Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone
Aim. The investigation of response of S. cerevisiae cells to the effect of ozone in different doses and determination of total antioxidant capacity (TAC) of these cells. Methods. TAC was estimated with the chemiluminescent method when introducing ozone into S. cerevisiae cells. The damage of cells w...
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| Cite this: | Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone / I.P. Goriacha, V.D. Zinchenko, T.S. Dyubko, E.A. Romodanova, A.L. Tatarets // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 299-304. — Бібліогр.: 16 назв. — англ. |
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Goriacha, I.P. Zinchenko, V.D. Dyubko, T.S. Romodanova, E.A. Tatarets, A.L. 2019-06-15T15:26:06Z 2019-06-15T15:26:06Z 2014 Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone / I.P. Goriacha, V.D. Zinchenko, T.S. Dyubko, E.A. Romodanova, A.L. Tatarets // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 299-304. — Бібліогр.: 16 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008A4 https://nasplib.isofts.kiev.ua/handle/123456789/154424 577.336:612 Aim. The investigation of response of S. cerevisiae cells to the effect of ozone in different doses and determination of total antioxidant capacity (TAC) of these cells. Methods. TAC was estimated with the chemiluminescent method when introducing ozone into S. cerevisiae cells. The damage of cells was determined by the methods of fluorescent microscopy and flow cytometry using fluorescent dye Square-460. Results. It was shown that under the ozone dosed administration into the S. cerevisiae cells suspension the chemiluminescence flash occurred in response to each new ozone doze. The flash amplitude slightly changes under low doses and decreases after achieving some total ozone dose, being characteristic for these cells. At the same time the damaged cells appear, the number of which augments with increasing the administered ozone dose. Conclusions. We assessed the TAC of S. cerevisiae cells, as the ozone dose, which may be neutralized by the systems of their antioxidant protection: 240 ± 20 nmol/106 cells. The S. cerevisiae cells are resistant to the effect of excessive ozone until its doses exceed the TAC value for these cells. Мета. Дослідження реакції клітин S. сerevisiae на дію озону в різних дозах і визначення загальної антиоксидантної ємності (ЗАОЄ) даних клітин. Методи. ЗАОЄ визначали хемілюмінесцентним методом за введення озону в клітини S. сerevisiae. Пошкодження клітин виявляли методами флуоресцентної мікроскопії та проточної цитофлуориметрії із застосуванням флуоресцентного барвника Square-460. Результати. Показано, що за дозованого введення озону у суспензію клітин S. сerevisiae виникає спалах хемілюмінесценції у відповідь на кожну нову дозу озону. Амплітуда спалаху мало змінюється за низьких доз і знижується після досягнення деякої характерної для даних клітин сумарної дози озону. При цьому з’являються пошкоджені клітини, кількість яких збільшується з ростом дози введеного озону. Висновки. Оцінено ЗАОЄ клітин S. сerevisiae як дозу озону, яку здатні нейтралізувати системи їхнього антиоксидантго захисту, що становить 240 ± 20 пмоль/106 кл. Клітини S. сerevisiae є стійкими до дії надлишкового озону за його доз, які не перевищують величину ЗАОЄ даних клітин. Цель. Исследование реакции клеток S. cerevisiae на действие озона в различных дозах и определение общей антиоксидантной емкости (ОАОЕ) данных клеток. Методы. ОАОЕ определяли хеми- люминесцентным методом при введении озона в клетки S. сerevisiae. Повреждение клеток озоном выявляли методами флуоресцентной микроскопии и проточной цитофлуориметрии с использованием флуоресцентного красителя Square-460. Результаты. Показано, что при дозированном введении озона в суспензию клеток S. сerevisiae возникают вспышки хемилюминесценции в ответ на каждую новую дозу озона. Амплитуда вспышки мало изменяется при низких дозах и снижается после достижения некоторой характерной для данных клеток суммарной дозы озона. При этом появляются поврежденные клетки, количество которых увеличивается с ростом дозы введенного озона. Выводы. Оценена ОАОЕ клеток S. сerevisiae как доза озона, которую способны нейтрализовать системы их антиоксидантной защиты, она составляет 240 ± 20 пмоль/106 кл. Клетки S. cerevisiae устойчивы к действию избыточного озона при его дозах, не превышающих величину ОАОЕ данных клеток. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Molecular and Cell Biotechnologies Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone Антиоксидантна ємність і стійкість до дії озону клітин Saccharomyces cerevisiae Антиоксидантная емкость и устойчивость к действию озона клеток Saccharomyces cerevisiae Article published earlier |
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Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone |
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Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone Goriacha, I.P. Zinchenko, V.D. Dyubko, T.S. Romodanova, E.A. Tatarets, A.L. Molecular and Cell Biotechnologies |
| title_short |
Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone |
| title_full |
Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone |
| title_fullStr |
Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone |
| title_full_unstemmed |
Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone |
| title_sort |
antioxidant capacity and sustainability of saccharomyces cerevisiae cells exposed to ozone |
| author |
Goriacha, I.P. Zinchenko, V.D. Dyubko, T.S. Romodanova, E.A. Tatarets, A.L. |
| author_facet |
Goriacha, I.P. Zinchenko, V.D. Dyubko, T.S. Romodanova, E.A. Tatarets, A.L. |
| topic |
Molecular and Cell Biotechnologies |
| topic_facet |
Molecular and Cell Biotechnologies |
| publishDate |
2014 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Антиоксидантна ємність і стійкість до дії озону клітин Saccharomyces cerevisiae Антиоксидантная емкость и устойчивость к действию озона клеток Saccharomyces cerevisiae |
| description |
Aim. The investigation of response of S. cerevisiae cells to the effect of ozone in different doses and determination of total antioxidant capacity (TAC) of these cells. Methods. TAC was estimated with the chemiluminescent method when introducing ozone into S. cerevisiae cells. The damage of cells was determined by the methods of fluorescent microscopy and flow cytometry using fluorescent dye Square-460. Results. It was shown that under the ozone dosed administration into the S. cerevisiae cells suspension the chemiluminescence flash occurred in response to each new ozone doze. The flash amplitude slightly changes under low doses and decreases after achieving some total ozone dose, being characteristic for these cells. At the same time the damaged cells appear, the number of which augments with increasing the administered ozone dose. Conclusions. We assessed the TAC of S. cerevisiae cells, as the ozone dose, which may be neutralized by the systems of their antioxidant protection: 240 ± 20 nmol/106 cells. The S. cerevisiae cells are resistant to the effect of excessive ozone until its doses exceed the TAC value for these cells.
Мета. Дослідження реакції клітин S. сerevisiae на дію озону в різних дозах і визначення загальної антиоксидантної ємності (ЗАОЄ) даних клітин. Методи. ЗАОЄ визначали хемілюмінесцентним методом за введення озону в клітини S. сerevisiae. Пошкодження клітин виявляли методами флуоресцентної мікроскопії та проточної цитофлуориметрії із застосуванням флуоресцентного барвника Square-460. Результати. Показано, що за дозованого введення озону у суспензію клітин S. сerevisiae виникає спалах хемілюмінесценції у відповідь на кожну нову дозу озону. Амплітуда спалаху мало змінюється за низьких доз і знижується після досягнення деякої характерної для даних клітин сумарної дози озону. При цьому з’являються пошкоджені клітини, кількість яких збільшується з ростом дози введеного озону. Висновки. Оцінено ЗАОЄ клітин S. сerevisiae як дозу озону, яку здатні нейтралізувати системи їхнього антиоксидантго захисту, що становить 240 ± 20 пмоль/106 кл. Клітини S. сerevisiae є стійкими до дії надлишкового озону за його доз, які не перевищують величину ЗАОЄ даних клітин.
Цель. Исследование реакции клеток S. cerevisiae на действие озона в различных дозах и определение общей антиоксидантной емкости (ОАОЕ) данных клеток. Методы. ОАОЕ определяли хеми- люминесцентным методом при введении озона в клетки S. сerevisiae. Повреждение клеток озоном выявляли методами флуоресцентной микроскопии и проточной цитофлуориметрии с использованием флуоресцентного красителя Square-460. Результаты. Показано, что при дозированном введении озона в суспензию клеток S. сerevisiae возникают вспышки хемилюминесценции в ответ на каждую новую дозу озона. Амплитуда вспышки мало изменяется при низких дозах и снижается после достижения некоторой характерной для данных клеток суммарной дозы озона. При этом появляются поврежденные клетки, количество которых увеличивается с ростом дозы введенного озона. Выводы. Оценена ОАОЕ клеток S. сerevisiae как доза озона, которую способны нейтрализовать системы их антиоксидантной защиты, она составляет 240 ± 20 пмоль/106 кл. Клетки S. cerevisiae устойчивы к действию избыточного озона при его дозах, не превышающих величину ОАОЕ данных клеток.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154424 |
| citation_txt |
Antioxidant capacity and sustainability of Saccharomyces cerevisiae cells exposed to ozone / I.P. Goriacha, V.D. Zinchenko, T.S. Dyubko, E.A. Romodanova, A.L. Tatarets // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 299-304. — Бібліогр.: 16 назв. — англ. |
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UDC 577.336:612
Antioxidant capacity and sustainability
of Saccharomyces cerevisiae cells exposed to ozone
I. P. Goriacha1, V. D. Zinchenko1, T. S. Dyubko1, 2, E. A. Romodanova3, A. L. Tatarets2
1Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine
23, Pereyaslavskaya Str., Kharkiv, Ukraine, 61015
2State Scientific Institution «Institute for Single Crystals», NAS of Ukraine
60, Lenin Ave., Kharkiv, Ukraine 61178
3National University of Pharmacy
53, Pushkinska Str., Kharkiv, Ukraine, 61002
irynagor@gmail.com; vd_zin@mail.ru
Aim. The investigation of response of S. cerevisiae cells to the effect of ozone in different doses and determina-
tion of total antioxidant capacity (TAC) of these cells. Methods. TAC was estimated with the chemiluminescent
method when introducing ozone into S. cerevisiae cells. The damage of cells was determined by the methods of
fluorescent microscopy and flow cytometry using fluorescent dye Square-460. Results. It was shown that under
the ozone dosed administration into the S. cerevisiae cells suspension the chemiluminescence flash occurred in
response to each new ozone doze. The flash amplitude slightly changes under low doses and decreases after
achieving some total ozone dose, being characteristic for these cells. At the same time the damaged cells appear,
the number of which augments with increasing the administered ozone dose. Conclusions. We assessed the TAC
of S. cerevisiae cells, as the ozone dose, which may be neutralized by the systems of their antioxidant protection:
240 ± 20 nmol/10
6
cells. The S. cerevisiae cells are resistant to the effect of excessive ozone until its doses exceed
the TAC value for these cells.
Keywords: oxidative stress, reactive oxygen species, ozone, S. cerevisiae, chemiluminescence.
Introduction. The impact of ozone on the functional
activity of microorganisms is multidirectional, depen-
ding on the ozone dose. For instance, low doses of ozo-
ne stimulate respiration and reproductive capability of
microorganisms, whereas high doses thereof inhibit the-
se processes and result in the death of cells [1, 2]. The
response of microorganisms to ozone follows the ge-
neral picture of the biological system response to the
stress. The effect of the vital functions stimulation using
low doses of the stimulant (for weak stress) is observed
for different living systems, from bacteria to plants,
animals and humans [3].
The adjustment of cells and organisms to different
types of stresses is of interest due to the response of the
living systems to the change in the environment, the stu-
dy of the effect of medical preparations on the human
organism, and the elaboration of the treatment methods.
Like other reactive oxygen species (ROS) – hydro-
gen peroxide, superoxide, etc., – ozone causes the oxi-
dative stress for the living systems, upsetting the pro-
and antioxidant balance therein [4]. This induces the
response of the biological system, which, as well as the
response to other kinds of stress, is non-linear and de-
pendent on the dose of ozone [5]. The character of the
dose dependence of the living system response to ozo-
ne is similar to the character of the response to the acti-
vity of other inductors of the oxidative stress.
The study of the adaptive response of S. cerevisiae
yeasts to the oxidative stress, caused by hydrogen pero-
xide, allowed determining three remarkable ranges of
hydrogen peroxide concentrations. After the pre-treat-
ment of cells with hydrogen peroxide in the concentra-
299
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 4. P. 299–304 doi: http://dx.doi.org/10.7124/bc.0008A4
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
tion of less than 0.5 mmol, the rate of their division and
growth increased by 15–30 % compared to the cells
which have not been pre-treated. The increase in the
concentration of hydrogen peroxide to 1 mmol led to the
decrease in the number of surviving cells up to 10 %
compared to the control, and the concentration of 3 mmol
led to the death of cells [6].
Three dose ranges were also observed while study-
ing the adaptive response of S. cerevisiae cells to the
impact of ozone: low doses (13–42) pmol/106 cells;
medium doses (42–240) pmol/106 cells; and high doses
– over 300 pmol/106 cells. The S. cerevisiae and E. coli
cells, treated with low doses of ozone (about 15
pmol/106 cells), restore the indices of division and
growth after their external freezing-thawing better than
the non-treated cells [7]. The impact of low doses of
ozone is explained by the hormesis – the «favorable»
effect of the stress factor on the organism [3]. Medium
doses of ozone cause reversible inhibition of the
division and growth of cells, whereas high doses lead to
their death.
It is known that the character of the cell response to
the stimulant (the activation of vital processes or their
inhibition and damage of cells) reflects the degree of
the damage to the living system homeostasis under the
effect of the stimulant as a stress factor. However, if the
effect of the stress factor is in the acceptable range in
terms of its intensity, the cell or the organism is capable
of adjusting and surviving [3]. When ozone is used as
an external stimulant, the degree of damage to the cell
homeostasis is determined by the dose of the introdu-
ced ozone or the intensity of the oxidative stress, cau-
sed by ozone [5].
The capability of cells to resist the oxidant effect,
causing the oxidative stress, is measured by the total an-
tioxidant status (TAOA) or the total antioxidant capaci-
ty (TAOC) of cells.
The terms TAOA and TAOC are often interchan-
ged, although they have different meanings. The term
«TAOC» is intended for the data on the number of free
radicals or ROI in the non-radical form, available for
the antioxidant system to interact, which preconditions
the duration of the antioxidant effect [8, 10].
The term TAOA describes the initial dynamics of
the antioxidant activity, characterized by the constant
value for the rate of the corresponding reaction [9]. In
our opinion, it would be more reasonable to measure
the TAOA of S. cerevisiae cells in order to determine
their resistance to the ozone effect. Similar approach is
widely used to monitor the antioxidant status of diffe-
rent biological objects [10, 11].
The aim of the current work is to study the resistan-
ce of S. cerevisiae cells to the effect of different doses
of ozone and to determine the TAOA of the abovemen-
tioned cells.
Materials and methods. The investigation was per-
formed using S. cerevisiae cells (Y-126, obtained via
the Research Institute of the Chemical Industry of the
Russian Federation from the Central Museum of Indust-
rial Microorganisms, the All-Russia Research Institute
of Genetics). The yeasts were grown in the slant wort
agar for 48 h at 30 °C and washed from the nutrient
medium with the physiological saline solution. The
physiological saline solution was prepared using analy-
tically pure sodium chloride («Reanal», Hungary). The
study was conducted using the suspensions with the
cell concentration of (5 � 0.1) �107 cells/ml. All the esti-
mations were performed at 20 ± 1 °C.
Ozone was obtained using the electrosynthesis,
flowing the gaseous oxygen through the barrier dischar-
ge ozone generator [12] and dissolving via barbotage in
the physiological saline solution at the temperature of
thawing ice. The concentration of the dissolved ozone
in the ozonous physiological saline solution (OPSS)
was determined using the Specord UV VIS spectropho-
tometer («Carl Zeiss», Germany), estimating the optic
density of the solution at the wavelength of 255 nm
(Hartley band) [13]. The ozone concentration in OPSS
was brought up to 1–5 mg/l depending on the dose of
the ozone, required for the introduction into the suspen-
sion of cells.
The chemiluminescence (CL) was observed on the
bioluminometer with the spectral sensitivity range of
300–380 nm [14]. The cuvette with the suspension of S.
cerevisiae cells in the concentration of 5 �107 cells/ml was
placed into the bioluminometer. The reaction was initia-
ted by the introduction of 0.1 OPSS with the required
ozone concentration into 0.5 ml of the cell suspension.
The burst of luminescence was registered using the com-
puter, connected to the bioluminometer via the interface.
In the work we used the fluorescent probe, a squarai-
ne dye Square-460 («SETA» BioMedicals, USA). The
300
GORIACHA I. P. ET AL.
solutions of the fluorescent dye were prepared using
the ethyl alcohol. The cells were dyed by 15-min in-
cubation in the medium with Square-460 dye in the con-
centration of 40�mol/l, with the subsequent washing of
the free dye during centrifugation for 3 min at 800 g.
The dye was introduced into the cells suspension after
their treatment with ozone. The distribution of cells, dy-
ed with Square-460, in terms of the fluorescence inten-
sity was studied using the flow cytofluorometer BD
FACS Calibur (USA). The fluorescence of cells was
excited by the argon laser (the wavelength of 488 nm)
and registered in the FL2 channel (the wavelength of
578 nm) where the fluorescence of the damaged and
non-damaged cells has more differences in terms of its
intensity compared to the channels FL1 and FL3 (the
wavelength of 530 and 650 nm, respectively).
When the results are presented in the SSC-FL2 coor-
dinate system, the damaged cells are registered in the
right part of the dot diagram, as their being dyed with
Square-460 causes higher fluorescence intensity com-
pared to that of non-damaged cells. The ratio of the da-
maged and non-damaged cells was estimated using
WinMDI 2.9 software.
The fluorescent microphotographs were obtained
using the Axio Observer Z1 confocal laser scanning mic-
roscope («Carl Zeiss»). The Filter set 10 («Carl Zeiss»)
with the excitation waves of 450–490 nm and the emis-
sion of 516–565 nm was used in the work.
The results are presented using the data of at least
five independent experiments. The statistical processing
of the obtained results was conducted using the Origin
8.5 software. The data were estimated using Student’s
t-test. The results are presented in the form of average
arithmetic values, their dispersion is described using
the average mean-square error (p < 0.05).
Results and discussion. The ability of the fluores-
cent dye Square-460 to stain the living and necrotic
cells differently was used to observe the cells damage.
Fig. 1 presents the fluorescent microphotographs of
Square-460-dyed S. cerevisiae cells.
It is evident that the probe dyes only the envelopes
of the non-damaged cells and has limited penetration
through the envelope, which is also confirmed by less
bright cytoplasm fluorescence (Fig. 1, A). Fig. 1, B, is
the image of the cells after the introduction of the ozone
dose of 280 pmol/106 cells. The field of vision contains
some cells, the cytoplasm of which has bright fluores-
cence, which we believe to be the indication of the pro-
be penetration into the cells due to the death of the cell.
Fig. 2 presents dot diagrams of the fluorescence of
S. cerevisiae cells, obtained using the flow cytofluoro-
meter, which illustrate the impact of different doses of
ozone on the cells integrity.
At the ozone doses of 36 pmol/106 cells (Fig. 2, B)
and 64 pmol/106 cells (Fig. 2, C) the image of dot dia-
grams does not differ from the control (Fig. 2, A). The
damaged cells constitute less than 10 ± 2 % of the total
population, which slightly changes under the impact of
the mentioned doses of ozone.
At the ozone dose of 240 pmol/106 cells (Fig. 2, D)
the number of the damaged cells is 25 ± 5 % of the total
population. The increase of the ozone dose to 320
pmol/106 cells (Fig. 2, E) leads to the increase in the
number of the damaged cells up to 70 ± 5 %. At the
dose of 2.2 �103 pmol/106 cells (Fig. 2, F) all the cells are
in the area, corresponding to that of the damaged cells.
301
ANTIOXIDANT CAPACITY AND SUSTAINABILITY OF S. cerevisiae CELLS EXPOSED TO OZONE
A B
10 �m10 �m
Fig. 1. S. cerevisiae cells, dy-
ed with Square-460: A – cells,
not treated with ozone; B –
cells after the ozone impact
in the dose of 280 pmol/10
6
cells
Fig. 3 demonstrates the dependence of the number
of the damaged S. cerevisiae cells on the ozone dose,
calculated on the basis of the processed dot diagrams of
fluorescence.
The number of the damaged cells is slightly varying
at the ozone doses of under 240 pmol/106 cells and is ra-
pidly increasing after the introduction of higher ozone
doses into the cell. Therefore, when exceeding some
threshold dose, ozone causes the cells damage, which is
registered by the increase in the fluorescence intensity
of Square-460-dyed cells.
To estimate the impact of ozone on S. cerevisiae
cells, we used the method of determining TAOC, based
on the analysis of the capability of cells to neutralize
the excess of the externally introduced ROS. For this
purpose a dosed amount of ROS in the form of ozone is
introduced into the cell, and the index of TAOC is esti-
mated as the maximal amount of ozone, capable of neu-
tralizing the antioxidant systems of the cell.
The method is based on our discovery of the pheno-
menon of CL of the living cells at the introduction of
ozone therein. The CL response is present at the intro-
duction of a weaker oxidant – hydrogen peroxide – into
the cells. The CL is not observed at the introduction of
ozone into the cells, inactivated by heating to 100 °C. We
have not found any reports in the available literature
about the observation of the living cells in response to
the introduction of low doses of ozone. In our opinion,
the emission of light in response to the introduction of
ozone into the cells is related to the processes of ROS
disposal by the systems of the antioxidant protection of
the cells. The clarification of the mechanism of this phe-
nomenon requires further studies. Fig. 4 demonstrates
the oscillographic images of the series of the bursts of
CL at the introduction of portions of the ozonous phy-
siological saline solution into the suspension of S. cere-
visiae cells.
The amplitude of the chemiluminescence response
of the cells to ozone is slightly changing at the ozone
dose in each portion of 30 pmol/106 cells, whereas the
total dose of the introduced ozone does not reach the
values much above 200 pmol/106 cells (Fig. 4, A). At a
higher ozone dose in each portion (240 pmol/106 cells)
the amplitude of the bursts decreases up to complete ab-
302
GORIACHA I. P. ET AL.
A B C
D E F
Fig. 2. The dot diagrams
of the fluorescence of S.
cerevisiae cells, dyed
with the probe Square-
460, illustrating the da-
mage of the cells, treated
with different doses of
ozone (pmol/10
6
cells):
A – control (no treatment
with ozone); B – 36; C –
64; D – 240; E – 320; F –
2.2 � 10
3
0 100 200 300 400 2000 2100 2200 2300
0
20
40
60
80
100
C
o
n
te
n
ts
o
f
c
e
ll
s
w
it
h
d
a
m
a
g
e
d
m
em
b
ra
n
es
,
%
Ozone dose, pmol Î3 /10
6
cell
Fig. 3. The dependence of the number of the damaged S. cerevisiae
cells on the ozone dose; p < 0.05 compared to the control
sence with the introduction of each subsequent portion
of ozone (Fig. 4, B).
Fig. 5 presents the dependence of the intensity of
the CL burst (area under the burst peak) on the total do-
se of the introduced ozone at the consecutive introduc-
tion of the portions of the ozonous physiological saline
solution into the suspension of S. cerevisiae cells.
In the range of low ozone doses I the amplitude of
the burst is less dependent on the dose compared to the
range of higher doses II. The salient point is used to
find the value of the maximal dose, when the amplitude
of the chemiluminescence burst starts decreasing ra-
pidly with the increase of the introduced ozone dose. In
this work the value of the maximal dose of ozone, found
therefrom, is accepted as TAOC.
For the S. cerevisiae cells this value was 240 ± 20
pmol/106 cells. It corresponds to some threshold in the
dose of exogenous ROS, the excess of which changes
the character of the response of S. cerevisiae cells to the
oxidative stress. In the range of doses under 240 pmol/
106 cells, the S. cerevisiae cells demonstrate their re-
sistance to the impact of ozone. In this dose range the
compensatory biological process, launched in response
to the short-term impairment of homeostasis, is revea-
led in the form of positive impact on the cells division
and growth – the hormesis [7]. It allows the assumption
that at such doses of ozone the reserves of the antioxi-
dant protection of cells are sufficient for the disposal of
the introduced ozone. At the ozone doses over 240
pmol/106 cells the cells damage is observed. The exces-
sive ozone is not disposed completely, there is an oxida-
tive stress, which has the potential to cause the damage
of the biologically significant molecules (nucleic acids
and proteins), as well as lipids [15]. Moreover, the ROI
accumulation plays an important role as a link of the
programmed cell death – apoptosis, and at the high le-
vel of ROS – necrosis, which is observed in the cells of
different types [16].
Conclusions. TAOC of S. cerevisiae cells was esti-
mated as the dose of ozone, which can be neutralized by
the systems of their antioxidant protection – 240 ± 20
pmol/106 cells. It was demonstrated that S. cerevisiae
cells are resistant to the impact of excessive ozone at the
doses, not exceeding the value of TAOC of these cells.
303
ANTIOXIDANT CAPACITY AND SUSTAINABILITY OF S. cerevisiae CELLS EXPOSED TO OZONE
Time, s
C
h
em
il
u
m
in
es
ce
n
ce
in
te
n
si
ty
,
au
A B
0
0.1
0.2
0.3
0.4
0 10 20 30 40 50 60
Time, s
0
0.1
0.2
0.3
0.4
0 10 20 30 40 50 60
C
h
em
il
u
m
in
es
ce
n
ce
in
te
n
si
ty
,
au
Fig. 4. The series of the chemiluminescence bursts at the consecutive introduction of the portions of the ozonous physiological saline solution into
the suspension of S. cerevisiae cells. The ozone dose at the introduction of each OPSS portion was 30 pmol/10
6
cells (A) and 240 pmol/10
6
cells (B)
I II
0
0,1
0,2
0,3
0,4
0,5
0 100 200 300 400 500 600
Ozone dose, pmol O3/10
6
cells
C
m
e
m
il
u
m
in
e
sc
e
n
c
e
in
te
n
si
ty
,
a
rb
.
u
n
it
s
Fig. 5. The dependence of the intensity of the chemiluminescence res-
ponse of S. cerevisiae cells on the ozone dose at the consecutive intro-
duction of the portions of the ozonous physiological saline solution in-
to the suspension of cells
Àíòèîêñèäàíòíà ºìí³ñòü ³ ñò³éê³ñòü äî 䳿 îçîíó êë³òèí
Saccharomyces cerevisiae
². Ï. Ãîðÿ÷à, Â. Ä. dzí÷åíêî, Ò. Ñ. Äþáêî, Å. Î. Ðîìîäàíîâà,
À. Ë. Òàòàðåöü
Ðåçþìå
Ìåòà. Äîñë³äæåííÿ ðåàêö³¿ êë³òèí S. ñerevisiae íà ä³þ îçîíó â ð³ç-
íèõ äîçàõ ³ âèçíà÷åííÿ çàãàëüíî¿ àíòèîêñèäàíòíî¿ ºìíîñò³ (ÇÀΪ)
äàíèõ êë³òèí. Ìåòîäè. ÇÀΪ âèçíà÷àëè õåì³ëþì³íåñöåíòíèì ìå-
òîäîì çà ââåäåííÿ îçîíó â êë³òèíè S. ñerevisiae. Ïîøêîäæåííÿ êë³-
òèí âèÿâëÿëè ìåòîäàìè ôëóîðåñöåíòíî¿ ì³êðîñêîﳿ òà ïðîòî÷-
íî¿ öèòîôëóîðèìåò𳿠³ç çàñòîñóâàííÿì ôëóîðåñöåíòíîãî áàðâ-
íèêà Square-460. Ðåçóëüòàòè. Ïîêàçàíî, ùî çà äîçîâàíîãî ââåäåí-
íÿ îçîíó ó ñóñïåíç³þ êë³òèí S. ñerevisiae âèíèêຠñïàëàõ õåì³ëþ-
ì³íåñöåíö³¿ ó â³äïîâ³äü íà êîæíó íîâó äîçó îçîíó. Àìïë³òóäà ñïà-
ëàõó ìàëî çì³íþºòüñÿ çà íèçüêèõ äîç ³ çíèæóºòüñÿ ï³ñëÿ äîñÿãíåí-
íÿ äåÿêî¿ õàðàêòåðíî¿ äëÿ äàíèõ êë³òèí ñóìàðíî¿ äîçè îçîíó. Ïðè
öüîìó ç’ÿâëÿþòüñÿ ïîøêîäæåí³ êë³òèíè, ê³ëüê³ñòü ÿêèõ çá³ëüøó-
ºòüñÿ ç ðîñòîì äîçè ââåäåíîãî îçîíó. Âèñíîâêè. Îö³íåíî ÇÀΪ
êë³òèí S. ñerevisiae ÿê äîçó îçîíó, ÿêó çäàòí³ íåéòðàë³çóâàòè ñèñ-
òåìè ¿õíüîãî àíòèîêñèäàíòãî çàõèñòó, ùî ñòàíîâèòü 240 ± 20
ïìîëü/10
6
êë. Êë³òèíè S. ñerevisiae º ñò³éêèìè äî 䳿 íàäëèøêîâîãî
îçîíó çà éîãî äîç, ÿê³ íå ïåðåâèùóþòü âåëè÷èíó ÇÀΪ äàíèõ êë³òèí.
Êëþ÷îâ³ ñëîâà: îêñèäàòèâèé ñòðåñ, àêòèâí³ ôîðìè êèñíþ,
îçîí, S. ñerevisiae, õåì³ëþì³íåñöåíö³ÿ.
Àíòèîêñèäàíòíàÿ åìêîñòü è óñòîé÷èâîñòü ê äåéñòâèþ îçîíà
êëåòîê Saccharomyces cerevisiae
È. Ï. Ãîðÿ÷àÿ, Â. Ä. Çèí÷åíêî, Ò. Ñ. Äþáêî, Ý. À. Ðîìîäàíîâà,
À. Ë. Òàòàðåö
Ðåçþìå
Öåëü. Èññëåäîâàíèå ðåàêöèè êëåòîê S. cerevisiae íà äåéñòâèå îçî-
íà â ðàçëè÷íûõ äîçàõ è îïðåäåëåíèå îáùåé àíòèîêñèäàíòíîé åì-
êîñòè (ÎÀÎÅ) äàííûõ êëåòîê. Ìåòîäû. ÎÀÎÅ îïðåäåëÿëè õåìè-
ëþìèíåñöåíòíûì ìåòîäîì ïðè ââåäåíèè îçîíà â êëåòêè S. ñere-
visiae. Ïîâðåæäåíèå êëåòîê îçîíîì âûÿâëÿëè ìåòîäàìè ôëóîðåñ-
öåíòíîé ìèêðîñêîïèè è ïðîòî÷íîé öèòîôëóîðèìåòðèè ñ èñïîëü-
çîâàíèåì ôëóîðåñöåíòíîãî êðàñèòåëÿ Square-460. Ðåçóëüòàòû.
Ïîêàçàíî, ÷òî ïðè äîçèðîâàííîì ââåäåíèè îçîíà â ñóñïåíçèþ êëå-
òîê S. ñerevisiae âîçíèêàþò âñïûøêè õåìèëþìèíåñöåíöèè â îò-
âåò íà êàæäóþ íîâóþ äîçó îçîíà. Àìïëèòóäà âñïûøêè ìàëî èç-
ìåíÿåòñÿ ïðè íèçêèõ äîçàõ è ñíèæàåòñÿ ïîñëå äîñòèæåíèÿ íåêî-
òîðîé õàðàêòåðíîé äëÿ äàííûõ êëåòîê ñóììàðíîé äîçû îçîíà.
Ïðè ýòîì ïîÿâëÿþòñÿ ïîâðåæäåííûå êëåòêè, êîëè÷åñòâî êîòî-
ðûõ óâåëè÷èâàåòñÿ ñ ðîñòîì äîçû ââåäåííîãî îçîíà. Âûâîäû. Îöå-
íåíà ÎÀÎÅ êëåòîê S. ñerevisiae êàê äîçà îçîíà, êîòîðóþ ñïîñîá-
íû íåéòðàëèçîâàòü ñèñòåìû èõ àíòèîêñèäàíòíîé çàùèòû, îíà
ñîñòàâëÿåò 240 ± 20 ïìîëü/10
6
êë. Êëåòêè S. cerevisiae óñòîé÷èâû
ê äåéñòâèþ èçáûòî÷íîãî îçîíà ïðè åãî äîçàõ, íå ïðåâûøàþùèõ
âåëè÷èíó ÎÀÎÅ äàííûõ êëåòîê.
Êëþ÷åâûå ñëîâà: îêñèäàòèâíûé ñòðåññ, àêòèâíûå ôîðìû êèñ-
ëîðîäà, îçîí, S. ñerevisiae, õåìèëþìèíåñöåíöèÿ.
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