太鼎生物科技/太鼎食安科技

Yeast strain, culture and sample preparation

彗星分析試驗----酵母菌培養及酵母菌樣品配製

The yeast Saccharomyces cerevisiae strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0ura3Δ0) [Brachmann et al., 1998]
was used throughout this work. This organism was maintained on standard solid yeast extract (1% w/v),
peptone (2% w/v), dextrose (2% w/v) and agar (2% w/v)
medium (YPD). For experiments, the yeast cells were grown in liquid YPD medium
at 30ºC using 500-mL or 50-mL Erlenmeyer flasks, with air-liquid ratio of 10:1
and agitation by a mechanical shaker at 200 revolutions per minute (rpm).
Careful preparation of cells for the comet protocol was necessary
for obtaining reproducible results. A liquid pre-culture of 5-10 mL was
inoculated with a small amount of yeast cells and incubated overnight. Cells
were then suspended in fresh medium to a density of 1.2*107 cells per milliliter. The cells were harvested after two
generations by centrifugation (2 min at 4500 g , 4ºC), washed twice with the same volume of
ice-cold deionized water and diluted back to the same
concentration in ice-cold S-buffer ( 1 M sorbitol, 25 mM
KH2PO4, pH 6.5).

Viability assay

彗星分析試驗----酵母菌活性分析

Yeast cells were serially diluted to 10-4 in deionized sterilized water and 100 μL were spread on
solid YPD medium. Hydrogen peroxide was immediately added to the undiluted suspension ( 5 mM
or 10 mM final concentration) and
incubated at 30ºC, 200 rpm. The same procedure was followed at different time points
and all plates were incubated at 30ºC for 48 h. Colonies were counted and
viability was calculated as percentage of colony forming units in relation to
the untreated sample.

The yeast comet assay

酵母菌彗星分析試驗----

Aliquots of this suspension with approximately 106 cells were harvested by
centrifugation (2 min at 18000 g ,
4ºC) and mixed with 1.5% (w/v) low melting agarose (in S buffer) containing
approximately 2 mg/mL of zymolyase (20T; 20000U/g). Eighty microliters of this
mixture were spread over an agarose-coated slide (slide coated with a water
solution

107 of 0.5% (w/v) normal melting agarose), covered with a
cover slip and incubated for 20 min at 30ºC for
cell wall enzymatic degradation, after which the cover slips were removed. All further procedures were performed in a cold
room at 4ºC. Slides were incubated in lysis
solution ( 30 mM NaOH, 1 M NaCl, 0.05% w/v laurylsarcosine, 50 mMEDTA,
10 mM Tris-HCl, pH 10) for 20
min in order to lyse spheroplasts. The slides were rinsed three times for 20
min each in electrophoresis buffer ( 30mM
NaOH, 10 Mm EDTA, 10 mM Tris-HCl, pH 10) to remove lysis
solution. Samples were 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 incubation in 76 and 96% ethanol. The slides were then
air-dried and were visualized immediately or stored at 4ºC for later observation.
For visualization in a fluorescence
microscope the slides were stained with ethidium bromide (10 μg/mL) and 20
representative images of each slide were acquired at magnification of 400× using a Leica Microsystems DM fluorescence microscope.
The images were analyzed with the

If the experiment involved pre-treatment with quercetin,
ursolic acid or plant extracts prior to addition of the genotoxic agent on the slide, 50 μL of
the natural compound or extract was placed on top
of the gel-embedded spheroplasts, covered with cover slip and incubated at 30ºC for 20 min. The cover slip was removed
and the slides were washed in S-buffer for 5 min.

 If the experimental setup required treatment with
hydrogen peroxide, about 80 uL of a hydrogen peroxide solution were placed on
top of the gel-embedded spheroplasts after incubation for cell wall
degradation. The solution was covered by a cover slip and incubated for 20 min
at 4ºC. After this incubation the slides were washed once with S buffer for 5
min before spheroplast lysis. For the study of DNA repair temperature dependence,
the procedure was performed with 5 mM
H2O2 and cells were
incubated at 0ºC, 16ºC, 30ºC or 37ºC during 0-60 min after the washing
step to allow DNA repair.Alternatively, incubations were done with cells
directly harvested from the culture (5 mL

aliquots of the cell suspension in 50 mL Erlenmeyer
flasks) for 20 min at 30ºC with different concentrations of the natural
compounds or plant extracts, washed and subsequently incubated with a hydrogen
peroxide solution for 20 min. At the end of this incubation, cells were washed,
embedded in low melting agarose containing zymolyase, spread on glass slides,
covered with cover slips and then incubated for 20 min to allow digestion of the cell wall by zymolyase. For background
DNA damage recovery, the comet assay was applied with extended incubation of
cells embedded in low melting agarose containing zymolyase to 90 min, instead
with the usual 20 min.Chemicals

All reagents were of analytical grade. Quercetin and
ursolic acid were obtained from Sigma and were dissolved in DMSO 1% (v/v) at
the specified concentrations. Sage water extracts (Salvia officinalis and Salvia
fruticosa) were a kind gift from Cristina Pereira-Wilson [Lima et al., 2005]