Biol. Pharm. Bull. 31(9) 1778—1785 (2008)
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Evaluation of Radioprotective Effects of Propolis and Quercetin on Human White Blood Cells in Vitro ´ ,a Ivan BASˇ IC´,a ¯ IKIC Vesna BENKOVIC´,*,a Nevenka KOPJAR,b Anica HORVAT KNEZˇEVIC,a Domagoj D c c c a Snjezˇ ana RAMIC´, Tomislav VICULIN, Fabijan KNEZˇEVIC´, and Nada ORSˇ OLIC´ a
Department of Animal Physiology, Faculty of Science, University of Zagreb; Rooseveltov trg 6, HR-10000 Zagreb, Croatia: b Institute for Medical Research and Occupational Health, Mutagenesis Unit; Ksaverska c. 2, HR-10000 Zagreb, Croatia: and c The University Hospital for Tumors; Ilica 197, HR-10000 Zagreb, Croatia. Received January 14, 2008; accepted June 12, 2008 This in vitro study aimed at investigating the possible radioprotective effects of natural substances propolis and quercetin on g -irradiated human white blood cells. The levels of primary DNA damage were studied by the alkaline comet assay, while the cytogenetic damage was evaluated using the analysis of structural chromosome aberration and cytokinesis-block micronucleus assay. The results obtained by all endpoints indicate acceptable toxicity profiles of propolis and quercetin in vitro, and also confirmed their radioprotective abilities. Propolis was found to be more effective in diminishing the levels of primary and more complex cytogenetic DNA damage in gamma-irradiated white blood cells. Data gathered in present study support the use of propolis and quercetin as non-toxic protective substances. However, to clarify the underlying mechanisms of their cyto/radioprotective activities, additional studies are necessary at both in vitro and in vivo levels. Key words
propolis; quercetin; radioprotection; in vitro; white blood cell; DNA damage
Growing clinical, toxicological and biochemical evidence supports the use of different natural products as adjunct treatment for patients exposed to radiation as well as in chemopreventive strategies. Propolis has gained popularity as a health natural product extensively used in food and beverages to improve human health and to prevent diseases such as inflammation, heart disease and even cancer.1) The wide spectrum of propolis activities was mainly attributed to the large number of flavonoids. In addition to flavonoids, propolis contains phenolic acids, esters, enzymes, vitamins and minerals.2—4) The flavonoids possess many biological properties being strong antioxidants and have antimicrobial, antiinflammatory/antialergic, antimutagenic, anticlastogenic and anticarcinogenic properties.1,5—7) Antioxidant activity of flavonoids is based on ability of direct scavenging of reactive oxygen, nitrogen and chlorine species, such as superoxide, hydroxyl radical, peroxyl radicals, hypochlorous acid, and peroxynitrous acid. Because of the high reactivity of the hydroxyl substituents of the flavonoids, radicals are made inactive. Flavonoids can also increase the function of the endogenous antioxidant enzyme systems.8) Furthermore, their antioxidant effects may be a result of a combination of radical scavenging and an interaction with enzyme functions. Growing evidences suggest that flavonoids prevent oxidative damage of DNA (single strand breaks, double strand breaks, oxidative damage to sugar and base residues, chromosomal aberration and mutation) and other cellular components. They may interact with cellular drug transport systems and transmembrane transport, interfere with cyclin-dependent regulation of the cell cycle, inhibiting telomerase, affecting signal transduction pathways, inhibiting cyclooxygenases and lypooxygenases, decreasing xantine oxidase, metalloproteinase and sulfotransferase activities.9) Quercetin is one of the most abundant dietary flavonoids present in fruits and vegetables and its average human daily intake in various countries is estimated to be approximately 25 mg.10) Based on the structure–activity relationships for an∗ To whom correspondence should be addressed.
e-mail:
[email protected]
tioxidant effects, quercetin appears to be one of the most active flavonoids.1) It has greatest pharmacological activities among the flavonoids and possesses potential therapeutic applications. In Ames test, quercetin is regarded as mutagenic, however, recent in vitro studies indicate that quercetin is protective against genotoxicants, and regarded as antimutagenic.11) The aim of present in vitro study on human white blood cells was to estimate radioprotective effects of natural substances propolis and quercetin. The effectiveness of tested compounds was evaluated by the alkaline comet assay, the analysis of structural chromosomal aberrations and cytokinesis-block micronucleus assay. Moreover, possible genotoxic effects of the compounds were also assessed on non-irradiated blood samples. MATERIALS AND METHODS Blood Sampling To overcome possible inter-individual variability in response to treatments, blood sample was obtained from one healthy male donor (age 35 years, nonsmoker) who gave an informed consent for participation in the study. The donor was selected according to current IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans (Albertini et al., 2000).12) Venous blood was collected under sterile conditions in heparinised vacutainer tubes (V5 ml, Becton Dickinson) containing lithium heparin as anticoagulant. Chemicals and Reagents. Ethanolic Extract of Propolis (EEP) Raw Croatian propolis was collected by scraping it off hive frames. The collected propolis samples were kept desiccated in the dark until analysis at room temperature. Ethanolic propolis extract (EEP) was prepared as proposed by Kosalec et al.13) Briefly, propolis (10 g) was crushed into small pieces in a mortar and mixed vigorously with 34.85 ml 80% (v/v) ethanol during 48 h at 371 °C. After extraction, the ethanolic extract of propolis was filtered through Whatman No. 4 paper and then lyophilized. Before use EEP was © 2008 Pharmaceutical Society of Japan
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dissolved in ethanol and further dilutions were made in water. The final concentration of ethanol was less than or equal to 1%. Ethanol (1%) was used as the solvent control. EEP was tested at dose of 100 m g ml1. The chemical profile of propolis from the northern hemisphere, often named as “poplar-type” propolis can be characterized by the three analytical parameters: total flavonol and flavone content, total flavanone and dihydroflavonol content, and total polyphenolics content. EEP used in this study contains: flavones and flavonols 1.60%, flavanones and dihydroflavonols 38.60%, total flavonoids 40.20%, total polyphenols 84.40%.14) Preparation of Flavonoids Quercetin dihydrate (QU)3,3,4,5,7-pentahydroxyflavone dihydrate (Fluka, BioChemica, Switzerland), was dissolved in ethanol and further dilutions were made in water. The final concentration of ethanol was less than or equal to 1%. It was tested in concentration 50 m M. Other chemicals and reagents if not specified were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). AET (2-(2-aminoethyl)isothiourea dihydrobromide; C3H9N3S2HBr) was used for comparison since it is previously established as chemical radioprotector.15) It was tested in concentration 5 m M. Experimental Design All experiments were performed in triplicate and the data were pooled. Blood samples (V5 ml) were pre-treated with for 30 and 120 min with test compounds: EEP (100 m g ml1), quercetin (50 m M) and AET (5 m M) at room temperature. These concentrations were selected by taking into account the survival and the cytotoxic effects found in previous in vivo dose range studies.16,17) Matched controls were also included: negative (peripheral blood non-pretreated with tested compounds) and solvent (peripheral blood non-pretreated with 1% ethanol). First subgroup of samples was selected as non-irradiated, and second as irradiated. Both irradiated and non-irradiated samples were used to prepare the slides for the alkaline comet assay and to establish lymphocyte cultures for the analysis of structural chromosome aberrations and cytokinesis-block micronucleus assay. Irradiation of Blood Samples Following pre-treatment, one blood sample from each experimental group was exposed to gamma rays from 60Co source (Alcyon, CGR-MeV, France) at room temperature. For this purpose, vacutainer containing blood sample was mounted in an acrylic phantom (dimensions: 202015 cm3), in depth of 5.5 cm and it was placed transversally to the axis of irradiation. Radiation field was 1515 cm2, and the distance between the surface of phantom and source of radiation was 80 cm. Total exposures to radiation lasted for 336 s, and the absorbed dose was 4 Gy. Mounting of a vacutainer in an acrylic phantom and the scheme of the radiation exposure are shown on Fig. 1. Irradiated blood samples were immediately stored on 4 °C and within 30 min transferred in laboratory where further analyses were performed. The Alkaline Comet Assay The comet assay was carried out under alkaline conditions, basically as described by Singh et al.18) Fully frosted slides were covered with 1% normal melting point (NMP) agarose. After solidification, the gel was scraped off from the slide. The slides were then coated with 0.6% NMP agarose. When this layer had solidi-
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Fig. 1. Mounting of a Vacutainer in an Acrylic Phantom (a) and the Scheme of the Radiation Exposure (b)
fied a second layer containing the blood sample (5 m l) mixed with 0.5% low melting point (LMP) agarose was placed on the slides. After 10 min of solidification on ice, slides were covered with 0.5% LMP agarose. Afterwards the slides were immersed for 1 h in ice-cold freshly prepared lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1% Nasarcosinate, pH 10) with 1% Triton X-100 and 10% dimethyl sulfoxide added fresh to lyse cells and allow DNA unfolding. The slides were then randomly placed side by side in the horizontal gel-electrophoresis tank, facing the anode. The unit was filled with freshly prepared electrophoretic buffer (300 mM NaOH, 1 mM Na2EDTA, pH 13.0) and the slides were set in this alkaline buffer for 20 min to allow DNA unwinding and expression of alkali-labile sites. Denaturation and electrophoresis were performed at 4 °C under dim light. Electrophoresis was carried out for the next 20 min at 25 V (300 mA). After electrophoresis, the slides were washed gently three times at 5-min intervals with a neutralisation buffer (0.4 M Tris–HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 m g ml1) and covered with a coverslip. Slides were stored at 4 °C in humidified sealed containers until analysis. To prevent additional DNA damage, handling with blood samples and all steps included in the preparation of slides for the comet analysis were conducted under yellow light or in the dark. Furthermore, to avoid possible position effects during electrophoresis two parallel replicate slides per sample were prepared. Each replicate was processed in a different electrophoretic run. Corresponding negative and solvent controls were also included. Slides were examined at 250 magnification using a fluorescence microscope (Zeiss, Germany), equipped with an excitation filter of 515—560 nm and a barrier filter of 590 nm. Microscope was connected through a black and white camera to a computer-based image analysis system (Comet Assay II, Perceptive Instruments Ltd., U.K.). Data of three independent experiments were pooled and altogether 300 comets per each experimental group were measured. Comets were randomly captured at a constant depth of the gel, avoiding the edges of the gel, occasional dead cells
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and superimposed comets. To avoid the potential variability, one well-trained scorer performed all scorings of comets. Three comet parameters: tail length (DNA migration), tail DNA % and tail moment were evaluated. Statistical analyses were carried out using Statistica 5.0 software (StatSoft, Tulsa, U.S.A.). Each sample was characterized for the extent of DNA damage by considering the mean (standard error of the mean), median and range for the comet tail lengths measured. Moreover, cells were further classified as either “undamaged” or “damaged” by considering threshold levels indicating the comets with a long-tailed nucleus (LTN), i.e., the length over the 95th percentile of the distribution of the tail lengths in control.19) In order to normalize distribution and to equalize the variances, a logarithmic transformation of data was applied. Multiple comparisons between groups were done by means of ANOVA on log-transformed data. Post-hoc analysis of differences was done by Scheffé test. Comparisons regarding the frequency of LTN comets were made using non-parametric c 2-test. The level of statistical significance was set at p0.05. Chromosome Aberration (CA) Test The chromosome aberration test was performed according to current IAEA guidelines.20) Blood samples were cultivated in standard Ham’s F-10 medium (Sigma) containing inorganic salts, all amino acids, vitamins and other components. Medium was supplemented with 20% foetal calf serum (Sigma), 10 m g ml1 of phytohaemagglutinin (Murex, Dartford, U.K.), 0.0013 g l1 of phenol red pH indicator (Sigma), 100 IU of penicillin (Sigma), and 100 IU of streptomycin (Sigma). Sodium bicarbonate was added to adjust the pH value. Medium included phosphate salts, and HEPES buffer that provides more effective buffering. Duplicate cultures per sample were set up and incubated at 371 °C for 48 h in humidified atmosphere with 5.0% CO2 (Heraeus Hera Cell 240 incubator). Corresponding negative and solvent controls were also included. To arrest dividing lymphocytes in metaphase, colchicine (0.004%) was added 3 h prior to the harvest. Cultures were centrifuged at 1000 rpm for 10 min, the supernatant was carefully removed, and the cells were resuspended in a hypotonic solution (0.075 M KCl) at 37 °C. After centrifugation for 10 min at 1000 rpm, the cells were fixed with a freshly prepared fixative of ice cold methanol/glacial acetic acid (3 : 1, v/v). Fixation and centrifugation were repeated several times until the supernatants were clear. Cells were pelleted and resuspended in a minimal amount of fresh fixative to obtain a homogeneous suspension. The cell suspension was dropped onto microscope slides and left to air-dry. Slides were stained with 5% Giemsa solution (Sigma). Preparations were made according to standard procedure. Slides were stained with 5% Giemsa solution (Sigma). All slides were coded and scored blindly at 1000 magnification under oil immersion. Data of three independent experiments were pooled and altogether 300 metaphases per each experimental point were scored. Frequency and types of structural chromosome aberrations (chromatid breaks, chromosome breaks, acentric fragments, dicentric, tricentric and ring chromosomes) were estimated. Statistical significance of the results obtained was evaluated by c 2 test. The level of statistical significance was set at p0.05. Cytochalasin-Blocked Micronucleus (CBMN) Assay Lymphocyte cultures were incubated according to standard
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protocol for CBMN assay.21) Corresponding negative and solvent controls were also included. Cytochalasin B in final concentration 6 m g/ml was added in culture at 44 h. MN scoring was performed on coded slides at 1000 magnification under oil immersion. Identification of MN, nuclear buds, nucleoplasmic bridges, apoptotic and necrotic cells was done using the criteria of Fenech et al.22) Data of three independent experiments were pooled and altogether 1500 binucleated (BN) cells per each experimental point were scored. Statistical significance of the results obtained was evaluated by c 2 test. The level of statistical significance was set at p0.05. RESULTS The Alkaline Comet Assay Results of the alkaline comet assay performed on non-irradiated blood samples pretreated with EEP, quercetin and AET for 30 min show some differences in all of three comet parameters compared to negative control, but most of them were not statistically significant (Table 1). Pre-treatments with tested compounds did not induce significant differences in the DNA migration compared to negative control, while their inter-group differences are highlighted in Table 1. On the other hand, AET induced statistically significant increase of DNA migration in leukocytes and the highest number of LTN compared to negative control (Table 1). The radiation dose of 4 Gy induced a significant increase of primary DNA damage in human white blood cells. As reported in Table 1, the mean values of all comet parameters: tail length, tail DNA % and tail moment recorded in irradiated negative control were higher compared to pre-treated irradiated samples. Pre-treatments with EEP, quercetin and AET (that lasted for 30 min) reduced the levels of overall DNA damage but in different degrees. Inter-group comparisons of differences in the values of tail intensity and tail moment between tested groups are explained more in detail in Table 1. Considering our observations the radioprotective effects of two tested compounds were in order EEPquercetin, in means of all comet parameters evaluated (Table 1). Compared to them, AET was more effective radioprotector in means of all comet parameters evaluated. Results of the alkaline comet assay performed on blood samples analysed after 120 min of in vitro incubation with EEP, quercetin and AET show that treatment did not affect the levels of DNA damage in most of non-irradiated samples in comparison with negative control. As reported in Table 2 the highest values of mean tail length and number of LTN were measured in samples pre-treated with AET followed by solvent control. They were significantly increased as compared to negative control (p0.05; ANOVA and c 2 test) (Table 2). Results of the alkaline comet assay performed on irradiated blood samples analysed after 120 min of in vitro incubation indicate increased levels of DNA damage in most of samples. As reported in Table 2, the highest mean value of tail length and the highest number of LTN were recorded in sample pre-treated with AET, followed by sample pre-treated with quercetin (p0.05, ANOVA and c 2 test). On the other hand, sample pre-treated with EEP showed the lowest values for mean tail length. The comparisons between samples analysed 30 min, re-
13.46 13.46 14.74 12.82 13.46
17.31 17.95 14.74 19.87 18.27
17.680.20b),f) 19.080.25b) 15.360.15b),d),e),f) 20.340.25b),c),e),f) 19.390.26
Med.
13.550.12 13.400.08 14.570.15b),d),e),f) 13.230.10 13.460.09
MeanS.E.
10.90 11.54 10.90 13.46 11.54
9.62 9.62 10.26 10.26 9.62
Min.
Tail length (m m)
30.77 46.15 32.69 45.51 61.67
42.31 21.79 46.79 25.64 18.59
Max.
203b),c),d),f) 238c),d) 102b),d) 280b) 256
18 21 69b),c),e),f) 18 20
No. of LTN
2.470.16b),c),d) 2.800.19b),c) 1.680.11b),d) 3.310.18b) 2.880.21
1.110.14 0.970.13 0.560.07b) 0.740.08 0.970.08
MeanS.E.
1.79 1.94 1.04 2.52 1.85
0.29 0.26 0.06 0.23 0.45
Med.
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
Min.
Tail intensity (DNA %)
28.64 26.00 13.10 18.42 34.18
17.64 20.95 10.84 11.03 10.59
Max.
0.350.02b),c),d) 0.420.03b),c) 0.220.01d) 0.510.03b) 0.420.03
0.140.02 0.110.01 0.070.01 0.090.01 0.120.01
MeanS.E.
0.25 0.29 0.01 0.38 0.28
0.04 0.03 0.01 0.03 0.06
Med.
Tail moment
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
Min.
3.49 3.50 1.76 2.48 3.41
3.85 1.48 1.18 1.06 1.22
Max.
13.46 12.82 14.10 13.46 13.46
16.67 18.27 19.87 16.99 17.31
17.110.21b),d),f) 18.700.18b),c),d),e) 20.060.19b),d) 17.780.24b) 17.870.17
Med.
13.780.11 13.160.09c),e) 14.150.08b) 13.750.12 13.430.08
MeanS.E.
11.54 10.90 13.46 11.54 11.54
10.26 8.97 9.62 9.62 9.62
Min.
Tail length (m m)
51.92 36.54 30.77 51.92 32.69
30.13 22.44 19.87 28.85 18.59
Max.
203b),c),f) 270b),c),d) 286b),d) 202b) 240
31f) 13c),d) 38b) 40b) 18
No. of LTN
1.900.17b),c) 2.690.22b) 2.690.22b),e) 2.570.22b) 3.240.22
1.220.13 1.630.15 0.400.06b),d),e),f) 1.950.18b),e) 0.900.08
MeanS.E.
0.98 1.43 1.43 1.36 2.19
0.27 0.58 0.06 0.75 0.44
Med.
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
Min.
Tail intensity (DNA %)
25.34 33.49 33.49 29.94 35.10
17.08 18.51 9.37 18.39 6.04
Max.
0.260.02b),c) 0.400.03b) 0.400.03b),d),e) 0.370.03b) 0.510.03
0.150.02 0.180.02 0.050.01d),e),f) 0.220.02 0.110.01
MeanS.E.
0.14 0.22 0.22 0.21 0.36
0.04 0.07 0.01 0.09 0.06
Med.
Tail moment
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
Min.
2.60 4.51 4.51 4.99 4.72
2.41 1.54 1.08 2.12 0.77
Max.
Negative and solvent non-irradiated, as well as irradiated controls were also included. a) Synthetic radioprotective substance (2-(2-aminoethyl)isothiourea dihydrobromide). Evaluation of primary DNA damage was made on triplicate samples (each consisted of 100 comets), i.e. 300 nuclei, were measured per each experimental point. Med., median; LTN, long tailed nuclei. Statistical evaluation of comet parameters was done on logarithmically transformed data using ANOVA with post-hoc Schéffe test. Statistical significance of LTN data was evaluated by c 2 test. Significant differences (p0.05) are: b) as compared to negative control; c) as compared to AET, d) as compared to solvent control; e) as compared to sample pre-treated with EEP; f) as compared to samples pre-treated with quercetin.
Non-irradiated samples EEP Quercetin AET Solvent control Negative control Irradiated samples EEP Quercetin AET Solvent control Negative control
Sample
Comet parameters
Table 2. Results of the Alkaline Comet Assay on Human Peripheral Blood Leukocytes Incubated in Vitro for 120 min Following Pre-treatment with EEP (100 m g ml1) Quercetin (50 m M) and AETa) (5 m M) and GammaIrradiation (4 Gy)
Negative and solvent non-irradiated, as well as irradiated controls were also included. a) Synthetic radioprotective substance (2-(2-aminoethyl)isothiourea dihydrobromide). Evaluation of primary DNA damage was made on triplicate samples (each consisted of 100 comets), i.e. 300 nuclei, were measured per each experimental point. Med., median; LTN, long tailed nuclei. Statistical evaluation of comet parameters was done on logarithmically transformed data using ANOVA with post-hoc Schéffe test. Statistical significance of LTN data was evaluated by c 2 test. Significant differences (p0.05) are: b) as compared to negative control; c) as compared to AET, d) as compared to solvent control; e) as compared to sample pre-treated with EEP; f) as compared to samples pre-treated with quercetin.
Non-irradiated samples EEP Quercetin AET Solvent control Negative control Irradiated samples EEP Quercetin AET Solvent control Negative control
Sample
Comet parameters
Table 1. Results of the Alkaline Comet Assay on Human Peripheral Blood Leukocytes Pre-treated in Vitro for 30 min with EEP (100 m g ml1), Quercetin (50 m M) and AETa) (5 m M) and Then Irradiated with GammaIrradiation (4 Gy)
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Table 3. Protective Potency of EEP (100 m g ml1), Quercetin (50 m M) and AET (5 m M) Administered in Vitro to Human Peripheral Blood Leukocytes Irradiatd with Gamma-Irradiation (4 Gy) Sample Non-irradiated samples EEP Quercetin AETa) Solvent control Negative control Irradiated samples EEP Quercetin AETa) Solvent control Negative control
Tail length
No. of LTN
Tail intensity
Tail moment
III (p0.05) III (p0.05) III (p0.05) III (p0.05) III (n.s.)
III (n.s.) III (n.s.) III (p0.05) III (p0.05) III (n.s.)
III (n.s.) III (p0.05) III (n.s.) III (p0.05) III (n.s.)
III (n.s.) III (p0.05) III (p0.05) III (p0.05) III (n.s.)
III (p0.05) III (p0.05) III (p0.05) III (p0.05) III (p0.05)
III (n.s.) III (p0.05) III (p0.05) III (p0.05) III (n.s.)
III (p0.05) III (n.s.) III (p0.05) III (p0.05) III (n.s.)
III (p0.05) III (n.s.) III (p0.05) III (p0.05) III (n.s.)
DNA damage was estimated by the alkaline comet assay in two sampling times: 30 (I) and 120 min (II) after irradiation. Non-irradiated samples were studied in parallel. Synthetic radioprotective substance (2-(2-aminoethyl)isothiourea dihydrobromide). LTN, long tailed nuclei. Statistical evaluation of data concerning tail length, tail intensity and tail moment was done on logarithmically transformed data using t-test for dependent samples. For the statistical evaluation of LTN data c 2 test was used. The level of statistical significance was set at p0.05; n.s., non-significant. a)
spectively 120 min after irradiation were also performed (Table 3). Corresponding non-irradiated samples were studied in parallel. In non-irradiated samples pre-treated with EEP, quercetin and AET different levels of DNA damage were observed, depending on the prolonged incubation time. In samples pre-treated with EEP an increase of mean tail length, tail intensity and tail moment was observed. On the other hand, in samples pre-treated with quercetin and AET decreased values for all three comet parameters were observed. In g -irradiated samples pre-treated with EEP, quercetin and AET also different levels of DNA damage were observed, indicating different radioprotective properties of compounds tested. The values of mean tail length in EEP and quercetin pre-treated samples analysed 120 min following irradiation statistically significant differed as compared to time point 30 min. As observed, chemical radioprotector AET offered acceptable radioprotection only at time point 30 min. As observed, prolonged incubation with AET, additionally contributed to the levels of primary DNA damage in irradiated samples (especially of LTN). Pre-treatment with quercetin, similarly to AET, but in much lesser extent, contributed to the formation of LTN comets in sample analysed 120 min following irradiation. In view of other two comet parameters analysed, EEP and quercetin showed decreased values of tail intensity and tail moment. Analysis of Structural Chromosome Aberrations in Lymphocytes In non-irradiated peripheral blood lymphocytes pre-treated with EEP, quercetin and AET no statistically significant differences in the total number of structural chromosome aberrations as well as aberrant cells compared to negative control were observed. The most frequent type of aberrations was chromatid break, followed by acentric fragments. In almost all samples analysed the prevalence of cells with one aberration was recorded (Table 4). Irradiation with 4 Gy induced significant increase of chromosomal damage in peripheral blood lymphocytes; especially abundant were acentric fragments and dicentric chromosomes (Table 4). Inter-group comparisons did not show statistically significant differences in total number of structural chromosome aberrations (Table 4). Statistically significant increase in the number of chromatid breaks was recorded in solvent control (35), while statistically sig-
nificant increase in the number of ring chromosomes was observed in quercetin pre-treated sample (16). In all samples analysed the prevalence of cells with more aberrations was recorded. Statistically significant increase in the number of cells with chromosome aberrations was observed only in the irradiated solvent control (Table 4). Cytokinesis-Block Micronucleus Assay In non-irradiated peripheral blood lymphocytes pre-treated with EEP, quercetin and AET slightly, but not statistically significant, increased total number of binuclear cells with MN compared to negative control was observed. Moreover, inter-group comparisons did not indicate any statistically significant differences (Table 5). In majority of samples analysed the prevalence of BN cells with one MN was recorded. According to scoring criteria of CBMN assay21) the total number of nuclear buds (NB), nucleoplasmic bridges (NPB), apoptotic and necrotic cells in all samples was also determined. Although inter-group comparisons showed some differences in the total number of NB and NPB, they were not statistically significant. It should be pointed out that in sample pretreated with quercetin no apoptotic cells were detected. They were recorded in all other samples analysed, even in negative control (Table 5). Necrotic cells were detected in all samples analysed, but pre-treatment with EEP caused significantly decreased number of necrotic cells as compared to negative control. Irradiation with 4 Gy induced formation of micronuclei, NB, NPB, apoptotic and necrotic cells in tested blood samples (Table 5). In majority of samples analysed the prevalence of BN cells with one and two MN was recorded. In g irradiated peripheral blood lymphocytes pre-treated with EEP and quercetin statistically significant decreases in the total number of binuclear cells with MN compared to negative control and AET were observed. Inter-group comparisons are reported in Table 5. It should be pointed out that all tested compounds induced significant decrease in number of BN cells with 4 MN. Considering our observations the radioprotective effects of tested compounds (based on the total number of MN) were, in order, EEPquercetinAET. Also, it should be pointed out that pre-treatment with AET significantly potentates the formation of MN (38.53%) in g -irradiated peripheral blood lymphocytes (Table 5).
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Table 4. Results of the Structural Chromosome Aberration (CA) Analysis in Peripheral Blood Lymphocytes Pre-treated in Vitro for 30 min with EEP (100 m g ml1), Quercetin (50 m M) and AETa) (5 m M) and Then Irradiated with Gamma-Irradiation (4 Gy) Average No. of CA per cell
S cells with aberrations No./(%)
9 5 7 6 3
0.030 0.016 0.023 0.020 0.010
9 (3.00%) 5 (1.67%) 6 (2.00%) 6 (2.00%) 3 (1.00%)
385 420 391 467 381
1.283 1.400 1.303 1.557 1.270
175 (58.3%) 183 (61.0%) 187 (62.3%) 196 (65.3%)b) 171 (57.0%)
No. of structural CA/300 cells Sample B1 Non-irradiated samples EEP 7 Quercetin 3 AET 7 Solvent control 5 Negative control 3 Irradiated samples (4 Gy) EEP 9 Quercetin 7 AET 11 Solvent control 35b),c),d),e) Negative control 3
S
B2
Ac
Dc
Tc
R
1 — — — —
1 2 — 1 —
— — — — —
— — — — —
— — — — —
3 4 3 6 3
255 270 261 266 237
111 122 107 147 127
1 1 4 6 1
6 16d) 5 7 10
Distribution of aberrant cells No./(%) 1 CA
1 CA
9 (100.0%) 5 (100.0%) 5 (83.3%) 6 (100.0%) 3 (100.0%) 54 (30.9%) 45 (24.6%) 55 (29.4%) 47 (24.0%) 51 (29.8%)
— — 1 (16.7%) — — 121 (69.1%) 138 (75.4%) 132 (70.6%) 149 (76.0%) 120 (70.2%)
Negative and solvent non-irradiated, as well as irradiated controls were also included. a) Synthetic radioprotective substance (2-(2-aminoethyl)isothiourea dihydrobromide). Evaluation structural chromosome aberrations (CA) was made on triplicate samples (each consisted of 100 cells), i.e. 300 metaphases were screened per each experimental point. B1, chromatid break; B2, chromosome break; Ac, acentric fragment; Dc, dicentric chromosome; Tc, tricentric chromosome; R, ring chromosome. Statistical significance of CA data was evaluated using c 2 test. Significant differences (p0.05) are: b) as compared to negative control; c) as compared to AET; d) as compared to sample pre-treated with EEP; d) as compared to sample pre-treated with quercetin.
Table 5. Results of the Cytokinesis-Block Micronucleus Assay on Human Peripheral Lymphocytes Pre-treated in Vitro for 30 min with EEP (100 m g ml1), Quercetin (50 m M) and AETa) (5 m M) and Then Irradiated with Gamma-Irradiation (4 Gy)
Sample
S MNed BN No./(%)
Non-irradiated samples EEP 25 (1.67%) Quercetin 17 (1.13%) AET 22 (1.47%) Solvent control 16 (1.07%) Negative control 15 (1.00%) Irradiated samples (4 Gy) EEP 369 (24.60%)b),c),d) Quercetin 423 (28.20%)b),c) AET 578 (38.53%)b),e) Solvent control 408 (27.20%)b) Negative control 520 (34.67%)e)
Distribution of BN with
S MN Average in No. of MN 1500 BN per BN
1 MN
2 MN
3 MN
4 MN
5 MN
24 13 22 15 11
1 3 — 1 4
— 1 1 — —
— — — — —
— — — — —
26 22 25 17 19
246 274 360 274 320
98 119 167 107 127
21 25 42 22 36
3 5 9 4
1 — — 1 1
522 607 856 575 831
36c),d),e),f)
Total No. of NB
NPB
APO
0.017 0.015 0.017 0.011 0.013
6 7 8 3 3
1 1 6 6 7
3 0b),c) 9e) 1 7
0.348 0.405 0.571 0.383 0.554
16 19 16 30 27
31 56b),c) 27 44 27
10 25c),e) 10 12 12
NC 2b) 5 7 8 11 17b) 62c),e),f) 25b),e) 9b) 49e)
Negative ad solvent non-irradiated, as well as irradiated controls were also included. a) Synthetic radioprotective substance (2-(2-aminoethyl)isothiourea dihydrobromide). Evaluation was made on triplicate samples (each consisted of 500 binuclear cells; BN), i.e. 3000 nuclei, were scored to determinate total number of micronuclei (MN) for each experimental point. MNed, micronucleated; NB, nuclear buds; NPB, nucleoplasmic bridges; APO, apoptosis; NC, necrosis. Statistical significance of data was evaluated using c 2 test. Significant differences (p0.05) are: b) as compared to negative control; c) as compared to AET; d) as compared to sample pre-treated with quercetin; e) as compared to solvent control; f) as compared to sample pre-treated with EEP.
In g -irradiated peripheral blood lymphocytes both EEP and quercetin caused lower but not significant number of NBs as compared to negative control. Inter-group comparisons showed that quercetin significantly contributed the formation of NPBs (56), apoptotic (25) and necrotic cells (62) as compared to AET. Also, it should be pointed out that pretreatment with EEP significantly reduced the number of necrotic cells as compared to negative control (Table 5). Other inter-group comparisons concerning the number of necrotic cells are explained more in detail in Table 5. DISCUSSION Current research was aimed at understanding the role of natural substances EEP and quercetin in ameliorating radiation induced DNA damage under in vitro condition and to compare it with a known chemical radioprotector AET. A battery of endpoints on human white blood cells was em-
ployed to investigate the levels of primary DNA damage as well as cytogenetic consequences fixed in the lymphocyte genome following pre-treatment with tested compounds and g -irradiation. The use of flavonoids as potential radioprotectors is of increasing interest because of their high antioxidant activity and abundance in the diet.23) Moreover, propolis is also well known for its medical effects, including anti-inflammatory, antiviral, immunostimulatory and carcinostatic activities. Many studies reported beneficial properties of propolis in vivo and in vitro.17,24—28) However, the evaluation of their findings is often complex since propolis represents a mixture of different bioactive compounds with various chemical compositions and pharmacological activities. Previous analyses have identified at least 200 compounds in propolis extracts, including fatty and phenolic acid, as well as esters, flavonoids, terpenes, aromatic aldehydes, alcohols, sesquiterpenes, b -steroids and naphtalens.29) With regard to the chem-
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ical profile EEP used in present study consists mostly (84.40%) of polyphenols.14) In this study the potential radioprotective properties of the ethanolic extract of Croatian propolis and quercetin, as its important constituent, on human blood cells in vitro were investigated. As known, ionizing radiation is a classic mutagen and the DNA of exposed cells undergoes single- or doublestrand breaks and damage to bases and sugars, ultimately leading to chromosomal aberrations.30) Genotoxic effects of ionizing radiation are also mediated through formation of free radicals and reactive oxygen species that additionally harm DNA and accordingly can cause cell damage and cell death.31) All above-mentioned lesions significantly contribute to the increased levels of primary DNA damage that could be detected by the alkaline comet assay. This sensitive and versatile technique was used in earlier studies with different flavonoids and polyphenolic compounds both in vitro and in vivo. Results obtained in these studies indicate both the beneficial effects of tested substances as well as sensitivity of the assay.32—39) The results of present alkaline comet study indicate favourable toxicity profiles of EEP and quercetin. Both compounds offered acceptable radioprotection in vitro when weighed against well-known chemical radioprotector AET. Our results support the findings by other authors, indicated possible toxic effects of the compound administered in doses needed for effective radioprotection.40—42) Although 30 min of in vitro pre-treatment with AET offered better radioprotection to irradiated leukocytes than other tested substances, with prolonged incubation time it significantly increased the levels of primary DNA damage. When compared EEP with quercetin, EEP was found to be better radioprotector, in terms of all comet parameters studied. The radioprotective properties of EEP were reported earlier in mice exposed to different doses of gamma radiation.16,43) It is believed that antioxidative activities of propolis are due to its high flavonoid content, which makes up approximately 25—30% of its dry weight.44) Good radioprotective properties of EEP used in the present study could be also attributed to the high flavonoid content, since it contained over 40% of total flavonoids.14) Radioprotective effectiveness of quercetin on DNA level was reported earlier.45) However, considering the results of our study, it is possible that following gamma-irradiation pro-oxidative effects of quercetin were also present. These observations agree with some of earlier reports by other authors.46—48) However, majority of earlier studies reports beneficial effect of quercetin on primary DNA damage. Duthie et al.35) observed that quercetin in combination with hydrogen peroxide substantially decreased the yield of DNA damage in human lymphocytes. Blasiak10) found out that quercetin decreased the extent of DNA damage evoked by cadmium and increased the effectiveness of the repair of these lesions. In order to precisely interpret the biological significance of the results obtained, we used a study design in which comet assay was joined with other biomarkers of DNA damage (e.g. chromosome aberration, micronuclei), as proposed by experts in the field.49) The difference between effects measured in the comet assay and cytogenetic tests is basically due to variations in the type of DNA alterations that the test system detects: the comet assay detects repairable DNA lesions or
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alkali-labile sites while cytogenetic tests detect fixed mutations which persist at least one mitotic cycle. The enzymatic repair systems can efficiently repair DNA damage and maintain the integrity and stability of the genome. Although the vast majority of primary lesions (especially single strand breaks) produced in DNA following irradiation is repaired by error free mechanisms in a few minutes to a couple of hours,18,50) some DNA damages always escape from enzymatic repair systems. Lesions that remain incompletely repaired or unrepaired, especially double strand breaks (which repair is much slower and more complicated), are converted into cytogenetic alterations that may be visualized in proliferating cells.51) Chromosomal aberrations (CA) are important biomarkers for quantitation of radiation-induced DNA damage.20) Radioprotective properties of propolis and flavonoids against DNA damage induced by gamma irradiation were not broadly investigated by means of structural CA analysis in vitro. Montoro et al.27) observed a significant decrease in the radiationinduced chromosome aberrations when used ethanol extract of propolis for different doses of irradiation, indicating possible dose dependence in the radioprotective effects on DNA. The results obtained in present study demonstrate that EEP and quercetin did not cause significant formation of structural CA in non-irradiated human peripheral lymphocytes in vitro. However, they differentially influenced the CA frequency in irradiated cells. While total number of structural CA in EEP pre-treated samples was similar, in sample pretreated with quercetin an increased total number of structural CA as compared to irradiated negative control was observed. This observation is in agreement with earlier findings by other authors46,52) who reported that quercetin caused chromosomal aberrations as well as sister chromatid exchanges, possibly mediated by topoisomerase II poisoning. It is well established that increased levels of chromosomal damage, especially an excess of acentric and dicentric chromosomes correlates with formation of micronuclei in treated cells. Micronucleus assay, as significant biomarker for evaluation of cytogenetic damage was employed in many earlier studies with different flavonoids both in vivo and in vitro. Some of them also reported dose-dependent radioprotective effect.23,53—59) In this study we used the CBMN assay simultaneously with criteria for scoring as suggested by Fenech et al.22) and also obtained interesting results. EEP and quercetin did not cause significant formation of MN, NB and NPB in non-irradiated samples as compared to negative control. The most important finding was that both of them also reduced the number of apoptotic and necrotic cells as compared to negative control. According to the results obtained, both EEP and quercetin diminished the total number of micronuclei in gamma-irradiated samples and their radioprotective properties were much better than those of AET. AET was found to be effective radioprotector both on non-proliferating cells (as measured by the alkaline comet assay) and on the chromosomes. However, the results of CBMN assay indicate its cytotoxicity that significantly limits its radioprotective value. This observation is also in agreement with previous reports by other authors.40,42) Given that micronuclei originate both from clastogenic and aneugenic impacts caused by ionizing radiation, our results point to cyto/radioprotective capacity
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EEP and quercetin both at the level of DNA and mitotic spindle. When compared EEP with quercetin, EEP was found to be better radioprotector, possibly due to its complex composition made of many bioactive substances. CONCLUSION Present in vitro study showed that EEP and quercetin offer a quite measurable protection against DNA damage caused by ionizing radiation. Data gathered here support their use as non-toxic protective substances. However, to clarify the underlying mechanisms of their cyto/radioprotective activities, additional studies are necessary. Acknowledgements This study was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Grants No. 022-0222148-2137 and No. 119-701255). REFERENCES 1) Middleton E. Jr., Kandaswami C., Theoharides T. C., Pharmacol. Rev., 52, 673—751 (2000). 2) Burdock G. A., Food Chem. Toxicol., 36, 347—363 (1998). 3) Banskota A. H., Tezuka Y., Kadota S., Phytother. Res., 15, 561—571 (2001). 4) Bankova V., J. Ethnopharmacol., 100, 114—117 (2005). 5) Nijveldt R. J., van Nood E., van Hoorn D. E., Boelens P. G., van Norren K., van Leeuwen P. A., Am. J. Clin. Nutr., 74, 418—425 (2001). 6) Manach C., Scalbert A., Morand C., Remesy C., Jimenez L., Am. J. Clin. Nutr., 79, 727—747 (2004). 7) Soobrattee M. A., Neergheen V. S., Luximon-Ramma A., Aruoma O. I., Bahorun T., Mutat. Res., 579, 200—213 (2005). 8) Galati G., O’Brien P. J., Free Rad. Biol. Med., 37, 287—303 (2004). 9) Halliwell B., Rafter J., Jenner A., Am. J. Clin. Nutr., 81(Suppl.), 268S—276S (2005). 10) Blasiak J., Polish. J. Environ. Studies, 10, 437—442 (2001). 11) Okamoto T., Int. J. Mol. Med., 6, 275—278 (2005). 12) Albertini R. J., Anderson D., Douglas G. R., Hagmor L., Hemminki K., Merlo F., Natarajan A. T., Norppa H., Shuker D. E. G., Tice R., Waters M. D., Aitio A., Mutat. Res., 463, 111—172 (2000). 13) Kosalec I., Bakmaz M., Pepeljnjak S., Knezevic S., Acta Pharm., 54, 65—72 (2004). 14) Kosalec I., Bakmaz M., Pepeljnjak S., Acta Pharm., 53, 275—285 (2003). 15) Sverdlov A. G., Mozzhukhin A. S., Pavlova L. M., Nikanorova N. G., Radiobiology, 9, 706—710 (1969). 16) Benkovic V., “Thesis (PhD),” Faculty of Science, University of Zagreb, 2006. 17) Orsolic N., Benkovic V., Horvat-Knezevic A., Kopjar N., Kosalec I., Bakmaz M., Mihaljevic´ Z., Bendelja K., Basic I., Biol. Pharm. Bull., 30, 946—951 (2007). 18) Singh N. P., McCoy M. T., Tice R. R., Schneider L. L., Exp. Cell Res., 175, 184—191 (1988). 19) Poli P., Buschini A., Spaggiari A., Rizzoli V., Carlo-Stella C., Rossi C., Toxicol. Lett., 108, 267—276 (1999). 20) I.A.E.A., International Atomic Energy Agency Technical Report Series 405, Cytogenetic Analysis for Radiation Dose Assessment. I.A.E.A., Vienna (2001). 21) Fenech M., Morley A. A., Mutat. Res., 147, 29—36 (1985). 22) Fenech M., Chang W. P., Kirsh-Volders M., Holland N., Bonassi S., Zeiger E., Mutat. Res., 534, 65—75 (2003).
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