Brewer’s Spent Grains Protects against Oxidative DNA Damage in Saccharomyces cerevisiae

Brewer’s spent grain (BSG), obtained from barley malt during brewing, contains high amounts of phenolic acids, predominantly ferulic and p-coumaric acids. The protective effects of BSG extracts against oxidative DNA damage induced by H2O2 in Saccharomyces cerevisiae cells were investigated using an optimized yeast comet assay and flow cytometry. The results indicated that BSG extracts from black malt exhibited a 5-fold reduction in the genotoxic effects of H2O2, compared to the 2-fold decrease by the BSG extracts from pilsen malts. Flow cytometry analysis with dichlorofluorescein diacetate demonstrated that the intracellular oxidation of S. cerevisiae is also reduced to approximately 50% in the presence of 20-fold diluted BSG extracts. BSG extracts obtained from pilsen and black malt types exert dose-dependent protective properties against the genotoxic effects induced by ROS and decrease intracellular oxidation of yeast cells.


Introduction
Brewer's spent grain (BSG) is the resultant residual solid fraction of barley malt after wort production for the brewing industry.BSG is a unique source of bioactive ingredients such as hydroxycinnamic acids ferulic and p-coumaric acids (Meneses, Martins, Teixeira, & Mussatto, 2013;Moreira, Morais, Barros, Delerue-Matos, & Guido, 2012;Moreira et al., 2013;Mussatto, Dragone, & Roberto, 2007).Several studies demonstrate that extracts containing hydroxycinnamic acids hold important health benefits, including antioxidant or free radical scavenging (Shahidi & Chandrasekara, 2010), anti-carcinogenic (Gomes et al., 2003), anti-inflammatory (Kim et al., 2012) and antigenotoxic (McCarthy et al., 2012) properties.Currently, BSG is used in animal feed as a source of protein and fiber, providing all the essential amino acids in combination with other nitrogen sources (Mussatto, Dragone, & Roberto, 2006).Furthermore, new applications of the by-products from the brewing industry would be an advantage due to the increasing costs for their disposal (Vieira et al., 2014).lead to the damage of cell components, including DNA (Henle, Luo, Gassmann, & Linn, 1996).In a situation of DNA oxidative damage, cell cycle is arrested for the activation of repair mechanisms and if damage cannot be repaired, mechanisms of programmed cell death are activated.The principal DNA repair pathways are base excision repair (BER) by removing oxidation damaged single bases and nucleotide excision repair (NER), which is involved in repairing bulky DNA lesions caused by ultraviolet light (Azevedo, Marques, Fokt, Oliveira, & Johansson, 2011).
The DNA damage can be evaluated using the comet assay, where the chromosomal DNA migration distance by electrophoresis correlates with the extent of DNA damage.The comet assay has been used for a variety of applications with several organisms (Rank, Syberg, & Jensen, 2009) and was also optimized for application in yeast cells (Saccharomyces cerevisiae) (Azevedo et al., 2011;Marques, Azevedo, Johansson, & Oliveira, 2011).Azevedo et al. (2011) reported an optimized comet assay protocol for yeast cells to evaluate DNA damage and oxidative damage caused by H 2 O 2 with high sensitivity and reproducibility.
Several studies have focused on the antioxidant properties of BSG (Bartolomé, Santos, Jiménez, del Nozal, & Gómez-Cordovés, 2002;Meneses et al., 2013), however, to our knowledge, only one has reported the antigenotoxic activity of BSG extracts.McCarthy et al. (2012) have showed phenolic BSG extracts are able to protect against DNA damage in human lymphocytic U937 cells using the comet assay.The current study aimed at investigating the protective effects of BSG phenolics against DNA oxidative damage in S. cerevisiae.Yeast S. cerevisiae has been chosen has an eukaryotic model to study the cellular response against stress damages due to biochemical and molecular similarities with human cells (Mager & Winderickx, 2005).A recent study by our group on the evaluation of antioxidant activity and identification of the major phenolic compounds of BSG from six malt types (pilsen, melano, melano 80, carared, chocolate and black) has revealed that pilsen BSG exhibits higher total phenolic content and antioxidant activity and black BSG, the lower (Moreira et al., 2013).Therefore, BSG samples used in this study were those whose differences were more pronounced, corresponding to BSG obtained from pilsen and black malts.We have examined whether these extracts could inhibit oxidative DNA damage induced by H 2 O 2 using a recently developed comet assay applied to yeast.

Chemicals and Samples
All reagents were of analytical grade and were purchased from Sigma-Aldrich, unless otherwise stated.BSG samples used in this work were kindly supplied by Unicer -Bebidas de Portugal, S.A. (S.Mamede de Infesta, Portugal) and obtained from barley malt samples supplied by Os Três Cervejeiros, Lda (Porto, Portugal).BSG samples investigated in this work were chosen according to the more pronounced differences in total phenolic content and antioxidant activity, evaluated in a previous work (Moreira et al., 2013), and correspond to BSG samples obtained from pilsen and black malts.Pilsen malt produces very light colored, clean and crisp worts and is used as base malt for all beer styles.The color ranges from 3.5 to 5.7 EBC units and kilning temperature between 80 to 85 ºC.Black malt is roasted for a higher period and the end temperature is very high (230 ºC), forming harsher flavors and EBC color ranges from 1350 to 1500 units.The BSG samples used for phenolics extraction correspond to the remaining solid fraction obtained following the removal of wort during the pilot scale production of beer in the brewing process.For wort production it was followed the procedure previously described (Moreira et al., 2013), and the obtained solid residue was frozen, lyophilized and then finely ground.

BSG Phenolic Extracts
Phenolics were extracted from dried BSG samples according to the microwave-assisted extraction (MAE) procedure previously optimized (Moreira et al., 2012).After MAE, the extracts were centrifuged and the pH of the supernatant was adjusted to pH 6.5 with HCl 6 M.After filtration through a cellulose filter (0.45 μm), the extracts were stored at -20 °C until further analysis.

Viability Assays
A pre-culture prepared in YPD medium, with a single yeast colony and grown overnight, was diluted to a density of 1.2 × 10 7 cells/mL in fresh medium and harvested by centrifugation (2 min at 5000 rpm, 4 ºC) after two generations.Cells were subsequently washed twice, each time with the same volume of sterilized deionized water.Pre-treatment with BSG extracts was made by ressuspending the pellet in the same volume of ½ diluted BSG extracts (1:1 (v/v) YPD 2-fold concentrated and BSG extract).Cells with BSG extracts were incubated at 30 ºC, 200 rpm for 90 min.Samples were harvested at 0, 20, 60 and 90 min, serially diluted to 10 -4 in sterilized deionized water and spread on YPD plates.Plates were incubated at 30 ºC for 48 h and the colonies were counted.Viability was calculated as percentage of colony-forming units (CFU) at each time point in relation to the beginning of the experiment (0 min).
As a statistically significant decrease in cells growth rate was observed at 90 min for black BSG extracts, another assay was carried out under similar conditions.Cells were prepared in the same way as for viability measurement, except that they were suspended in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4), instead of YPD medium.In PBS, cells are not able to grow and a lower value of viability may be related to a cytotoxic effect.Survival rates were calculated as percentage of CFU, assuming 100% survival for cells of the suspension before any treatment.

Analysis of DNA Damage by the Yeast Comet Assay
DNA damage was assessed by a recently developed yeast comet assay (Azevedo et al., 2011).Briefly, cell walls were digested with 2 mg/mL zymolyase (20,000 U/g; ImmunO TM -20 T), incubated at 30 ºC for 20 min and collected by centrifugation at 5000 rpm, 4 ºC for 2 min.Spheroplasts were then suspended in 1 mL S buffer (1 M sorbitol, 25 mM KH 2 PO 4 , pH 6.5) and 80 µL of this suspension were distributed by microtubes and collected by centrifugation at 5000 rpm, 4 °C for 2 min.Each pellet was suspended in BSG extracts diluted in water and exposed to the oxidant solution (10 mM H 2 O 2 ) for 20 min at 4 ºC.Treated spheroplasts were collected from suspension by centrifugation at 5000 rpm, 4 ºC for 2 min and then embedded in 1.5% (w/v in S buffer) low melting agarose (LMA) at 35 ºC and distributed by glass slides (slide coated with a water solution of 0.5% w/v normal-melting agarose), covered with a coverslip and placed on ice to solidify.Following exposure of the cells to the H 2 O 2 treatment, the slides were incubated in the lysing buffer (30 mM NaOH, 1 M NaCl, 0.05% w/v laurylsarcosine, 50 mM EDTA, 10 mM Tris-HCl, pH 10) for 20 min in order to lyse spheroplasts.Samples were incubated in electrophoresis buffer (30 mM NaOH, 10 mM EDTA, 10 mM Tris-HCl, pH 10) for 20 min and then submitted to electrophoresis in the same buffer for 10 min at 0.7 V/cm.After electrophoresis, the slides were incubated in neutralization buffer (10 mM Tris-HCl, pH 7.4) for 10 min, followed by consecutive 10 min incubations in 76% and 96% (v/v) ethanol.The slides were then dried at room temperature and were visualized immediately or stored at 4 ºC until observation.For visualization in a fluorescence microscope (Leica Microsystems DM fluorescence), the slides were stained with GelRed (10 µg/ mL; Biotium) and representative images were acquired at magnification of 400× in order to obtain at least 20 random comets per sample that were analyzed with the CometScore version 1.5 software for the tail length (expressed in μm).Error bars represent variability between the mean of at least three different slides obtained from biologically independent experiments.

Flow Cytometry Analysis
Cells were prepared as described for cell viability measurements, except that they were diluted to a density of 1.0 × 10 6 cells/mL and suspended in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4), instead of YPD medium.Five hundred microlitres of untreated cells were removed for autofluorescence measurement.Dichlorofluorescein diacetate (H 2 DCFDA; Sigma-Aldrich) (50 µM final concentration) was added as a fluorochrome to the reminder of the cells and cell suspension was further incubated at 30 ºC, 200 rpm for 1 h in the dark, washed twice with the same volume of PBS and distributed in aliquots for the different assay conditions.Cells were treated with diluted BSG extracts, 5 mM H 2 O 2 , or both and incubated at 30 ºC, 200 rpm for 20 min.Twenty thousand cells of each sample were analyzed by flow cytometry in an Epics XLTM cytometer (Beckman Coulter) equipped with a 15 mW argon-ion laser emitting at 488 nm.Green fluorescence was collected through a 488 nm blocking filter, a 550 nm long-pass dichroic and a 225 nm bandpass filter.Data were analyzed and histograms were made with the Flowing software version 2.5.0.

Statistical Analysis
All results are the mean and standard deviation (SD) values of at least three independent experiments.Differences in means were detected using one-way ANOVA and Tukey's test.The software employed for statistical analysis was Graphpad Prism, version 5 for Windows.Asterisks indicate differences considered statistically significant: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001, when compared to the respective control.jas.ccsenet.

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Discussion
This work clearly demonstrates that BSG extracts obtained from pilsen and black malt types exert dose-dependent protective effects against the genotoxic effects induced by H 2 O 2 in yeast cells.However, oxidative protective activity of pilsen BSG extract was less effective comparing to black BSG extract (Figure 2), suggesting a less antigenotoxic activity.Beneficial properties of BSG extracts may be related to bioactive compounds with antioxidant activity, which prevent DNA oxidative damage induced by strong reactive ROS (Bellion et al., 2010).Therefore, the differences observed for pilsen and black BSG extracts can be ascribed to the different composition of the extracts.Ferulic and p-coumaric acids are well known phenolic compounds present in high concentrations in BSG (Moreira et al., 2013).Moreover, previous studies have demonstrated that ferulic acid (FA) protects DNA against oxidative damage caused by H 2 O 2 , as measured by the comet assay (Wang, Sun, Cao, Song, & Tian, 2008).Recently, McCarthy et al. (2012) also demonstrated that FA at 1 µg/mL leads to a 50% reduction of DNA oxidative damage in U937 cells treated with H 2 O 2 using the comet assay.In a study conducted by Wang et al. (2008) and also applying the comet assay, the FA ester of oligosaccharides present in wheat bran were able to protect against H 2 O 2 induced DNA damage in normal human peripheral blood lymphocytes.In all these works the observed effects were mainly attributed to the antioxidant capacity of the FA moiety.
The results reported herein for yeast comet assay did not directly correlate with previously reported data (Moreira et al., 2013), which demonstrated that pilsen BSG extract exhibited the highest antioxidant activity and higher phenolic content.This difference can be partially be explained by the oxidative mechanism and radical system involved in antioxidant evaluation, which was shown to interfere in the evaluation of the antioxidant activity of a selected sample (Shahidi & Chandrasekara, 2010).The radical systems used in the experiments carried out in our previous work (Moreira et al., 2013) were based on in vitro assays, while the comet assay is developed in yeast cells involving a high complex chemical environment.Additionally, the observed differences pilsen and black BSG extracts regarding preventive DNA oxidative damage may be attributed to melanoidins compounds.Melanoidins are generated during malt kilning through the Maillard reaction and are, therefore, mainly present in black malts.Melanoidins can be defined as brown high molecular weight compounds formed in the last stages of the Maillard reaction of thermally treated foods.Several studies reported that these high molecular weight compounds may chelate iron and potentially inhibit the metal-dependent processes leading to DNA damage (Coghe, Gheeraert, Michiels, & Delvaux, 2006;Inns, Buggey, Booer, Nursten, & Ames, 2007).In agreement, the antioxidant properties of BSG extracts can be attributed to chemical components, which may have a scavenging effect on H 2 O 2 and/or an induction of oxidative stress response and/or induction of DNA damage repair.The mechanism of the protective action of the bioactive compounds is not clearly understood.
The commonly accepted mechanism is the scavenging of ROS by polyphenols since they are well-known due to their strong antioxidant activity.However, mechanisms involving metal binding have also been proposed (Lopes, Schulman, & Hermes-Lima, 1999;Perron, García, Pinzón, Chaur, & Brumaghim, 2011;Perron, Hodges, Jenkins, & Brumaghim, 2008).Perron et al. (2008) have confirmed that polyphenol compounds, such as catechin, quercetin, gallic, protocatechuic and vanillic acids inhibit DNA damage by a mechanism involving iron binding.They reported that the iron oxidation observed upon binding to polyphenol compounds may result in an iron (III) complex that cannot be reduced by cellular reductants to catalytically generate a hydroxyl radical.Therefore, the determination how iron coordination controls the antioxidant activity of these extracts is important to better understanding their biological activity.
Antioxidant activity assessed by flow cytometry assays demonstrated that oxidative damage in yeast cells can be reduced to approximately 50% in the presence of 20-fold diluted BSG extracts.These results strongly suggest that BSG extracts reduce intracellular ROS in yeast cells, supporting the hypothesis the same extracts possess ROS scavenging properties and/or efficiently recycle endogenous scavenger cellular proteins such as glutathione, thioredoxin, superoxide dismutase, catalase, and/or induce the pentose phosphate pathway activity in regenerating NADPH (Azevedo et al., 2011).

Conclusions
The results from the current study revealed that BSG extracts from pilsen and black malts may have strong in vivo antioxidant activity protecting Saccharomyces cerevisiae cells against oxidative DNA damage.Black BSG extracts exhibited a 5-fold reduction in the genotoxic effects of H 2 O 2 , while pilsen BSG extracts exhibited a 2-fold reduction.Antioxidant activity assessed by flow cytometry assays demonstrated that the H 2 O 2 oxidant activity can be reduced to approximately 50% in yeast cells in the presence of 20-fold diluted BSG extracts.

Table 1 .
Treatment of S.cerevisiae cells with BSG extracts decreased the intracellular oxidative damage by H 2 O 2 .Yeast spheroplasts were simultaneously treated with 20-, 50-, 75-and 100-fold diluted BSG extracts in PBS and 5 mM H 2 O 2 .The negative control (H 2 O) reflects the amount of DNA damage in cells without exposure to BSG extracts and H 2 O 2 .The 20-fold diluted BSG extract reflects the amount of DNA damage in cell only treated with BSG extracts.Oxidative damage was analyzed by flow cytometry (see Materials and Methods).Fluorescence average ± SD are from three independent experiments (*** represents p < 0.001)