Subscriber access provided by MONASH UNIVERSITY
Article
New Cell-Based Assay Indicate Dependence of Antioxidant Biological Activity on the Origin of Reactive Oxygen Species Martin D. Dimitrov, Margarita G. Pesheva, and Pencho V. Venkov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf401045w • Publication Date (Web): 15 Apr 2013 Downloaded from http://pubs.acs.org on April 17, 2013
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 33
Journal of Agricultural and Food Chemistry
New Cell-Based Assay Indicates Dependence of Antioxidant Biological Activity on the Origin of Reactive Oxygen Species
*
Martin D. Dimitrov ; Margarita G. Pesheva ; Pencho V. Venkov
Sofia University St.”Kliment Ohridski”, Faculty of Biology, Department of Genetics Dragan Tzankov str, Sofia 1407, Bulgaria
*
Corresponding Author, Sofia University St.”Kliment Ohridski”, Faculty of Biology,
Department of Genetics, Dragan Tzankov str, Sofia,1407 Bulgaria, +3598167261, email:
[email protected] ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1 2
ABSTRACT
3
The mobility of Ty1 transposon in Saccharomyces cerevisiae was found to vary
4
proportionally to the level of ROS generated in cells which provides the possibility to
5
determine antioxidant activity by changes in a cellular process instead of using chemical
6
reactions. The study of propolis, royal jelly and honey with the newly developed Ty1antiROS
7
test reveals an inverse exponential dependence of antioxidant activity on increased
8
concentrations. This dependence can be transformed to proportional by changing the source
9
of ROS: Instead of cell-produced to applied as hydrogen peroxide. The different test-
10
responses are not due to excess of added hydrogen peroxide as evidenced by the exponential
11
dependence found by usage of yap1∆ tester cells accumulating cell generated ROS. Results
12
indicate that the activity of antioxidants to oxidative radicals depends on the origin of ROS
13
and this activity is elevated for cell-generated ROS compared to ROS added as reagents in the
14
assay.
15 16
Key words: Ty1antiROS test, Ty1 transposition, exponential, proportional test response
17 18 19 20 21 22 23 24 25
2 Environment ACS Paragon Plus
Page 2 of 33
Page 3 of 33
Journal of Agricultural and Food Chemistry
26 27
INTRODUCTION
28
Reactive oxygen species (ROS) including superoxide anion (O2.- ), hydrogen peroxide
29
(H2O2), hydroxyl radical (HO-) and singlet oxygen (1O2) are oxygen derived products and
30
their accumulation into cells can lead to oxidative stress associated with chronic infections,
31
cardio-vascular disease, cancer, Alzheimer’s disease and age-related functional decline1,2.
32
Cells possess protective systems of different antioxidants including enzymes, glutathione and
33
vitamins. Fruits, vegetables, whole grains and honey bee products contain variety of
34
compounds with antioxidant activity and epidemiological studies evidenced that their regular
35
consumption is associated with a reduced risk of developing cancer and cardiovascular
36
disease3.
37
In order to be biologically meaningful, antioxidant compounds must be able to either 1)
38
help protect an organism along the body surface (external or internal surfaces), 2) enter into
39
the blood stream and help reduce the level of free radical damage or 3) be able to enter into
40
living cells and protect the cellular interior from free radical damage.Recently, it has been
41
recognized that the mechanisms of action of antioxidants in living cells go beyond the
42
activity to scavenge free radicals or to activate antioxidative enzymes4 and can have effects on
43
regulation of gene expression, carcinogenesis, modulation of enzyme activity and signal
44
transduction pathways, as well as stimulation of the immune system5.
45
The increasing interest in the role of free radicals in the pathogenesis of human diseases
46
and the benefits of consumption foods with antioxidant properties has led to an increased
47
necessity to develop new techniques and testing systems to measure antioxidants in vitro and
48
in vivo. Considering the biological effect of antioxidants, these methods have to take into
49
account the main characteristics and complex nature of antioxidants action. The first major
50
problem that has to be considered in this field is that free radicals are extremely reactive and
3 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
51
consequently short lived. Any produced in the cell oxidative radical reacts at or close to its
52
source of formation6 making unreliable the estimation of antioxidants in cellular lysates or
53
extracts. Another question that need to be addressed in determination the biological effects of
54
antioxidants is their bioavailability and metabolism5,7. In some cases the original antioxidant
55
compounds may be metabolized such that original chemical structures never actually reach
56
the target whereas its metabolites may do so. It becomes crucial to have detailed knowledge
57
what exactly compound can accumulate on the target places and how active is it.
58
Consequently, antioxidant activity needs to be assayed by methods taking into account the
59
ability of antioxidants to penetrate cells and its availability on target subcellular structures in
60
active form necessary to neutralize oxidative radicals shortly after their generation. Few of
61
now existing tests for determination of antioxidant activity reach these criteria.
62
In vitro assays for measuring antioxidant activity are based on chemical reactions
63
resulting from direct interaction between antioxidant and oxidative radicals4,7. Linear dose
64
dependence between antioxidant concentration and activity has been found in the test tube
65
assays. Most in vitro tests are designed to measure one specific ROS and none of the existing
66
assays truly reflects the total antioxidant capacity of a particular sample. For this purpose, the
67
usage of batteries of in vitro tests has been recommended7. Although these chemical in vitro
68
antioxidant assays are easy, fast, cheap and suitable for measuring antioxidant properties of
69
food and dietary supplements, they are conducted under nonphysiological conditions and the
70
obtained results can not be extrapolated to the in vivo situation.
71
The opposite alternative is given by the in vivo tests with animal models and human
72
studies which are of extreme importance because they detect the effect an antioxidant can
73
have on a whole organism. Antioxidant containing food is consumed during these trails and
74
the antioxidant status is studied in blood or tissues samples using in vitro assays8,9.
75
Assessment of the relative contribution of individual antioxidants to the total antioxidant
4 Environment ACS Paragon Plus
Page 4 of 33
Page 5 of 33
Journal of Agricultural and Food Chemistry
76
capacity requires separate specific assays. The significant advantages of in vivo assays are
77
that they estimate not only the penetration of antioxidants into cells but also the passage of
78
antioxidants through the intestinal tract and their circulation in blood vessels in an active
79
form. Unfortunately, the in vivo assays are time consuming, very expensive and not
80
applicable to study large number of samples. Therefore, there is a need for cell-based test
81
systems to allow trustful and relevant antioxidant research prior to animal studies and human
82
clinical trails.
83
Cell-based tests represent assays in which an in vitro reaction between oxidative radicals
84
and antioxidants is carried out in live cells. Different cellular models have been published that
85
made use of various cells in culture5,10,11 or human blood cells12, Basically, host cells are
86
saturated with the antioxidant containing sample by incubation in the corresponding cell-
87
culture media, washed and the intracellular ROS level is increased by addition of H2O2.
88
Hydrogen peroxide readily crosses cell membranes through the aquaporins13 and numerous
89
studies14 evidenced a dose-dependent increase of ROS following exposure of cells to
90
exogeneously added H2O2. In the different cell-based assays concentration of H2O2 between 1
91
mM11 and 167 mM12 were used to achive the necessary initial level of ROS in cells. Some of
92
these concentrations are unphysiological and toxic for cell15 and their impact on results
93
obtained will be considered in Discussion section. The antioxidant in the test product
94
neutralizes part of H2O2 and the remaining peroxides can be measured. Where it was studied,
95
a linear dependence of antioxidant activity on concentration was found indicating
96
stoichiometric interactions between oxidants and antioxidants in chemical reactions11,12. The
97
main advantages of cell-based assays are that they directly measure the penetration of the
98
studied antioxidant into cells and the ability of the original compound or its metabolites to
99
neutralize oxidative radicals inside the living cells. In a recent publication12 the comparison of
5 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
100
in vitro and cell-based antioxidant methods clearly showed the advantages of cell-based
101
assays as more relevant way to study biological systems.
102
In this communication we demonstrate that the mobility of the Ty1 transposon in S.
103
cerevisiae cells is activated proportionally to the level of ROS generated in cells thus
104
providing the possibility to measure antioxidant activity by estimation the decrease of Ty1
105
transposition rate. Contrary to all other in vitro and most cell-based assays using chemical
106
reactions, the newly developed Ty1antiROS test determines quantitatively the activity of
107
antioxidants using a cellular process, the Ty1 transposition. We used the Ty1antiROS test to
108
study antioxidant properties of honey bee products and found that the activity of antioxidants
109
to oxidative radicals depends on the origin of ROS. This activity is clearly elevated for cell-
110
generated ROS compared to ROS added as reagents in the assay.
111 112
MATERIALS AND METHODS
113
Materials and Chemicals. Water solutions of honey bee products (origin Bulgaria) were
114
used since it has been shown that the water soluble fractions contain all antioxidant
115
compounds and exhibit higher antioxidant activity compared to the ethanol extracts16.
116
Commercially available honey, propolis and royal jelly were obtained from the Bulgarian
117
Association of Honey Producers. Honey and royal jelly were dissolved in sterile water while
118
propolis was first treated with dimethylsulphoxide for 10 minutes and then diluted with 9
119
parts the initial volume of sterile water. Stock solutions were filter sterilized and kept at +4°C
120
until used.
121
All carcinogens, including hexavalent chromium (as CrO3), disodium hydrogen arsenate
122
(Na2HAsO4), N-acetyl-L-cysteine and hydrogen peroxide were from Sigma Aldrich
123
(Germany). The S9 metabolite mix was from Microbiological Associates (Rockville. USA).
6 Environment ACS Paragon Plus
Page 6 of 33
Page 7 of 33
Journal of Agricultural and Food Chemistry
124
The components for the nutritional media used to cultivate yeast cells were from Difco Chem.
125
Co (USA).
126
Strains and Cultivation Procedures. The Saccharomyces cerevisiae 551 strain with
127
genotype MATα ura3-167 his3∆200:TymHIS3AI sec53 rho+ (National Bank for Industrial
128
Microorganisms and Cell Cultures, Sofia, Bulgaria, Cat № 8719) was used as tester strain in
129
the Ty1antiROS assay. This strain is a derivative of S. cerevisiae DG114117 and has a Ty1
130
element marked with the indicator gene HIS3AI that allows the determination of Ty1
131
transposition in the genome as a whole. The SEC53 gene in S. cerevisiae 551 has been
132
replaced with a sec53 mutation which destroys the barrier function of cell wall and evidence
133
for a generally increased permeability of 551 cells were presented18. The rho- mutants of S.
134
cerevisiae 551 were obtained by ethidium bromide treatment19. The isogenic strain S.
135
cerevisiae551yap1∆ has a disrupted YAP1 gene and was obtained by integrative
136
transformation of 551cells with the yap1::hisG-URA3-hisG cassette
20
.
137
Strains were cultivated at 30°C on a rotary water bath shaker in YEPD liquid rich medium
138
(1% bacto yeast extract, 2% bactopepton, 2% dextrose. pH=6,8) to exponential phase of
139
growth corresponding to 5-7x107 cells/ml and then used in the experiments. The media for
140
cultivation of yeast cells have been made according to published protocols19.
141 142
Determination of Ty1 Transposition Rate. The Ty1 transposition test was performed with
143
S. cerevisiae 551 strain as described18 and used to study the dependence between activation of
144
Ty1 transposition and increase of ROS level. Briefly, experimental cells treated for 30
145
minutes with carcinogen to induce the mobility of Ty1 transposon were collected, suspended
146
in fresh YEPD medium and cultivated at 20°C for 16h to complete the initiated transposition
147
events. When 551 rho- mutants were used as tester cells, the treatment with carcinogen was
148
omitted and 0,5mM H2O2 (final concentration) was added for a period of 30min. Appropriate
7 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
149
dilutions of cells were plated to determine survivals (on YEPD) and number of His+
150
transposants (on SC-His). Ten SC-His plates for each concentration of carcinogen were plated
151
with suspension’s aliquots corresponding to a total of about 108 cells surviving the procedure.
152
The number of His+ transposants counted on the SC-His plates was in the range of 100-600 in
153
samples trated with different concentrations of carcinogen and about 20 in the control
154
untreated samples indicating the background level of spontaneous Ty1 transposition. Each
155
transposition event of the marked Ty1 in tester strains gives rise to one histidine prototrophic
156
colony on selective medium and the number of His+ transposants is a quantitative measure for
157
the frequency of transposition of the marked Ty1 transposon17. Median transposition rates
158
were determined and results are presented as fold increase of Ty1 transposition rate related to
159
the control sample taken as fold increase of 1,0. When metabolic activation was needed to
160
convert pre-carcinogens in their active forms, S9 mix was added to samples 60 min before
161
treatment with genotoxins.
162 163
Quantitative Assay for Superoxide Anions. We used an assay for superoxide anions
164
determination as adapted for S. cerevisiae cells21. The assay is based on reduction the
165
tetrazolium dye XTT (2,3-bis(2-methoxynitro-5-sulfophenyl)-5-[(phenyl-amino-carbonyl]-
166
2H-tetrazolium hydroxide). XTT is taken up only by living cells where it is reduced by O2.- to
167
water soluble orange-colored formazans. A molar extinction coefficient of 2.16 x 104 M-1 S-1
168
for XTT at 470 nm has been estimated which allows determination the quantity of O2.- per
169
one live cell. The superoxide anions assay was performed immediately after the treatment
170
with carcinogens and before the cultivation of cells at 20°C in the transposition test. When
171
necessary, ROS were scavenged by addition of N-acetyl-L-cystein (60mM final
172
concentration) to the cell-suspension 60 min before the treatment with carcinogen. Results
173
obtained are presented as pM O2.- /cell ± SD.
8 Environment ACS Paragon Plus
Page 8 of 33
Page 9 of 33
Journal of Agricultural and Food Chemistry
174 175
The Ty1antiROS Test for Determination of Antioxidant Activity. The Ty1antiROS assay
176
is based on the inhibition by antioxidants of the Ty1 transposition process induced
177
proportionally to the level of ROS generated by treatment of tester cells with carcinogens. A
178
typical protocol of the Ty1antiROS test is given below.
179 180
S. cerevisiae 551 tester cells were grown at 30°C in YEPD liquid medium to a density corresponding to 5-7x107cells/ml.
181
The culture is divided into 4 ml aliquots and growing cells are treated for 60min at 30°C
182
with increasing concentrations of the substance studied for antioxidant activity. Water
183
insoluble substances are dissolved in dimethylsulphoxide or ethanol and used in volumes not
184
exceeding a final concentration of 5% the assay volume. The control samples are treated with
185
the same volume of the solvent.
186
Cells are washed by centrifugation, suspended in the same volume of fresh YEPD
187
medium containing the inducer of Ty1 transposition and cultivated for 30 min at 30°C in
188
water bath shaker. Hexavalent chromium (CrVI) at a final concentration of 5mM is the
189
preferred inducer of Ty1 transposition because is a direct carcinogen, has a high ROS
190
generating capacity (e.g. is a powerful inducer of Ty1 mobility) and gives a good
191
reproducibility of results with in-run and between different days. Very similar results have
192
been obtained by usage of other inducers of Ty1 transposition, such as 12 mM
193
methylmethansulphonate (MMS) or 0,16 mM benzo(a)pyrene (B(a)P).
194
Cells are collected by centrifugation, suspended in the same volume of fresh YEPD
195
medium and cultivated at 20°C to determine the Ty1 transposition rate (see “Determination of
196
Ty1 transposition rate”).
197
Data obtained for Ty1 transposition rates are plotted against the concentrations of the
198
studied antioxidant and the IC50 value is calculated. The IC50 value is the amount (mkg/ml,
9 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
199
mg/ml for extracts or µM/ml, mM/ml for purified substances) inhibiting 50% of Ty1
200
transposition rate in the control sample.
201
The IC50 value measurement by this protocol is relevant for final concentrations of CrVI,
202
MMS and B(a)P at levels of 5mM, 12mM and 0,16mM, respectively. These concentrations of
203
carcinogens induced a fold increase of Ty1 transposition rate equal to 10,0±1,0. When usage
204
of other carcinogens or concentrations is required, the obtained IC50 values change according
205
to the Ty1 fold increase in the control samples. In such cases results are related to the IC50
206
values obtained at Ty1 fold increase of 10,0 using the formula:
207 208
IC 50 =
10 * IC A 50 A
where: “A” is the Ty1 fold increase in the control sample of the experiment with Ty1 fold
209
increase different of 10;
210
“ICA50”is the corresponding IC50 value, obtained in the same experiment ;
211
“IC50” is the calculated value, related to a Ty1 fold increase equal to 10,0.
212 213
Statistical Analysis. All results were presented as mean ±SD from 4-10 independent
214
experiments. Comparison between two means were performed using unpaired student’s t
215
tests. The 0.05 probability level was chosen as the point of statistical significance throughout.
216 217
RESULTS
218
Proportional Dependence Between Activation of Ty1 Transposition and Increase of ROS
219
Level in S. cerevisiae. Previously, it was found that ROS have an independent and key role in
220
the induction of Ty1 transposition20. Here we extended this study by determination the
221
dependence between ROS level and Ty1 transposition rate into cells treated with different
222
concentrations of carcinogens. Table 1 and Figure 1 show the results obtained with B(a)P,
223
CrVI and MMS. The increase of carcinogen’s concentrations was associated with an increase
10 Environment ACS Paragon Plus
Page 10 of 33
Page 11 of 33
Journal of Agricultural and Food Chemistry
224
of both, superoxide anions level and Ty1 transposition rate. A linear dependence exists
225
between enhanced O2.- and Ty1 transposition. The similarity of O2.- levels necessary to
226
achieve a certain fold increase of Ty1 transposition indicates that a specific activation of Ty1
227
transposition rate is due to a specific level of ROS and is not dependent on the chemical
228
structure of the carcinogens. The different carcinogens are not equally powerful ROS
229
generators. For instance, a 10 fold increase of Ty1 transposition rate was achieved at O2.-
230
levels in the range 0,9-1,0pM/cell generated by 0,16 mM B(a)P, 5mM CrVI or 12mM MMS.
231
Results similar to these shown on Table 1 have also been obtained with other carcinogens,
232
such as dichloromethane, tetrahydrofuran and arsen (data not shown), indicating that the
233
proportional dependence between the activation of Ty1 transposition and the increase of ROS
234
level is a property common to S. cerevisiae cells treated with different carcinogens. The
235
finding that Ty1 transposition rate is proportional to the level of ROS generated in S.
236
cerevisiae cells was used to develop the Ty1antiROS method for determination of antioxidant
237
activity based on the inhibition of Ty1 transposition process by the studied antioxidant (see
238
Materials and Methods).
239 240
Antioxidant Capacity of Honey Bee Products Determined With the Ty1antiROS Test.
241
Antioxidant activity has been found in all honey bee products11,22. Most studies have been
242
performed by using propolis (in vitro and cell-based tests) and honey (in vitro and in vivo
243
tests). Royal jelly has been rarely studied and a weak antioxidant activity was reported using
244
in vitro methods23 while subsequent studies with a cell-based assay11 showed absence of
245
antioxidant activity. The antioxidant capacities of honey bee products, especially of honey,
246
have been recently reviewed 24,25.
247
We studied propolis, royal jelly and honey with the Ty1antiROS test and the results
248
obtained for propolis are shown on Figure 2. S. cerevisiae cells were treated with increased
11 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
249
concentrations of propolis, ROS were generated by treatment with CrVI and the rate of Ty1
250
transposition was determined. The inhibition of Ty1 transposition rate with increasing the
251
concentrations of propolis follows a curve (Figure 2a) which can be transformed into a strait
252
line by changing the scale for Ty1 transposition rate to logaritmic one (Figure 2b). The Ty1
253
transposition decreased log linearly in the range 0-2 mkg/ml propolis. Higher concentrations
254
of propolis possessed a total inhibiting activity to Ty1 transposition process indicating a
255
complete scavenging of ROS into cells. This was confirmed by measurements of O2·- : While
256
control (0 mkg/ml propolis) samples showed 1.050 ± 0.110 pM O2·-/cell, the samples treated
257
with 2 mkg/ml propolis showed only 0.035 ± 0.011 pM O2·-/cell which is similar to the basal
258
level of 0.048 ± 0.005 pM O2·-/cell in S. cerevisiae. The study of royal jelly and honey in the
259
Ty1antiROS test gave also a dependence of activity on concentration manifested by
260
exponentially declined curve (data not shown) and the calculated IC50 values showed the
261
following rank order for antioxidant activity: propolis ›royal jelly › honey (Table 2). Almost
262
equal IC50 values, were found when MMS was used, instead of CrVI, demonstrating that the
263
usage of different carcinogens as ROS generators did not influence the results obtained with
264
the Ty1antiROS assay.
265
The exponentially decreased rate of Ty1 transposition to increased concentrations
266
suggested higher antioxidant activity in the product studied by the Ty1antiROS test compared
267
to the proportional dependence that has been found with most of the other cell-based assays.
268
This higher activity may be due to the complex nature of the studied honey bee products
269
which are extremely rich on sugars, vitamins, proteins and other bioactive molecules. The
270
control experiment however made with the superoxide anions scavenger N-acetyl-L-cystein
271
(used instead of propolis) showed also a log liner dependence (Figure 2) with a calculated
272
IC50 of 14mM. This result evidenced that the observed exponential decrease of Ty1
273
transposition is due to the antioxidant activity of the studied honey bee products.
12 Environment ACS Paragon Plus
Page 12 of 33
Page 13 of 33
Journal of Agricultural and Food Chemistry
274
Together, the results obtained in the Ty1antiROS test proved an inverse exponential
275
dependence of antioxidant activity to concentrations which suggests a high intracellular
276
activities of antioxidants when studied with the Ty1antiROS test. This high activity may be
277
due to the cellular mechanism of the Ty1antiROS test which has been studied in some details.
278 279
Cellular Mechanism of the Ty1antiROS Assay. The main difference between Ty1antiROS
280
test and the other cell-based assays is the way ROS levels are increased into tester cells. In our
281
assay ROS production is induced into tester cells, while in most other tests H2O2, added to the
282
test system as an assay’s reagent, penetrates tester cells and increases intracellular ROS level.
283
We have studied the different responses, which antioxidants may have to the ROS produced
284
by the tested cells and to exogeneously added H2O2.
285
More than 95% of ROS in S. cerevisiae cells are produced by leakage of electrons along
286
the mitochondrial oxidative phosphorylation chain. The rho- mutants, representing large
287
deletions of mitochondrial DNA genes involved in oxidative phosphorylation can not
288
generate oxidative radicals and rho- cells are devoided of ROS26. We isolated rho- mutants of
289
the S. cerevisiae551 strain and used them as testers in the Ty1antiROS assay. Since rho- cells
290
can not produce ROS the treatment with carcinogen in the assay was omitted and H2O2 was
291
added to supply the missing ROS into tester cells. In preliminary experiments we found that
292
treatment of S. cerevisiae551rho- cells with 0,5mM H2O2 induced a 10 fold increase of Ty1
293
transposition rate which is similar to the values found by treatment with CrVI (5mM) or
294
MMS (12mM). We conducted the Ty1antiROS test to measure antioxidant activity of
295
propolis and royal jelly using S. cerevisiae551 rho- cells as testers and raised the level of ROS
296
by addition of H2O2 at a final concentration of 0,5 mM instead of inducing ROS generation by
297
treatment with a carcinogen. The results obtained (Figure 3) showed a gradual decline of Ty1
298
transposition rate with increasing the concentration of the honey bee product. The antioxidant
13 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
299
activity was proportional to the concentration of studied products and is manifested with a
300
strait line.
301
The calculated IC50 values were 3.30 ± 0.78 mkg/ml for propolis and 55.6 ± 3.5 mkg/ml
302
for royal jelly which are about 5 fold higher than the IC50 values obtained for the same
303
samples in the Ty1antiROS assay conducted with cell-generated ROS (Table 2). This result
304
indicated that in Ty1antiROS test the change of ROS origin from cell-produced to added as a
305
reagent is associated with a change in the activity of studied antioxidants towards a less
306
effective neutralization of oxidative radicals and consequently to higher IC50 values.
307
During the adaptation of the Ty1antiROS assay to the rho- tester cells we noticed that the
308
addition of H2O2 at final concentration of 0.5mM necessary to achieve a 10 fold increase of
309
Ty1 transposition rate corresponded to an intracellular O2.- level of 3.421 ± 0.822 pM/cell.
310
This O2.-
311
generated after treatment of tester rho+ cells with carcinogens (Table 1) in order to induce a
312
10 fold increase of Ty1 transposition rate. We considered the possibility that an intracellular
313
ROS excess in the case of H2O2 addition may contribute to the Ty1antiROS test response. To
314
study this possibility we created conditions in which tester cells accumulate high amounts of
315
ROS that are cell-generated.
value is significantly higher compared to O2.- levels of about 1.00 pM/cell
316
S. cerevisiae has been shown to have distinct protective oxidative stress responses to
317
superoxides and peroxides27. The YAP1 gene encodes a transcription factor which binds to
318
AP-1 sites in promoters of target genes involved in the defense response against H2O228.
319
Mutants deleted for YAP1 gene (yap1∆) accumulate ROS in the cells due to the absence of an
320
active detoxifying system. We used a strain with deleted YAP1 gene (S. cerevisiae 551 yap1∆)
321
as tester in the Ty1antiROS assay to study the activity of antioxidants from propolis into cells
322
with accumulated cell-generated ROS. Since S. cerevisiae551yap1∆ showed increased
323
sensitivity to treatment with ROS generators, MMS at the low concentrations of 4mM was
14 Environment ACS Paragon Plus
Page 14 of 33
Page 15 of 33
Journal of Agricultural and Food Chemistry
324
used to induce a high level ROS production in the Ty1antiROS test. The S.
325
cerevisiae551yap1∆ cells treated for 30 min with MMS (4mM) increased Ty1 mobility 27
326
folds and propolis applied at increasing concentrations inhibited the Ty1 transposition rate in
327
these cells with a log dependence on concentration (Figure 4) as it was found in the test
328
performed with the original tester strain S. cerevisiae 551 (Figure 2). These results suggested
329
that the excess of ROS is not the major reason for the different kind of Ty1antiROS test
330
responses if oxidative radicals are produced into tester cells. When ROS are cell-generated,
331
antioxidants are more effective in neutralization of oxidative radicals which is demonstrated
332
by an exponential dependence of antioxidant activity on concentration. The effectiveness of
333
antioxidants seems lower if ROS are accumulated into tester cells following an exposure to
334
extracellularly added H2O2.
335 336
DISCUSSION
337
One of the major reasons for the dramatic expansion of antioxidant research since the
338
mid-1990’s was the accumulation of data evidencing a casual link between oxidative stress
339
and carcinogenesis2,29. It was found that carcinogens are powerful generators of ROS in
340
different cells30,31, including S. cerevisiae32,33. One of the effects of carcinogen-increased ROS
341
level in S. cerevisiae
342
communication we provide evidence for a proportional dependence between the carcinogen-
343
induced ROS level and the Ty1 transposition rate. This observation promoted the
344
development of a new yeast cell-based assay for quantitative measurement the antioxidant
345
activity. The Ty1antiROS test is based on the reduction by antioxidants of Ty1 transposition
346
rate induced by treatment of tester cells with a carcinogen. The study of several antioxidant’s
347
concentrations gives a dependence of antioxidant activity on concentration and the possibility
348
to calculate IC50 value for the studied antioxidant. The IC50 value corresponds to a 50%
is the activation of Ty1 transposon mobility20 and in this
15 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
349
inhibition of ROS level measured in the Ty1antiROS test as a 50% inhibition of the Ty1
350
transposition rate. The principal difference between the Ty1antiROS test and the other cell-
351
based assays is that the antioxidant activity is determined using a cellular process, and not a
352
chemical reaction. The Ty1 transposition is quantitatively measured by the number of
353
colonies on selective medium related to the total number of survived cells, assuring that
354
antioxidant activity is determined within live cells. This characteristics supposed that the
355
measurement of antioxidant activity with Ty1antiROS test means determination the effect
356
antioxidants have only on oxidative radicals that are physiologically active and able to trigger
357
cellular processes, such as the Ty1 transposition process. In most of the other cell-based
358
assays the antioxidant activity is determined on all available in tester cells oxidative species
359
irrespective of their physiological state and by means of chemical reactions.
360
The study of several honey bee products with Ty1antiROS test reveals an inverse
361
exponential dependence of antioxidant activity on concentration which can be transformed to
362
a proportional dependence by changing the origin of ROS from cell-generated to externally
363
applied as an assay’s reagent. An exponential dependence designates a higher antioxidant
364
activity compared to a proportional dependence and IC50 values differ about 5 fold between
365
the two kinds of determinations. The only parameter that has been changed between the two
366
trails is the origin of ROS and the different responses of the Ty1antiROS test most likely are
367
due to the different ROS: cell-generated or added as a chemical. The simplest explanation of
368
this observation would be that in the cell antioxidants differentiate the two kinds of ROS
369
being more effective to cell-generated oxidative species. However, data for the existence of
370
different interactions in vivo between antioxidants and oxidative radicals are not presented till
371
now and until firm data will be obtained, this explanation remains completely hypothetical.
372
Linear34 and nonlinear35 but not exponential responses of antioxidant activity to
373
antioxidant concentration have been found with other cell-based tests in the study of different
16 Environment ACS Paragon Plus
Page 16 of 33
Page 17 of 33
Journal of Agricultural and Food Chemistry
374
natural products including honeybee products. A proportional dependence of antioxidant
375
activity on concentration of honey bee products was also found with the Ty1antiROS assay
376
when tester cells were depleted of ROS (rho- tester, Figure 3) and oxidative radicals were
377
added in form of H2O2. These assay’s conditions are very similar to those in other tests where
378
extracellular addition of H2O2 is used to achieve intracellular ROS level high enough to study
379
antioxidant activity of different foods or components11,12. The concentrations of H2O2 required
380
for sufficient initial ROS level into the different cell-based tests are high, unphysiological and
381
toxic for cells15. The high levels of H2O2 cause rapid autoinactivation of catalase by
382
conversion the active enzyme- H2O2 complex I to the inactive complex II36,37, followed by
383
insufficient decay of H2O2. Some of the oxidative radicals such as O2.- can not cross cell-
384
membranes, remains inside cells38 and accumulate ROS. Contrary to what is widely believed
385
recent studies
386
biomembranes and the permeability of plasma membrane to peroxides is limited. In such case
387
the removal of H2O2 introdiced into cells by addition to the assay mix is decreased and
388
peroxides will also accumulate into cells. As a result, artificially high levels of intracellular
389
ROS due to the extracellular addition of H2O2 and not to cell-generated ROS have to be
390
detoxified by the studied antioxidants which results in higher IC50 values, about 5 fold higher
391
in the case of Ty1antiROS test, performed with a rho- tester strain. The error in determination
392
of antioxidant activity would be even greater in assays using cell-cultures. It has been found
393
that antioxidants, mainly polyphenolic compounds, interact with commonly used cell-culture
394
media components to generate high levels of H2O2 which account for some of the reported
395
effects10.
396
antioxidants22,25, we checked if the studied here propolis, royal jelly and honey exert pro-
397
oxidant effects on the nutritional media used to cultivate S. cerevisiae cells. We were unable
398
to find any oxidation using 3 fold higher concentrations than those used in the Ty1antiROS
39
Since
show that in S. cerevisiae cells
honey
bee
products
contain
H2O2 does not freely defuse across
polyphenolic
17 Environment ACS Paragon Plus
compounds
as
main
Journal of Agricultural and Food Chemistry
399
assay (data not shown). This observation confirms previous studies40 showing that quercetin
400
does not promote the production of H2O2 when added to media for cultivation of yeast cells.
401
Different activity of antioxidants to oxidative radicals were found in in vitro studies using
402
cyclic voltammetry to investigate antioxidant/oxidant interactions41. It has been shown that
403
the immediate and frequent interactions characteristic for the short lived reagents can be
404
changed to slow and not frequent interactions when reagents showing higher stability are
405
used. Although results obtained in vitro not always correspond to the in vivo situation, it is
406
relevant to mention that cell-generated ROS are extremely unstable and short lived while
407
H2O2 is a relatively stable chemical6.
408
All these data fit well with the results obtained in the present study and suggest the
409
following cellular mechanism of the Ty1antiROS test. The cell-generated ROS are
410
biologically active and induce the Ty1 transposition proportionally to their concentration. The
411
antioxidants in honey bee products taken up in advance scavenge ROS involved in triggering
412
the Ty1 transposition process showing a high effectiveness against physiologically active
413
ROS as evidenced by the exponential dependence of antioxidant activity on concentrations.
414
When ROS level is elevated in tester cells with compromised mitochondrial function (rho-
415
mutants) by addition of H2O2, the oxidative radicals accumulate inside the cells at high level
416
due to low permeability of plasma membrane and inactivation of catalase responsible for
417
H2O2 decay. These ROS are more stable and not very active as inducers of Ty1 transposition
418
since 5 fold higher levels are required to activate the Ty1 transposition at rates found after
419
induction of ROS production into cells. The neutralization of these ROS levels requires higher
420
amounts of antioxidants because their interaction with oxidants may be slower, not so
421
frequent and therefore less effective. As a consequence, the dependence of antioxidant
422
activity on concentration is changed to proportional and the calculated IC50 values are higher.
18 Environment ACS Paragon Plus
Page 18 of 33
Page 19 of 33
Journal of Agricultural and Food Chemistry
423
Considering the shortcomings and advantages of the Ty1antiROS test it is obvious that
424
this assay can not compete with most of the other in vitro or cell-based assays because is very
425
time consuming. Although Ty1antiROS test is easy to perform and does not require special
426
laboratory equipment or training of personnel, results with this test are obtained in 6 days and
427
the test is not automated as a high-throughput assay. The main advantage of Ty1antiROS test
428
consists in measuring antioxidant activity by following the changes of a cellular process, the
429
Ty1 transposition, instead of using chemical reactions. Transposones are ubiquitous, from
430
E.coli to humans, and changes in their transposition are regulated by hundreds of gene-
431
functions involved in the main cellular processes42. In Ty1antiROS test the oxidative species
432
are produced by tester cells and the interaction with antioxidant takes place in live cells. The
433
assay shows good sensitivity as evidenced by its ability to detect antioxidant activity in royal
434
jelly (Table 2) which is often impossible with some other cell-based assays11. These
435
characteristics make the Ty1antiROS test a new highly sensitive cell-based assay which gives
436
results very close to the in vivo situation, and suitable to study the cellular mechanisms of
437
interactions between oxidative radicals and antioxidants. The advantages of utilization yeast
438
cells as a model to screen in vivo for natural antioxidants have been demonstrated
439
previously40 in a study showing that quercetin protects cells from H2O2
440
mechanism independent of its metal-chelating properties and the induction of antioxidant
441
defense systems.
stress by a
442
The Ty1antiROS test is not the first assay measuring antioxidant activity by utilizing a
443
cellular function. Recently, several bacterial cell-based systems for intracellular antioxidant
444
activity screening using green fluorescence protein have been developed43,44 and the methods
445
of assessment of antioxidant capacity were critically reviewed 45. Although the question for
446
the cellular mechanism of oxidant/antioxidant interactions was not addressed in these studies,
447
some of the data shown on the tables indicate an exponential dependence between antioxidant
19 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
448
concentration and function similar to that found with the Ty1antiROS test. A significant
449
advantage in the field was the development of a high-throughput reporter gene assay to test
450
the influence of natural compounds on promoter activities of rat catalase and human
451
gluthathione peroxidase and superoxide dismutase in V79 cells46. The usage of such assays
452
will allow easy and fast study of oxidant/antioxidant interactions in higher eukaryotic cells.
453 454 455 456 457 458 459
Acknowledgements We thank R. Akada (Tokiwadai, Japan) for the cassette for disruption of YAP1 gene and G. Jensen (NIS Labs, OR, USA) for critical reading and comments on the manuscrips.
460 461 462 463 464
LITERATURE CITED 1. Ames, B.N.; Gold, L.S. Endogeneous mutagens and the causes of aging and cancer. Mutat. Res. 1991, 250, 3-16.
465 466 467
2. Liu, R.H.; Hotchkiss, J.H. Potential genotoxicity of chronically elevated nitric oxide: A review. Mutat. Res.1995, 339, 73-89.
468 469 470
3. Temple, N.J. Antioxidants and disease: More questions than answers.Nutr. Res. 2000 20,449-459.
471
20 Environment ACS Paragon Plus
Page 20 of 33
Page 21 of 33
472 473
Journal of Agricultural and Food Chemistry
4. Magalhaes, L.M.; Segundo, M.A.; Reis, S.; Lima, J. Methodological aspects about in vitro evaluation of antioxidant properties. Anal. Chim. Acta. 2008 613,1-19.
474 475 476
5. Liu, R.H.; Finely, J. Potential cell culture models for antioxidant research. J. Agric. Food. Chem. 2005, 53, 4311-4314.
477 478 479
6. Holley, A.E.; Cheeseman, K.H. Measuring free radicals reactions in vivo. British. Med. Bull. 1993, 49,494-505.
480 481
7. Prior, R.L.; Wu, X.; Schaich, K. Standardized methods for the determination of
482
antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food.
483
Chem. 2005, 53, 4290-4302.
484 485
8. Wayner, D.; Burton, G.; Ingold, K.; Barcly, L.; Locke, S. The relative contribution of
486
vitamin E, urate, ascorbate and proteins to the total peroxyl radical-traping antioxidant
487
activity of human blood plasma. Biochem. Biophys. Acta. 1987, 924,408-419.
488 489
9. Sapakal, V.D.; Shikalgar, T.S.; Chadge, R.V.; Adnaik, R.S.; Naikwade, N.S.; Magdun
490
C.S. In vivo screening of antioxidant profile: a review. J. Herbar. Med. and Tox. 2008,
491
2,1-8.
492 493
10. Long, L.H.; Clement, M.V.; Halliwell, B. Artefacts in cell culture:rapid generation of
494
hydrogen peroxide on addition of (-)- epigallocatechin, (-)- epigallocatechin gallate,
495
(+)- catechin and quercetin to commonly used cell-culture media. Biochem. Biophys
496
Res. Commun. 2000, 273,50-5.
21 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
497 498
11. Nakajima, Y.; Tsuruma, K.; Shimazawa, M.; Mishima, S.; Hara, H. Comparison of
499
bee products based on assays of antioxidant capacity. BMC Copl. Alternative.
500
Medicine .2009, 9,4-13.
501 502
12. Honzel, D.; Carter, G.; Redman, K.A.; Schauss, A.G.; Endres, J.R.; Jensen, G.S.
503
Comparison of chemical and cell-based antioxidant methods for evaluation of foods
504
and natural products: generating multifaced data by parallel testing using erythrocytes
505
and polymorphonuclear cells. J. Agric. Food. Chem. 2008, 56,8319-8325.
506 507
13. Henzel, T.; Steudel, E. Transport and metabolic degradation of hydrogen peroxide in
508
Chara corralina: model calculation and measurements with the pressure probe suggest
509
transport of H2O2 across water channels. J. Exp. Bot. 2000, 51,2053-2066.
510 511
14. Temple, M.D.; Perrone, G.G.; Dawes, I.W. Complex cellular responses to reactive oxygen species. Trends. Cell. Biol. 2005, 15,319-326.
512 513
15. Ou, P.; Wolf, S.P. A discontinuous method for catalase determination at “near
514
physiological” concentrations of H2O2 and its application to the study of H2O2 fluxes
515
within cells. J. Biochem. Biophys. Methods. 1996, 31,59-67.
516 517
16. Volpert, R.; Elstner, E.F. Biochemical activities of propolis extracts. I. Standardization
518
and antioxidant properties of ethanolic and aqueous derivatives. Z. Naturforsch
519
[C].1993, 48,851-857.
520
22 Environment ACS Paragon Plus
Page 22 of 33
Page 23 of 33
521 522
Journal of Agricultural and Food Chemistry
17. Curcio, J.M.; Garfinkel, D.G. Single step selection for Ty1 element retrotransposition. Proc. Natl.Acad. Sci. USA 1991, 88,936-940.
523 524
18. Pesheva, M.; Krastanova, O.; Staleva, L.; Dentcheva, V.; Hadzhitodorov, M.; Venkov,
525
P. The Ty1 transposition assay: a new short-term test for detection of carcinogens. J.
526
Microb. Methods. 2005, 61,1-8.
527 528 529
19. Sherman, F.; Fink, G.R.; Hicks, G.B. Methods in yeast genetics: a laboratory manual. Cold Spring Harbor Laboratory Press. 2001, Plainview, NY.
530 531
20. Stoycheva, T.; Pesheva, M.; Venkov, P. The role of reactive oxygen species in the
532
induction of Ty1 retrotransposition in Saccharomyces cerevisiae. Yeast. 2010, 27,259-
533
267.
534 535
21. Stamenova, R.; Dimitrov, M.; Stoycheva, T.; Pesheva, M.; Venkov, P.; Tsvetkov, Ts.
536
Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by
537
improper cryopreservation. Cryobiology. 2008, 56,241-247.
538 539 540
22. Khalil, M.I.; Sulaiman, S.A.; Boukraa, L. Antioxidant properties of honey and its role in preventing health disorder. The Open Neutraceuticals Journal. 2010, 3,6-16.
541 542 543
23. Kuwabara, Y.; Hori, Y.; Yoneda, T.; Ikeda, Y. The antioxidant properties of royal jelly. Jpn. Pharmacol. Ther. 1996, 24, 63-67.
544
23 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
545
24. Alvarez-Suarez, J.M.; Giampieri, F.; Battino, M. Honey as a sourse of dietary
546
antioxidants : structures, bioavailability and evidence of protective effects against
547
human hronic diseases. Curr.Med.Chem. 2013, 20,621-638.
548 549
25. Kucuk, M.; Kolayli, S.; Karaoglu, S.; Ususoy, E.; Baltaci, C.; Candun, F. Biological
550
activities and chemical composition of three honeys of different types from Anatolia.
551
Food. Chem. 2007, 100,526-534.
552 553
26. Rasmussen, A.K.; Chatterjec, A.; Razmussen, L.J.; Singh, K.V. Mitochondrial
554
mediated nuclear mutator phenotype in Saccharomyces cerevisiae. Nucl. Acids. Res.
555
2003, 31, 3909-3917.
556 557 558
27. Jamieson, D.J. Saccharomyces cerevisiae has distinct adaptive responses to both hydrogen peroxide and menadione. J. Bacterial. 1992, 174,6678-6681.
559 560
28. Ngujen, D.T.; Alarco, A.M.; Raymond, M. Multiple Yap1p-binding sites mediate
561
induction of the yeast major facilitator FLR1 gene in response to drugs oxidants and
562
alkylating agents. J. Biol .Chem. 2001, 276, 1138-1145.
563 564 565
29. Block, G.; Patterson, B.; Subar, A. Fruit, vegetables and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer. 1992, 18,1-29.
566 567 568
30. Bernstein, H.; Bernstein, C.; Payne, C.M.; Dvorakova, K.; Garewal, H. Bile acids as carcinogens in human gastrointestinal cancers. Mutat. Res. 2005, 589,47-65.
569
24 Environment ACS Paragon Plus
Page 24 of 33
Page 25 of 33
570 571
Journal of Agricultural and Food Chemistry
31. Toyoyka, T.; Ibuki, Y. DNA damage induced by coexposure to PAHs and light. Envir. Tox. Pharm. 2007, 23,256-273.
572 573
32. Brennan, R.J.; Schiestl, R.H. Free radicals generated in yeast by the Salmonella
574
testnegative carcinogens benzene, thiourea and auramine. Mutat. Res. 1998, 403,65-
575
73.
576 577 578
33. Herrero, E,; Ros, J.; Bellí, G. Cabiscol, E. Redox control and oxidative stress in yeast cells. Biochem. Biophys. Acta. 2008, 1780,1217–1235.
579 580 581
34. Kim, D-O.; Lee, H.Y.; Lee, C.Y. Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J. Agric. Food. Chem. 2002, 50,3713-3717.
582 583
35. Alia, M.; Mateos, R.; Ramoss, A.; Leucumberri, E.; Bravo, L.; Grya, L. Influence of
584
quercentin and rutin on grouth and antioxidant defense system of a human hepatoma
585
cell line (HepG2). Eur. J. Nutr. 2006, 45,19-28.
586 587 588
36. Bonnichen, R.K.; Chance, B.; Theorell, H. Catalase activity. Acta Chem. Scand. 1947, 1,685-709.
589 590 591
37. Aebi,H. Catalase. Methods of enzymatic analysis. H.V. Bergmeyer Ed.Wienheim Verlag-Chemie,Germany 1987, 237-283
592 593 594
38. Lynch, R.I.; Fridovich, I. Permeation of the erythrozite stroma by superoxide radical. J.Biol.Chem. 1978, 253, 4697-4699.
25 Environment ACS Paragon Plus
Journal of Agricultural and Food Chemistry
595 596
39. Branco, M.R.; Marinho, H.S.; Cyrne, L.; Antunes, F. Decrease of H2O2 plasma
597
membrane permeability during adaptation to H2O2 in Saccharomyces cerevisiae,
598
J.Biol.Chem. 2004, 279,6501-6506
599 600
40. Belinha, I.; Amorin, M.A.; Rodriges, P.; de Freites, V.; Morados-Ferreira, P.; Matens,
601
N.; Costa, V. Quercentin increases oxidative stress resistance and longevity in
602
Saccharomyces cerevisiae. J.Agric.Food Chem. 2007, 55,2446-2451.
603 604
41. Ahmed, S.; Shakeel, F. Antioxidant activity coefficient, mechanism and kinetics of
605
different derivatives of flavones and flavonones towards superoxide radical. Czech. J.
606
Food. Sci . 2012, 30,153-163.
607 608
42. Stoycheva, T.; Pesheva, M.; Dimitrov, M.; Venkov, P. The Ty1 retrotransposition
609
short-term test for selective detection of carcinogenic genotoxins. Carcinogen First
610
Edition.; M.,Pesheva; M.,Dimitrov; T., Stoycheva Eds.; INTECH: Croatia, 2012, 83-
611
110.
612 613 614
43. Mitchell, R,J,; Gu, M.B.; Construction and characterization of novel dual stressresponsive bacterial biosensors. Biosens. Bioelectron. 2004, 19,977-985.
615 616
44. Lu, C.; Albino, C.R.; Benley, W.E.; Rao, G. Quantitative and kinetic study of
617
oxidative stress regulons using green fluorescence protein.Biotechnol. Bioeng. 2005,
618
89,574-587.
619
26 Environment ACS Paragon Plus
Page 26 of 33
Page 27 of 33
620 621
Journal of Agricultural and Food Chemistry
45. Niki, E. Antioxidant capacity: which capacity and how to assess it? J.Berry Res. 2011, 1, 169-176
622 623
46. Ullman, K.; Weincrierz, A.M.; Muller, C.; Thierbach, R.; Steege, A.; Toyokuni, S.;
624
Steinberg, P. A high-throughput reporter gene assay to prove the ability of natural
625
compounds to modulate gluthatione peroxidase, superoxide dismutase and catalase
626
gene promoters in V79 cells. Free Radic. Res. 2008, 42,746-753.
627 628
Figure Legend
629 630
Figure 1. Proportional dependence between Ty1 transposition rate and superoxide anions
631
level
632
Ty1 transposition rate and O2.- level were determined in S. cerevisiae 551 cells treated for
633
30 minutes with increasing concentrations (see Table 1) of sixthvalent chromium (-●-),
634
methylmethansulfonate (-x-), or benzo(a)pyrene (-○-).
635 636
Figure 2. Ty1antiROS test of propolis
637
S. cerevisiae 551 cells were treated with increasing concentrations of propolis (–x–) or
638
NAC (–o–), ROS level was induced with CrVI and Ty1 transposition rate (fold increase)
639
was determined. The curve linear dependence of Ty1 fold increase on concentration of
640
propolis or NAC (Figure 2a) is transformed to strait linear ( Fugure 2b) after changing
641
the scale for Ty1 fold increase to logarithmic one. Calculated IC50 values are 0,7mkg/ml
642
for propolis and 14 mM for NAC. Means ± SD from 8 experiments (p