)). Humans are exposed to pyrogallol through ingestion of tea and coffee  but also from degradation of gallic acid in colon . Phloroglucinol is found as a monomer of phlorotannins in brown algae, which is increasing in the human diet . Myricetin is a naturally occurring flavonol characterized by having a pyrogallol structure in the B ring as well as a 4-oxo function with an unsaturated bond between the 2 and 3 carbons within the C ring and the presence of hydroxyl groups at C3 and C5  (Figure 1(a)). In the human diet, myricetin is commonly found in tea, berries, and red wine . In this study, we investigated the effect of myricetin, pyrogallol, and phloroglucinol on yeast resistance to oxidative stress.
2. Materials and Methods
All reagents and chemicals used were of analytical grade. Sodium or potassium phosphates, riboflavin, and H2O2 were purchased from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO), myricetin, pyrogallol, phloroglucinol, and nitroblue tetrazolium were purchased from Sigma (Sintra, Portugal). Phenolic compounds were dissolved in DMSO at a 200 mM stock concentration and stored at −80°C. Solutions were prepared in ultrapure water (Milli-Q).
2.2. Yeast Strains and Growth Conditions
Saccharomyces cerevisiae cells (Euroscarf, Germany) used in this study were BY4741 (Matα, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0; parental strain), sod1Δ (BY4741 sod1Δ::KanMX4), and sod2Δ (BY4741 sod2Δ::KanMX4). Yeast cells were grown in YPD medium [1% (w/v) yeast extract, 2% (w/v) bactopeptone, and 2% (w/v) glucose] or in synthetic complete (SC) drop-out medium containing 2% (w/v) glucose, 0.67% (w/v) yeast nitrogen base without amino acids supplemented with the appropriate amino acids (80 mg His L−1, 400 mg Leu L−1, and 80 mg trp L−1), and nucleotides (80 mg Ura L−1). Cultures were maintained in an orbital shaker, at 26°C and 120 rpm, with a ratio of flask volume/medium volume of 5 : 1.
2.3. Oxidative Stress Resistance Assays
Yeast cells were grown to the exponential phase (OD600 = 0.5-0.6) in YPD medium, pretreated with polyphenols (myricetin, pyrogallol, or phloroglucinol at 300 μM) or equal volume of DMSO (vehicle) for 15 min, and subsequently exposed to 1.5 mM H2O2 for 1 hour. Cell viability was determined by dilution plate counts on YPD medium containing 1.5% agar. Colonies were counted after growth at 26°C for 3 days. Viability was expressed as the percentage of colony-forming units (CFU).
2.4. Intracellular Oxidation
The oxidant-sensitive probe 2′,7′-dichlorodihydrofluorescein (H2DCF-DA) (Molecular Probes) was used to measure intracellular oxidation. Yeast cells grown to the exponential phase in YPD medium and pretreated with polyphenols for 15 min were subsequently exposed to 1.5 mM H2O2 for 1 hour in the absence or presence of 25 μM H2DCF-DA. Cells were spun down (4,000 rpm, 4 min), washed twice, and suspended in filtered phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4). Fluorescence was measured in FL-1 channel (excitation and emission wavelength at 488 nm and 525 nm, resp.) in a Becton-Dickinson FACSort flow cytometer. Autofluorescence was analyzed in cells untreated with H2DCF-DA. Data was acquired from a total of 10,000 events/samples. BD CellQuest Pro Software was used for data acquisition and FlowJo Software for data analysis.
2.5. Protein Carbonylation
Protein extracts were prepared in 50 mM potassium phosphate buffer (pH 7.0) containing protease inhibitors (Complete, Mini, EDTA-free Protease Cocktail Inhibitor Tablets; Roche Applied Science), by vigorous shaking of the cell suspension, in the presence of glass beads, for 5 min. Short pulses of 1 min were used, with 1 min intervals on ice. Protein content was estimated by the Lowry method, with bovine serum albumin as a standard. Protein carbonylation assays were performed by slot-blot analysis, as previously described , using rabbit IgG anti-dinitrophenyl (DNP) (Sigma) at a 1 : 5,000 dilution as the primary antibody and goat anti-rabbit IgG-peroxidase (Sigma) at 1 : 5,000 as the secondary antibody. Immunodetection was performed by chemiluminescence, with a kit from GE Healthcare (RPN 2109). Quantification of bands was performed by densitometry.
2.6. Glutathione Levels and Enzymatic Activities
All the procedures were carried out at 4°C. Yeast cells were harvested by centrifugation. Glutathione levels were measured by the method of Tietze , as described in a previous work . For enzyme activities, yeast extracts were prepared as described for the analysis of protein carbonylation. The activity of catalase and SOD was analyzed in situ, after separation of proteins (50 μg) by native polyacrylamide gel electrophoresis (PAGE), as described previously [23, 24]. Quantification of bands was performed by densitometry.
2.7. Chronological Lifespan
Overnight cultures in SC medium were diluted to OD600 = 0.5 and grown to the stationary phase for 3 days (in the case of BY4741 and sod1Δ cells) or for 1 day (in the case of sod2Δ cells). Then, the compounds (300 μM myricetin or pyrogallol) or DMSO (vehicle; volume identical to compounds) were added to the cultures (day 0). These cells were kept in culture media at 26°C and viability was analyzed at indicated times by standard dilution plate counts on YPD medium containing 1.5% agar. Colonies were counted after growth at 26°C for 3 days and viability was expressed as the percentage in CFU relative to day 0.
2.8. Statistical Analysis
Analysis was performed in GraphPad Prism. Data are expressed as the mean values ± standard error of the mean (SEM) of at least three independent experiments. The 0.05 probability level was selected as the point of statistical significance. Values of oxidative stress resistance assays were analyzed by one-way ANOVA and compared by Dunnett’s multiple comparisons test. Intracellular ROS and protein carbonyls were analyzed by two-way ANOVA and compared by Sidak’s multiple comparisons test. Statistical analysis of total and oxidized glutathione levels and the ratio was performed by two-way ANOVA, Sidak’s multiple comparisons test () for comparison of values between treatments in each condition (control or H2O2), and multiple -tests using the Holm-Sidak method for corrections () for comparison of values between control and H2O2 for all treatments. Lifespans were compared by Student’s -test.
3.1. Myricetin and Pyrogallol Increase Hydrogen Peroxide Resistance in Saccharomyces cerevisiae
To assess the effect of myricetin, pyrogallol, and phloroglucinol on oxidative stress resistance, exponential phase yeast cells were pretreated with these compounds individually (300 μM) or DMSO (control) for 15 min and subsequently exposed to 1.5 mM H2O2 for 1 hour. The presence of polyphenols per se (in the absence of H2O2) did not affect cell viability, intracellular oxidation, or protein oxidation. Myricetin and pyrogallol, in contrast with phloroglucinol, increased cell viability from 33% (in control cells) to 64% and 51%, respectively (Figure 1(b)). To investigate if H2O2 resistance induced by these polyphenols was correlated with a decrease in oxidative stress markers, intracellular ROS levels were measured by flow cytometry using cells labeled with an oxidant-sensitive probe, H2DCF-DA (Figures 2(a)-2(b)), and protein oxidation was assessed through the analysis of protein carbonyl content (Figure 2(c)). In control cells, exposure to H2O2 caused a 10-fold increase in intracellular ROS and a 3-fold increase in protein carbonylation. Myricetin and pyrogallol, but not phloroglucinol, significantly decreased H2O2-induced intracellular oxidation and protein carbonylation.
3.2. Myricetin and Pyrogallol Do Not Affect the Activity of Superoxide Dismutase or Catalase
To investigate if the protective effect of myricetin or pyrogallol was associated with an induction of antioxidant defenses, the activity of superoxide dismutase and catalase was determined. Consistent with published data , SOD activity decreased 31% in control cells (DMSO-treated) exposed to H2O2 (Figure 3(a)). Pretreatment with the phenolic compounds did not affect basal SOD activity or prevent its decrease upon exposure to H2O2. Catalase is not expressed in exponential phase cells  and, therefore, its activity was not detected in control cells. Moreover, it was not induced in cells treated with the tested compounds (data not shown). These results indicate that the increase of oxidative stress resistance in cells pretreated with myricetin or pyrogallol did not result from the induction of SOD and catalase.
3.3. Myricetin Suppresses H2O2-Induced Glutathione Oxidation
The tripeptide glutathione (GSH) is the most abundant low-molecular weight thiol that serves to maintain a reduced intracellular environment . To assess the effect of myricetin, pyrogallol, and phloroglucinol on redox homeostasis, glutathione levels were determined in cells exposed to H2O2 (Figures 3(b)–3(d)). In control cells, after exposure to H2O2, total glutathione levels () decreased 37% whereas GSSG levels increased 70%, increasing the ratio between GSSG and . Similar results were observed in cells pretreated with phloroglucinol, which is consistent with the fact that this compound did not affect oxidative stress resistance. Myricetin and pyrogallol per se (in the absence of H2O2) decreased levels. However H2O2-induced glutathione depletion was lower in cells pretreated with these compounds, comparing with DMSO-treated cells. Moreover, the increase in the levels of GSSG and in the ratio induced by H2O2 was suppressed by myricetin, but not by pyrogallol. This is consistent with our data showing that oxidative stress resistance in cells pretreated with myricetin was higher than the observed in pyrogallol pretreated cells.
3.4. Myricetin Increases the Chronological Lifespan of sod2Δ Mutant Cells
Aging has been associated with an increase in intracellular oxidation and accumulation of oxidative damage . Mitochondria are a major source of ROS and its dysfunction has been implicated in aging [29, 30]. Mitochondria contain several antioxidant enzymes, including the superoxide dismutases Sod1p (CuZnSOD) that is present in the mitochondrial intermembrane space (and cytosol) and Sod2p (MnSOD) located in the mitochondrial matrix. Cells lacking Sod1p or Sod2p exhibit a decreased chronological lifespan associated with the accumulation of oxidative damage  (Figures 4(a)-4(b)). The protective effect of myricetin and pyrogallol against oxidative stress caused by H2O2 led us to assess its effect on the chronological lifespan (CLS) of parental cells and of sod1Δ and sod2Δ mutant cells. Parental cells showed a time-dependent loss of cell viability, which was not affected by pretreatment with myricetin, pyrogallol, or phloroglucinol (Figure 4(a)). These phenolic compounds also did not affect the lifespan of sod1Δ cells (data not shown). However, myricetin significantly increased the CLS of sod2Δ cells (Figure 4(b)), suggesting that this compound exerts a protective effect that is particularly relevant in cells that have a decreased capacity to scavenge superoxide radicals within the mitochondrial matrix. Consistently, myricetin decreased protein carbonylation in aged sod2Δ cells, although it had a modest effect in parental cells (Figures 4(c)–4(e)). In contrast, pyrogallol and phloroglucinol did not extend the CLS of sod2Δ cells (Figure 4(b)).
Mitochondria play an important function during oxidative stress. Indeed, ρ0 petite strains, which lack mitochondrial DNA, and cells deficient in electron transport chain function are sensitive to H2O2 [32, 33]. A recent study showed that H2O2 increases the mitochondrial production of superoxide radicals, which have a protective effect at low concentrations . However, high concentrations of superoxide radicals are detrimental. In agreement, sod2Δ cells were sensitive to H2O2 (Figure 5). We also assessed the effect of polyphenols in sod2Δ cells exposed to H2O2. The results show that pyrogallol pretreatment slightly increased H2O2 resistance of sod2Δ cells, although to levels below those observed in parental cells. In contrast, myricetin and phloroglucinol did not affect H2O2 resistance in these mutants (Figure 5).
The increased production of ROS and RNS together with the decrease of antioxidant defenses has been implicated in the pathogenesis of numerous diseases and aging . Thus, a diet containing natural compounds with antioxidant properties, such as phenolic compounds, may be beneficial to human health. The antioxidant activity of these compounds is determined by structural features, including the number and position of hydroxyl groups, polarity, solubility, and reducing potential [35, 36]. In this study, we used the yeast Saccharomyces cerevisiae to assess in vivo the antioxidant capacity of the flavonol myricetin and two simple phenols, pyrogallol and phloroglucinol. Myricetin was the most effective in increasing H2O2 resistance in yeast, whereas phloroglucinol had no protective effect. Consistently, H2O2-induced intracellular oxidation and protein carbonylation decreased in cells pretreated with myricetin and pyrogallol but not with phloroglucinol. Pyrogallol and phloroglucinol contain three hydroxyl groups in the ortho- and metaposition, respectively, of a benzene ring. The vicinal positions of hydroxyl groups in pyrogallol result in a lower bond dissociation energy of O–H, facilitating the donation of hydrogen to free radicals . In accordance with that, our results show that pyrogallol, in contrast with phloroglucinol, increased the viability of yeast cells exposed to H2O2. Myricetin, which contains a pyrogallol structure in the B ring, provided an even higher resistance. Our results are in accordance with data demonstrating the importance of the pyrogallol structure for the bioactivity of phenolic compounds . Our data is also consistent with several reports showing a protective effect of myricetin against oxidative stress in mammalian cells. For instance, myricetin decreases H2O2-induced DNA damage in Caco-2 and HepG2 cells  and decreases tert-butyl hydroperoxide-induced protein oxidation and lipid peroxidation in erythrocytes from T2DM patients .
Being redox-active compounds, phenolic compounds can also act as prooxidants and, therefore, induce stress responses leading to an increase in the levels of cellular antioxidant defenses [41, 42]. Our results indicate that this mechanism does not contribute to the protective effects of myricetin and pyrogallol in yeast, since these compounds did not increase intracellular oxidation or affect catalase and SOD activities under the conditions used in this study. We have previously observed that hydrogen peroxide resistance in yeast incubated with quercetin is also not associated with prooxidant effects or modulation of antioxidant defenses . In contrast, other reports showed that catalase activity increases in yeast treated with resveratrol and catechin, enhancing cellular resistance to oxidative stress .
Glutathione is an important cellular small molecule responsible for the maintenance of redox homeostasis . The reduced form (GSH) mediates H2O2 decomposition catalyzed by glutathione peroxidase  giving rise to oxidized glutathione (GSSG) which is then reduced to GSH by glutathione reductase . Glutathione has also important functions in detoxification of toxic compounds  and in the protection of proteins from oxidation through glutathionylation . Thus, glutathione oxidation is a biomarker of oxidative stress. In control (DMSO-treated) cells, exposure to H2O2 led to an increase in GSSG levels that, concomitantly with glutathione depletion, resulted in a higher ratio. In cells pretreated with myricetin, H2O2-induced glutathione oxidation and the increase in the ratio were suppressed, which is consistent with the reduction of intracellular oxidation. Pretreatment with pyrogallol, which had a lower protective effect comparing with myricetin, did not prevent glutathione oxidation. These results suggest a correlation between the protective effect of myricetin and maintenance of glutathione redox status. Treatment with both myricetin and pyrogallol per se led to a decrease in total GSH levels, which may result from the formation of GS-compound adducts mediated by glutathione-S-transferases. Indeed, these adducts have been reported for quercetin [41, 46] and glutathione-S-transferase activity may be induced by these phenolic compounds, similarly to the effects of coumarin .
High levels of ROS have been implicated in aging in yeast and higher eukaryotes [48, 49]. The accumulation of oxidative damage leading to neuronal death is associated with age-related diseases such as Alzheimer’s and Parkinson diseases . Therefore, a diet replete in phytochemicals with antioxidant activity, characteristic of a Mediterranean diet, reduces the functional decline associated with aging and age-related disorders, increasing health span [51, 52]. Several studies showed an increase of the lifespan of yeast cells incubated with phenolic compounds. Resveratrol and phloridzin, a major apple compound, increase yeast replicative lifespan by mechanisms associated with the activation of the sirtuin Sir2p [53, 54]. Moreover, quercetin and apple polyphenolic fractions increase yeast chronological lifespan [13, 55]. Here, we report that although myricetin does not affect the chronological lifespan of parental and sod1Δ cells, it extends the lifespan of yeast cells lacking the mitochondrial superoxide dismutase, which are known to exhibit a very short lifespan . In contrast, pyrogallol did not extend the CLS of sod2Δ cells. These results suggest that myricetin may be more effective in protecting aged cells that have high intracellular ROS levels and oxidative damage, especially in the mitochondria. In agreement with that, several reports show protective effects of myricetin in mitochondria. For instance, the protective effect of L-ascorbic acid against paraquat, a generator of superoxide radicals, is more pronounced in sod2Δ than in parental cells . In addition, myricetin decreased the generation of H2O2 in isolated mouse skeletal muscle mitochondria  and decreased the depolarization of the inner mitochondrial membrane potential in C6 glial cells exposed to oxygen-glucose deprivation  and it was the most efficient among other phenolic compounds in the protection of mouse brain mitochondria against toxicity induced by methyl mercury . Notably, myricetin was unable to protect sod2Δ cells against high doses of H2O2 whereas pyrogallol slightly increased the oxidative stress resistance of these mutants. It is likely that the excessive oxidative stress in sod2Δ cells treated with high doses of H2O2 overwhelms the protective effects of these compounds.
In summary, our data show that myricetin and, to a lesser extent, pyrogallol, increased yeast resistance to H2O2. This protective effect was correlated to a reduction in intracellular oxidation and protein carbonylation and maintenance of ratio. However, changes in catalase or superoxide dismutase activities were not associated with the protective effects. Furthermore, myricetin attenuated the shortened CLS of yeast cells lacking the mitochondrial superoxide dismutase (sod2Δ mutants).
|PAGE:||Polyacrylamide gel electrophoresis|
|RNS:||Reactive nitrogen species|
|ROS:||Reactive oxygen species|
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Vanda Mendes was supported by a FCT (Fundação para a Ciência e Tecnologia) Fellowship (SFRH/BD/78474/2011).
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