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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

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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


1 2

ABSTRACT

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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-

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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

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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

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assay.

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Key words: Ty1antiROS test, Ty1 transposition, exponential, proportional test response

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2 Environment ACS Paragon Plus

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INTRODUCTION

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Reactive oxygen species (ROS) including superoxide anion (O2.- ), hydrogen peroxide

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(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.

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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

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consumption is associated with a reduced risk of developing cancer and cardiovascular

36

disease3.

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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

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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.

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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

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problem that has to be considered in this field is that free radicals are extremely reactive and



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consequently short lived. Any produced in the cell oxidative radical reacts at or close to its

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source of formation6 making unreliable the estimation of antioxidants in cellular lysates or

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extracts. Another question that need to be addressed in determination the biological effects of

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antioxidants is their bioavailability and metabolism5,7. In some cases the original antioxidant

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compounds may be metabolized such that original chemical structures never actually reach

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the target whereas its metabolites may do so. It becomes crucial to have detailed knowledge

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what exactly compound can accumulate on the target places and how active is it.

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Consequently, antioxidant activity needs to be assayed by methods taking into account the

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ability of antioxidants to penetrate cells and its availability on target subcellular structures in

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active form necessary to neutralize oxidative radicals shortly after their generation. Few of

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now existing tests for determination of antioxidant activity reach these criteria.

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In vitro assays for measuring antioxidant activity are based on chemical reactions

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resulting from direct interaction between antioxidant and oxidative radicals4,7. Linear dose

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dependence between antioxidant concentration and activity has been found in the test tube

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assays. Most in vitro tests are designed to measure one specific ROS and none of the existing

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assays truly reflects the total antioxidant capacity of a particular sample. For this purpose, the

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usage of batteries of in vitro tests has been recommended7. Although these chemical in vitro

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antioxidant assays are easy, fast, cheap and suitable for measuring antioxidant properties of

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food and dietary supplements, they are conducted under nonphysiological conditions and the

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obtained results can not be extrapolated to the in vivo situation.

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The opposite alternative is given by the in vivo tests with animal models and human

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studies which are of extreme importance because they detect the effect an antioxidant can

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have on a whole organism. Antioxidant containing food is consumed during these trails and

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the antioxidant status is studied in blood or tissues samples using in vitro assays8,9.

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Assessment of the relative contribution of individual antioxidants to the total antioxidant


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capacity requires separate specific assays. The significant advantages of in vivo assays are

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that they estimate not only the penetration of antioxidants into cells but also the passage of

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antioxidants through the intestinal tract and their circulation in blood vessels in an active

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form. Unfortunately, the in vivo assays are time consuming, very expensive and not

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applicable to study large number of samples. Therefore, there is a need for cell-based test

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systems to allow trustful and relevant antioxidant research prior to animal studies and human

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clinical trails.

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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

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made use of various cells in culture5,10,11 or human blood cells12, Basically, host cells are

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saturated with the antioxidant containing sample by incubation in the corresponding cell-

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culture media, washed and the intracellular ROS level is increased by addition of H2O2.

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Hydrogen peroxide readily crosses cell membranes through the aquaporins13 and numerous

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studies14 evidenced a dose-dependent increase of ROS following exposure of cells to

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exogeneously added H2O2. In the different cell-based assays concentration of H2O2 between 1

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mM11 and 167 mM12 were used to achive the necessary initial level of ROS in cells. Some of

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these concentrations are unphysiological and toxic for cell15 and their impact on results

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obtained will be considered in Discussion section. The antioxidant in the test product

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neutralizes part of H2O2 and the remaining peroxides can be measured. Where it was studied,

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a linear dependence of antioxidant activity on concentration was found indicating

96

stoichiometric interactions between oxidants and antioxidants in chemical reactions11,12. The

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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

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neutralize oxidative radicals inside the living cells. In a recent publication12 the comparison of



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in vitro and cell-based antioxidant methods clearly showed the advantages of cell-based

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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

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study antioxidant properties of honey bee products and found that the activity of antioxidants

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to oxidative radicals depends on the origin of ROS. This activity is clearly elevated for cell-

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generated ROS compared to ROS added as reagents in the assay.

111 112

MATERIALS AND METHODS

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Materials and Chemicals. Water solutions of honey bee products (origin Bulgaria) were

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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.

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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

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(Na2HAsO4), N-acetyl-L-cysteine and hydrogen peroxide were from Sigma Aldrich

123

(Germany). The S9 metabolite mix was from Microbiological Associates (Rockville. USA).


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The components for the nutritional media used to cultivate yeast cells were from Difco Chem.

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Co (USA).

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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

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the Ty1antiROS assay. This strain is a derivative of S. cerevisiae DG114117 and has a Ty1

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element marked with the indicator gene HIS3AI that allows the determination of Ty1

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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

.

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Strains were cultivated at 30°C on a rotary water bath shaker in YEPD liquid rich medium

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(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

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events. When 551 rho- mutants were used as tester cells, the treatment with carcinogen was

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omitted and 0,5mM H2O2 (final concentration) was added for a period of 30min. Appropriate



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.

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The number of His+ transposants counted on the SC-His plates was in the range of 100-600 in

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samples trated with different concentrations of carcinogen and about 20 in the control

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untreated samples indicating the background level of spontaneous Ty1 transposition. Each

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transposition event of the marked Ty1 in tester strains gives rise to one histidine prototrophic

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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

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the control sample taken as fold increase of 1,0. When metabolic activation was needed to

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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

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tetrazolium dye XTT (2,3-bis(2-methoxynitro-5-sulfophenyl)-5-[(phenyl-amino-carbonyl]-

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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

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for XTT at 470 nm has been estimated which allows determination the quantity of O2.- per

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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

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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

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obtained are presented as pM O2.- /cell ± SD.


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The Ty1antiROS Test for Determination of Antioxidant Activity. The Ty1antiROS assay

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is based on the inhibition by antioxidants of the Ty1 transposition process induced

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proportionally to the level of ROS generated by treatment of tester cells with carcinogens. A

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typical protocol of the Ty1antiROS test is given below.

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S. cerevisiae 551 tester cells were grown at 30°C in YEPD liquid medium to a density corresponding to 5-7x107cells/ml.

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The culture is divided into 4 ml aliquots and growing cells are treated for 60min at 30°C

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with increasing concentrations of the substance studied for antioxidant activity. Water

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insoluble substances are dissolved in dimethylsulphoxide or ethanol and used in volumes not

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exceeding a final concentration of 5% the assay volume. The control samples are treated with

185

the same volume of the solvent.

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Cells are washed by centrifugation, suspended in the same volume of fresh YEPD

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medium containing the inducer of Ty1 transposition and cultivated for 30 min at 30°C in

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water bath shaker. Hexavalent chromium (CrVI) at a final concentration of 5mM is the

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preferred inducer of Ty1 transposition because is a direct carcinogen, has a high ROS

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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

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been obtained by usage of other inducers of Ty1 transposition, such as 12 mM

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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,



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mg/ml for extracts or µM/ml, mM/ml for purified substances) inhibiting 50% of Ty1

200

transposition rate in the control sample.

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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

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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

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increase different of 10;

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“ICA50”is the corresponding IC50 value, obtained in the same experiment ;

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“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

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tests. The 0.05 probability level was chosen as the point of statistical significance throughout.

216 217

RESULTS

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Proportional Dependence Between Activation of Ty1 Transposition and Increase of ROS

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Level in S. cerevisiae. Previously, it was found that ROS have an independent and key role in

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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

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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


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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.-

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levels in the range 0,9-1,0pM/cell generated by 0,16 mM B(a)P, 5mM CrVI or 12mM MMS.

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Results similar to these shown on Table 1 have also been obtained with other carcinogens,

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such as dichloromethane, tetrahydrofuran and arsen (data not shown), indicating that the

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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.

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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



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.


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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



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


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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



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


Page 16 of 33

Page 17 of 33


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


compounds

as

main


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.


Page 18 of 33

Page 19 of 33


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



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

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599 600

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603 604

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605

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606

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607 608

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609

short-term test for selective detection of carcinogenic genotoxins. Carcinogen First

610

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611

110.

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615 616

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617

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618

89,574-587.

619


Page 26 of 33

Page 27 of 33

620 621


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

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