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 H2O2 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 H2O2 and 15µM menadione caused a significative 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 H2O2 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, H2O2 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 findings, 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 H2O2 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, H2O2 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 H2O2 is non-existent in the mutant and partial in the wild type. This indicates that tucum protection against H2O2 is dependent on YAP1, but not in the case of menadione.
Table 1
Effects of tucum-do-cerrado extract on growth and cell viability of wild type (BY4741) and mutant (yap1Δ) Saccaromyces cerevisiae strains.
Medium
|
Liquid
|
Solid
|
Tucum(µg)
|
1 mMH2O2
|
15 uMMD
|
1 mM H2O2
|
15 uMMD
|
|
BY4741
|
yap1Δ
|
BY4741
|
yap1Δ
|
BY4741
|
yap1Δ
|
BY4741
|
yap1Δ
|
50
|
+++
|
0
|
+++
|
0
|
n.d.
|
n.d.
|
n.d.
|
n.d.
|
100
|
+++*
|
0
|
+++*
|
0
|
++
|
0
|
++
|
+
|
150
|
+++*
|
0
|
+++*
|
0
|
+
|
0
|
+++
|
+++
|
0, no effecton restoration of growth or cell viability; +, some protective effect; ++, medium protective effect; +++, restoration of growth or cell viability to the level in the absence of the oxidant; * indicates a delay in growth; n.d., not determined. All data compared to the control samples corresponding to yeast cells growth in YPD medium alone. |
Saccharomyces cerevisiae has different responses to stress caused by menadione and H2O2[25,26]. The antioxidant response of these cells is adapted to react to different levels of hydrogen peroxide by activating expression of genes coding for antioxidant enzymes such as SOD1 and SOD2 that are targeted by YAP1[5,27]. Menadione can increase superoxide and H2O2 concentrations[28] in the mutant strain, therefore the protection afforded by tucum extract in menadione stressed cells, is probably partially through an YAP1 independent pathway.