Bactris Setosa Mart. (Tucum-do-cerrado) Aqueous Extract Increases Growth and Viability in Saccharomyces Cerevisiae BY4741 and YAP1Δ Under Stress Caused by Menadione and Hydrogen Peroxide

Yeast cells from Saccharomyces cerevisiae can increase endogenous antioxidant response when stressed to prevent cell death. YAP1 is a transcription factor responsible to activate genes that encoding antioxidant enzymes such as superoxide dismutase and catalase and can be an important key to protect these cells. Tucum-do-cerrado (Bactris setosa Mart.) is a Brazilian fruit rich in polyphenols and bioactive compounds mainly found in the peel. This study investigated cell growth and viability using S. cerevisiae wild type and yap1 ∆ strains exposed to tucum-do-cerrado peel aqueous extract and hydrogen peroxide (H 2 O 2 ) and menadione induced oxidative stress. Yeast cells from BY4741 and yap1 ∆ were exposed to different concentrations of tucum extract, menadione and hydrogen peroxide separated and together in mixed groups for 20h and measured for growth curve. For colony survival yeast cells were exposed to these compounds for 72h in ágar plates and colonies were counted. Results showed that aqueous extract of tucum-do-cerrado was capable to recover BY4741 density of cells stressed with both menadione and H 2 O 2 but not for yap1 ∆ strain. Besides, higher concentrations of the extract demonstrated a delay in cell growth. Colony survival showed that the exposition to tucum extract resulted in colony recover in BY4741 yeast cells but not for mutant yap1 ∆ strains which maintained low viability even with high extract concentration. In conclusion, despite S. cerevisiae antioxidant response to menadione and H 2 O 2 is different, the protection afforded by tucum extract in H 2 O 2 stressed cells, is probably through an YAP1 pathway.


Introduction
Saccharomyces cerevisiae antioxidant response is carried out through several mechanisms, including metaloproteins, which protect the cells from iron and copper toxicity, and the product of the CUP1 gene, which protects cells against menadione-induced stress. S. cerevisiae also has cytoplasmic membranes with high content of saturated fatty acids, that are more resistant to lipid peroxidation, and vitamin C, both of which may provide an e cient protection from stress [1]. Yeast cells have both, enzymatic and non-enzymatic antioxidant systems [2]. The main enzymes are superoxide dismutase (SOD), catalase and peroxidases, such as cytochrome c peroxidase and glutathione peroxidase (GPX). The non-enzymatic defense system includes scavenger molecules such as glutathione [3].
Another key mechanism used by yeast against oxidative stress is regulation of the endogenous antioxidant system by transcription factors. While in animal cells this mechanism mainly involves the transcription factor Nrf-2 (NF-E2-related factor 2), that activates genes that encoding antioxidant enzymes [4], S. cerevisiae has the transcription factor YAP1 (Yeast Activator Protein 1) that plays similar role when cells face oxidative stress situations. Yeast cells have sensors that detect low levels of hydrogen peroxide and activate the thioredoxin and glutathione pathways, which activate YAP1, this process is known as antioxidant and redox signaling [5]. YAP1 activation occurs by its nuclear export regulation and this process is mainly mediated by Chromosomal Maintenance 1 (Crm1), also known as Exportin 1, which exports YAP1 to the cytoplasm in normal conditions [6]. However, under stress conditions both cysteine-rich domains located in N and C terminal parts of the YAP1 protein constitute disul des between the N-terminal Cys303 and the C-terminal Cys598, mainly in response to oxidation caused by hydrogen peroxide (H 2 O 2 ). As a result of this structural change, interaction between YAP1 and Cmr1 is inhibited, which causes the nuclear accumulation of YAP1 and its consequent activation [7].
A decrease in cellular growth can be used by yeast as a physiological response to prevent oxidative stress caused by molecules such as H 2 O 2 and menadione [8]. It has been shown that menadione increases H 2 O 2 production and it is responsible for the generation and accumulation of reactive oxygen species (ROS), that affect the morphology and viability of yeast cells [9,10].
Tucum-do-cerrado (Bactris setosa Mart.) is a Brazilian fruit and is known for its antioxidant properties in vitro and in vivo [11,12]. The fruit has bioactive compounds such as vitamin C, quercertin, catechin, peonidin, anthocyanins and avonoids, mainly found in the peel, that also showed major antioxidant activity in vivo and in vitro when compared to the pulp [11][12][13][14][15][16]. It is not known if these compounds can in uence yeast cell growth and the YAP1 pathway. Therefore, the aim of this study was to evaluate cell growth and viability using a S. cerevisiae strain that is unable to express YAP1 and the correspondent wild type strain exposure to tucum-do-cerrado peel aqueous extract and H 2 O 2 and menadione induced oxidative stress.

Extract of tucum-do-cerrado
Fruits were obtained from a local merchant from "Fazenda Grama", Teresópolis de Goiás -GO/Brazil. Fruits were washed with distilled water and peel was manually removed and lyophilized. For preparation of the crude aqueous extract, 1g of pulverized peel was mixed with 10 ml of distilled water. The contents were shaken for 16 h at 4ºCand ltered through a 0.22 µm lter. The aqueous extract was stored in a -20 ºC freezer until use.

Yeast strains
S. cerevisiae cells wild type BY4741 (MATa his3 leu2 met15 ura3) and mutant yap1∆ (wich doesn't contain the yap1 transcription factor) were used for growth and survival experiments. Strains were provided by Dr. Marcos Dias Pereira from Chemistry Institute of Federal University of Rio de Janeiro, Brazil.

Cell culture and growth
S. cerevisiae cells were maintained in Yeast extract, Peptone, Dextrose (YPD) medium composed by 1% yeast extract, 2% peptone, 2% dextrose and 2% ágar at 4ºC. Yeast growth were performed in YPD liquid medium at 28ºC and 200 rpm. Growth evaluation for further experiments was performed by optical density (OD 600 ) at 600 nm.

Yeast dry weight curve
Inoculum for each yeast strain (BY4741 and yap1∆) were prepared using 50ml of YPD medium and 1 colony each strain. After 18h incubation at 28ºC and 200 rpm, 2ml of inoculum was transferred to 2ml tubes previously weighed. Samples were centrifuged at 10.000 rpm for 5 min at room temperature. The supernatant was discarded and yeast cells were heated at 60ºC until reach stable weight. For number of cells (mg/ml) were used. Diluitions of 20, 25, 50 and 100 were made for each strains and absorbance readed in spectrophotometer at 600 nm. Angular coe cient was calculated and used to adjust number of cells to be used on further analysis.

Growth curve
One inoculum for each yeast strain (BY4741 and yap1∆) were prepared using 10ml of YPD medium and 1 colony of each strain. After 18h incubation at 28ºC and 200 rpm, absorbance was at 600 nm and cells were adjusted to 40 µg/mL [17] in a nal volume of 1ml for every experimental group. Two control groups were made, one just with yeast cells and medium and one added with 1% (v/v) ethanol. Aqueous extract of tucum were added in several concentrations (10, 25, 50, 100 and 150 µg/ml), menadione (5 and 15 µM) and hydrogen peroxide (0.5 and 1 mM) to form other experimental groups. These compounds were tested separated and together in mixed groups. Cells were plated at 96 well plates and growth measured each 30min during 20h at Synergy HTX Multi-Mode Microplate Reader 600nm. Data was collected on Gen5 software.

Cell Viability
Inoculum (BY4741 and yap1∆) were prepared using 10ml of YPD medium and 1 colony for each strain. After 18h incubation at 28ºC and 200 rpm, absorbance was measured at 600 nm and cells were adjusted to 40 µg/mL [17] in to a 5ml nal volume for every experimental group. Aqueous extract of tucum were added at 50 and 150µg/ml concentrations, menadione at 5µM and hydrogen peroxide at 0.5mM to form other experimental groups. Next, groups were incubated for 24h at 28ºC and 200 rpm and after this time cells were washed with PBS buffer x1, diluted in YPD medium at 10 0 , 10 − 1 , 10 − 2 e 10 − 3 and 10µl plated in YPD medium solid 2% ágar (w/v) for each group. Plates were incubated in bacteriological incubator for 72h at 28ºC. After this time colonies were counted and 10 − 3 dilution was used for analysis.

Statistical Analysis
Results are expressed as mean ± SEM. Samples were compared using one-way analysis of variance (ANOVA) and the post hoc test of Bonferroni. For statistics signi cance, P-value < 0.05 was used. Analysis were made by software GraphPad Prism v6. For yeast dry weight curve software Microsoft Excel was used.

Results
The yeast cell growth coe cient found in dry weight curve and used to calculate cell concentration for both yeast strains (BY4741 and yap1∆) was 0.5 (not shown). Both yeast strains were exposed to stress with menadione and H 2 O 2 in varied concentrations, as showed in Fig. 1, BY4741 strain stress were signi cative (p < 0.05) when exposed to H 2 O 2 and decrease cell concentration in both strains were dose dependent demonstrating the potential to slow cell growth in addition to reduction of cell density after exposition time. All concentrations for both menadione and H 2 O 2 had a signi cant impact (p < 0.05) on yap1∆ strain cells. Ethanol (1%) did not show potential to reduce growth. These results were used to select concentrations to further analysis.
When exposed to different concentration of tucum-do-cerrado extract (Fig. 2) cells from BY4741 and yap1∆ were not signi catively affected in cell density. In higher concentrations (100 and 150 µg/ml) was observed a delay in cell growth. Results for menadione (15µM) and H 2 O 2 (0.5mM) induced stress show that cells with all tucum extract concentrations (10, 25, 50, 100 and 150 µg/ml) managed to recover cell density at the end (Fig. 3). Unlike wild type S.cerevisiae cells, yap1∆ strain had a signi cative (p < 0.05) decrease in cell density for both stressors but did not recover when exposed to tucum extract in any tested concentrations (Fig. 3).
Colony count results showed rst that cell viability on BY4741 and yap1∆ were signi catively reduced (p < 0.05) at 15µM menadione and 0.5mM H 2 O 2 (Fig. 4) corroborating the result found in growth curves.
When exposed to 50 and 150µg/ml of tucum extract, BY4741 yeast viability was not affected but for yap1∆ strain all concentrations of tucum extract signi cantly reduced colony viability (Fig. 4). However, the lowest concentration of tucum indicated a major effect in reducing mutant colony cells viability.
Tucum extract showed in a dose-dependent way a protection effect for menadione 15µM in wild type and mutant yeast colony viability (Fig. 4). In a different way, exposition to 5.0mM H 2 O 2 resulted in colony viability recover in BY4741 yeast cells but did not showed the same result for mutant yap1∆ strains which maintained low viability even with high tucum extract concentration (Fig. 4).

Discussion
In our study, S. cerevisiae cells were treated with different compounds, including ethanol, the vehicle used to dissolve menadione. This was done because ethanol can reduce yeast cell viability of yeasts when in high concentrations [18]. Yeast cells BY4741 and yap1Δ, in the presence of ethanol, grew in a similar way as cells in the presence of YPD medium only. Therefore, we can exclude any ethanol effect in yeasts treated with menadione.
Previous studies showed that wild type (BY4743 and BY4741) S. cerevisiae have their growth reduced with 1to 4 mM H 2 O 2 and 150µM to 0.75mM menadione [8,19]. Hydrogen peroxide can reduce BY4741 yeast colony formation as well, at 2.5mM concentration [20]. We found that 0.5mM H 2 O 2 and 15µM menadione caused a signi cative reduction in cell grown in both wild (BY4741) and mutant (yap1∆) S. cerevisiae, which indicates that the BY4741 strain is sensitive to lower concentrations of H 2 O 2 than the concentrations investigated [8,20,21]. The yap1∆ strain was not investigated in this same context by other authors. The observed increased deleterious effects of the oxidants, H 2 O 2 and menadione, on yap1Δ, when compared with its wild type counterpart BY4741, demonstrates the important role of this transcription factor for antioxidant protection.
Secondary plant metabolism compounds have been extensively investigated for their antioxidant potencial. Curcumin, when used at the concentrations of 50 and 150µg/ml, delays S. cerevisiae cell growth, without reducing cell density, after 20 hours of exposure [22]. Pomegranate juice (100µl/ml) increases yeast cell density after 72 hours, but also delay the cell cycle [23]. According to our ndings, concentrations of 100 and 150µg/ml of tucum-do-cerrado aqueous extract had similar effects on BY4741 and yap1Δ yeast strains, indicating that some tucum-do-cerrado compounds may delay cell growth progression without causing cell death. A propolis alcoholic extract was shown to be nontoxic to wild type S. cerevisiae at the concentrations of 50 and 100µg/ml which is similar to the concentrations we used in our study. Such concentrations were toxic to the mutant yap1Δ strain while had no effect on the wild type yeast strain. [24]. Some foods rich in polyphenols have been shown to maintain normal cell growth even when cells are stressed with H 2 O 2 or menadione [19,22,23]. In the same experimental condition, tucum extract was effective on improve cell growth on wild S. cerevisiae in concentrations such as 10, 25 and 50µg/ml. The higher concentrations (100 and 150µg/ml) also improved cell growth but delayed cell growth progression and maintained cell density slight lower than control group. Moreover, tucum extract was not able to increase cell growth on mutant stressed cells from S. cerevisiae, suggesting that YAP1 gene expression may be involved in protection mechanism on this strain. Table 1 is a summary of the main results obtained in this study. In the experiments made in liquid medium, tucum extract showed no protection from the action of both, H 2 O 2 and menadione in the mutant yeast strain, but restored growth to the level in the absence of the oxidants in the wild type strain. Regarding the results in solid medium, the tucum extract showed partial protection from the action of menadione in the mutant and in the wild type strain. Protection against H 2 O 2 is non-existent in the mutant and partial in the wild type. This indicates that tucum protection against H 2 O 2 is dependent on YAP1, but not in the case of menadione.

Con ict of Interest Statement:
The authors certify that they have NO a liations with or involvement in any organization or entity with any nancial or non-nancial interest in the subject matter or materials described in this manuscript. Funding: Renata Cristina da Silva was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

Ethics declaration:
This study had no ethical approval required. Additionally, each of the authors con rms that this manuscript has not been previously published and is not under consideration by any other journal.
Data availability: All data generated or analysed during this study are included in this manuscript.