1. Introduction. Biología Celular, Fisiología y Inmunología. U.A.B. Dietéticos Intersa. Plaza Dr. Seres nº 13, Torreserona, Lleida.

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1 Evaluation of radioprotection properties of propolis by chromosomal alterations, cell proliferation kinetics, mitotic index and sister chromatid exchange. Alegria Montoro 1, Joan Francesc Barquinero 2, Miguel Almonacid 1, Ricardo Tortosa 1, LLeonard Barrios 2,Vicenta Sahuquillo 1, Juan Serrano 3, Maribel Saiz 3, Miguel Ramos 4, Gumersindo Verdú 4, Juan Ignacio Villaescusa 1. 1 Servicio de Protección Radiológica. Hospital Universitario la Fe. Valencia 2 Servicio de Dosimetría Biológica, Departamentos de Biología Animal, Vegetal y Ecología, y Biología Celular, Fisiología y Inmunología. U.A.B. 3 Dietéticos Intersa. Plaza Dr. Seres nº 13, Torreserona, Lleida Departamento de Ingeniería Química y Nuclear. UPV. Camino de Vera s/n Abstract. A consequence of ionizing radiation is the induction of chromosomal alterations. This causality relation involves that chromosomal alterations can be considered a good indicator of the radiological damage. Some chemical agents can modulate the tissue response to radiation. These compounds are useful because they show certain selectivity, protecting the healthy tissues (radioprotectors) or increasing the sensibility of tissues to radiations (radiosensibilizators). Propolis substance has showed radioprotection properties which are performed in the following study. Propolis is a product of extraordinary interest for both medicine and pharmaceutical industry, since it is assumed to show diverse beneficial health effects. Among many other attributes of EEP (propolis ethanolic extract), it exhibits antioxidant and radical free scavenger properties. In a previous study, human peripheral blood lymphocytes were exposed to 2 Gy of γ rays in presence and absence of EEP, and the analysis showed a reduction in the frequency of dicentrics and rings, with a maximum protection of 50%. The proposed concentration for radioprotection would be between µg ml -1. The cytotoxic effect has been evaluated analyzing the EEP effect in the cellular division cycle. Propolis ethanolic extract (EEP) has been obtained and samples of peripheral blood have been cultured in the presence of increasing concentrations of EEP. In order to quantify it, two indexes have been used, the mitotic index and cell proliferation index. For both indexes the cytotoxic effect takes place from 750 µg ml-1 concentrations onwards. Similar results were obtained for the analysis of chromosomal aberrations. Finally, propolis effect in lymphocytes by sister chromatid exchange test has been presented for higher concentrations of EEP. KEYWORDS: propolis, radioprotectors, dicentric, cell cycle 1. Introduction Many natural and synthetic chemicals substances have been investigated in the past for their efficiency to protect against radiation-induced damage in biological systems [1]. Propolis is a strongly adhesive resinous substance, collected, transformed and used by bees to seal holes in their honeycombs [2]. Propolis exhibits a broad spectrum of biological activities such as, anticancer, antioxidant, antiinflammatory, antibiotic, antifungal, anti-hepatotoxic and radioprotector. Finally, a difference from most radioprotector substances is that propolis doesn t produce side effects and is considered safe in low doses [3]. Radioprotector effect of propolis was demonstrated in a previous study [4] where human peripheral blood lymphocytes were exposed to 2 Gy of γ rays, in the presence and absence of EEP, with a reduction of 50% when the frequency of dicentrics were considered. Once radioprotector effect was demonstrated, authors considered whether a concentration-dependent protection takes place in lymphocytes of blood samples exposed to 2 Gy and increasing concentrations of EEP. Results indicate that the maximum protectible fraction for dicentrics is around 44% [5]. Presenting author, gverdu@iqn.upv.es

2 Cytogenetic alterations in peripheral blood lymphocytes, such as chromosomal aberrations (CAs) and sister chromatid exchanges (SCE) have long been applied in surveillance of human genotoxic exposure and early effects of genotoxic carcinogens. The use of these biomarker assays is based on the fact that most established human carcinogens are genotoxic in short-term tests and capable of inducing chromosomal damage. The relevance of chromosomal aberrations as a biomarker has been further emphasized by epidemiological studies suggesting that a high frequency of chromosomal aberrations is predictive of an increased risk of cancer. Other cytogenetic test, SCE, is a highly sensitive parameter for evaluating human occupational and environmental human exposure to mutagenic and carcinogenic agents. CAs are structural aberrations comprising chromosome and chromatid type breaks and rearrangements and SCE are interchanges of DNA replication products between sister chromatids at apparently homologous loci, suggested to represent homologous recombination repair of DNA double strand breaks [6]. The cell proliferation kinetics (PI) and mitotic index (MI) are recognized biomarkers in biological monitoring to evaluate lymphocyte proliferation in population as well as to evaluate normal or tumour cells [7]. There are few reports in literature about the genotoxic effect of propolis. Several authors [8] investigated in vitro anticarcinogenic potential of propolis using MI. Blood samples were exposed to increasing concentrations of propolis and differences between control and exposed groups were found. Taking into account MI as biomarker of genotoxic effect, propolis did not produce carcinogenic effect. Another cytogenic biomarker like SCE was evaluated [9]. Authors concluded that increasing concentrations of propolis in in vitro blood samples could have genotoxic effects in high concentrations. It has been reported [10] that the highest tested concentration displayed a significant increase in the frequency of CAs. However, no significant differences in MI were observed between cultures treated with and without propolis extract, concluding that the concentrations used in this work were not cytotoxic. Recent studies [11 a, b ] demonstrate the radioprotective effect of propolis in mice, moreover, genotoxic effect were also measured with comet assay in non-irradiated mice. Authors conclude that water and ethanolic extracts of propolis used were not cyto/genotoxic. The aim of this study is the analysis of several biomarkers such as chromosome aberrations and sister chromatid exchange. In addition to the cytogenetic analysis, the effect of EEP in the cell proliferation kinetics by the proliferation index is analyzed. Also, in order to detect the cytotoxic effect, the mitotic index is determined. 2. Material and Methods 2.1 Extraction procedure 10, 20, 60, 125, 250, 375, 500, 1000 mg of Pure extract of Propolis (Dietéticos Intersa S.A, Spain) was mixed with 10 ml of ethanol (EtOH) 95%. The solutions were kept one week at 4ºC and then centrifuged. The supernatant was filtered (0.22 µm) to eliminate the impurities and the wax, then 250 µl of this solution was added to 12 ml of peripheral blood samples to reach EEP of 20, 40, 120, 250, 500, 750, 1000 and 2000 µg.ml -1. Peripheral blood samples treated with the same final concentration of the solvent (0.3M EtOH) and another one without any treatment were used as controls. 2.2 Culture Conditions Peripheral blood samples were collected in sterile vacutainer tubes containing lithium heparin as anticoagulant. Separate cultures were set up for each treatment by mixing 0.75 ml of whole blood with 5 ml of PB-Max Kariotip medium (Gibco, Barcelona). Cultures were incubated 48 hours for CA analysis and 72 hours for the rest techniques. In order to analyze exclusively first division metaphases a final concentration of 12 µg.ml -1 of bromodeoxyuridine (Sigma, USA) was present since the set up of the cultures. 150 ml of Colcemid (Gibco, Barcelona) was added 2 hours before harvesting. Once prepared, slides of two-day-old were stained with the Giemsa counterstained of the fluorescence plus Giemsa stain technique. 2.3 Cytogenetic and genotoxic assessment

3 2.3.1 Chromosomal aberrations Two-to three-day old slides were stained with Fluorescence plus Giemsa stain technique [12]. Chromosomal analysis was carried out exclusively on first-division metaphases containing 46 centromeres out of one hundred cells. Chromosome abnormalities were classified as follows: dicentric chromosomes and rings, which were only considered when an acentric fragment was present. Acentric fragments, not associated with dicentric and ring chromosomes, were classified as extra acentric fragments. Translocations were only recorded when the morphology of the derivative chromosome was clearly indicative of this kind of rearrangement. Other abnormalities such as chromatid breaks (ctb) and gaps were also included Sister chromatid exchange Old two days slides were treated 20 min in Hoechst at room temperature. The slides were washed with distillated water. The slides were covered with 2xSSC and treated with UV light (300 W) for 2 min. Once washed, the slides with water and dried 30 min, the slides were stained with Giemsa for 5 min. The incidence of SCE was determined from the analysis of 30 second division metaphase for each concentration Mitotic index The mitotic index (MI) was calculated, for each concentration, as the proportion of metaphase for 1000 cells. The relative mitotic index (RMI) was evaluated as: The inhibition of mitotic index was evaluated as: Cell Proliferation Kinetics RMI = [MI treated/mi control] x 100. (1) [MI treated x 100/MI control]. (2) The proportion of first (M1), second (M2) and third (M3) (Figure 1) dividing cells was obtained from 100 consecutive metaphases, for each concentration. The proliferation index (PI) was calculated according to the formula (Rojas et al., 1993): The relative proliferation index (RPI) was evaluated as: [PI = (M1 + 2M2 + 3M3)/100]. (3) RPI = [PI treated/pi control] x 100. (4) Figure 1: Metaphases at first (a), second (b) and third (c) division respectively. a) b) c)

4 3. Results The endpoint evaluated were chromosomal aberrations, sister chromatid exchange, mitotic index and cell proliferation kinetics and results are shown in this order. The effects of propolis in the frequency of different chromosomal aberrations when human lymphocytes were exposed to different concentrations of EEP is shown in Table 1. The most frequent CAs were acentrics. Other chromosomal aberrations were found although no dicentric chromosomes were observed. The data obtained in the analysis of 100 metaphases per concentration indicates a statistically significant increase when lymphocytes were treated with EEP. The differences were significant from 750 µg.ml -1, where the increase in comparison with previous concentration was statistically significant for acentrics (0.12 ± vs 0.02 ± 0.014, p<0.01) and for total alterations (0.14 ± vs 0.02 ± 0.014, p<0.01). Results show that most of the breaks detected were of chromatid type. Table 1: Chromosome alterations incidence in human lymphocytes exposed to different concentrations after 100 cells analyzed. EEP [µg.ml -1 ] N (a) Dic (b) R (c) Ace (d) Other (e) Ctb (f) Gaps TA (g) (a) N: cells scored (b) Dic: dicentrics (c) R: rings (d) Ace: acentrics (e) Other: include translocations, inversions, etc. (f) Ctb: chromatid breaks (g) TA: total alterations. When cytotoxicity of the EEP on lymphocyte cultures was analyzed through mitotic index, a significant decrease was found for higher concentrations (> 750 µg.ml -1 ) (Table 2). Cells cultured in the presence of 20, 40, 120, 250 and 500 µg.ml -1 of EEP exhibited mean mitotic indices (4.31, 4.06, 4.05, 4.08, 4.11, respectively), with no differences respect control sample (4.26). However, a significant and concentration-dependent decrease in mitotic index was observed in cultures treated with 750, 1000 and 2000 µg.ml -1 of EEP (3.66, 2.60 and 1.67, respectively) (Figure 2). Also, a decrease rate of MI was obtained by Ozkul et al. (2005) when human lymphocytes were exposed to increasing concentrations of propolis. Proliferation index in control and in cultures exposed to µg.ml -1 of EEP, showed no differences. Although, EEP exhibited a significant and concentration-dependent decrease in PI in concentrations between µg.ml -1 (Figure 3). Table 2: Mitotic and proliferation indexes, percentage of cells in first (M1), second (M2) and third (M3) division, in human peripheral lymphocytes cultured during 72 h in the presence of various concentrations of EEP. Also, Table shows de RMI and RPI for each concentration.

5 EEP [µg/ml] Mitotic index (%) RMI Proliferation Index RPI SCE/cell Control 4.26 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.97 Figure 2: Effect of different concentrations of EEP on the mitotic index (MI) of cultured human lymphocytes. Data are presented as percentage of inhibition of the MI. Results for control cultures are taken as 100%. 6,0 5,0 4,0 3,0 MI 2,0 1,0 y = -0,0013x + 4,3293 R 2 = 0,9179 0, Concentration (µg.ml -1 ) Figure 3. It represents the effect of different concentrations of EEP on proliferation index (PI) of cultured human lymphocytes. 2,00 1,50 1,00 PI 0,50 y = -0,0002x + 1,4746 R 2 = 0,7709 0, Concentrations (µg.ml -1 )

6 When the relative indexes, RMI and RPI, the RMI are compared the RMI reduced up to 39 % whereas the RPI is reduced up to 68 % at maximum concentration of EEP. A reduction in both indexes on concentrations higher than 750 µg/ml is observed. The decrease of MI and PI indexes against the increasing concentration of propolis allow the possibility of looking fir a correlation between the two indexes, showing a negative slope line (Figure 4). Figure 4. Correlation between mitotic index and proliferation index from cultures untreated and treated with different concentrations of EPP. 1,70 1,50 1,30 Proliferation index 1,10 0,90 0,70 0,50 0,30 0, , % Inhibition IM % Inhibition MI The data on SCE/cell has been compiled in Table 2. The induction of SCE at all concentrations was significantly (p<0.05) only when lymphocytes were treated with higher concentration (12.08 ± 0.97). 4. Discussion There is an interest to identify and develop non-toxic radioprotective compounds. Propolis is atoxic [13], however more studies should be focused on investigating its pharmacokinetic, toxicity and biodisponibility. In order to complete the in vitro study of radioprotective properties of propolis extract, our work focuses on the cyto-genotoxic potential of propolis using different well-known biomarkers. Propolis and their constituents may be relevant to protect against radiation damage, including anti-inflammatory, antioxidant, antiproliferative, antitumoral, and apoptotic activities. It has not been tested enough in the required way for conventional pharmaceuticals. Preclinical screening of synthetic compounds or natural products to evidence antitumor activity generally relies on evaluating only a single parameter, usually cell death. However, more accurate information can be obtained with the use of two biological endpoints, such as the MI and PI assays. For instance, an inhibition of the MI can be interpreted as the arrest of cells at any moment during interphase. On the other hand, PI allows us to differentiate between drugs, which induce cell death, and those, which have a cytostatic effect by delaying any phase of the cell cycle [7]. Our data indicates, that lymphocytes cultured in the presence of propolis at concentrations greater than 750 µg.ml -1, exhibited a significant and concentration dependent decrease of MI and PI. These results could be due to a transient blockage at one or more points in the cell cycle. Some interpretations might be based on the antiproliferative effect of the EEP in the cell cycle.

7 The correlation between mitotic and proliferation indexes shows a negative slope line, that is characteristic of a cytostatic activity of the EEP. According to these results, a dose-related decrease of the PI could be interpreted as a potential capacity of EEP to interact directly with DNA. There is a dose related delay of the PI. This kind of agents is known to be clinically most effective as antineoplasic agents cycle [7]. The main result of our research, using four well-established and sensitive biomarkers, was the ideal concentration in in vitro studies to use propolis as a radioprotector. The concentration would be would be 500 µg.ml -1. To our knowledge, there have been no published studies investigating genotoxic effect of propolis with all these biomarkers together. Several authors [14] observed this antiproliferative effect of crisina (component of propolis) as a cell cycle-specific that causes a delay in the G 1 -S transition. A similar reduction in the MI and PI indexes has been described for concentrations greater than 20 mm of Melatonin [15]. In this study, it was also observed a concentration dependent decrease of both indexes. Recently, It has been reported [16] that extracts of Paico, plant used in natural medicine, decreased the MI but do not modify the PI. In the results presented in the PHD thesis [17] a maximum level of protection around 40 % against 2-Gy dose of gamma rays was observed. But this concentration produces a reduction of the RMI up to 61 % and RPI up of 76 %, being the whole inhibition up to 39 % and up to 68% respectively. This means that the use of propolis as radioprotector agent must have a concentration of 500 µg.ml -1, where MI and PI haven t significantly reduced. Several authors [10] also demonstrated the mutagenic effect of propolis in Chinese hamster ovary cells with the highest concentration used in this work. Several authors [8] have analyzed other biomarkers such as micronucleus (MN), and have found a increase in the rate of MN with different concentrations of propolis. The same work reported that increasing SCE rates showed that propolis could have genotoxic effects in high concentrations. These results are consistent with our study. Further studies are necessary to evaluate efficiency using standard extracts of propolis, and identify the bioactive compounds responsible of the radioprotective property. 5. Acknowledgements This study has been partially funded by Dietéticos Intersa S.A. of Barcelona. We acknowledge also Hospital Universitario la Fe of Valencia for the invaluable in this work. REFERENCES [1] Nair, C.K.K., et al. Radioprotectors in radiotherapy, J Radiat Res, 42 (2001) 21. [2] Burdock, G.A., et al. Review of the Biological Properties and Toxicity of Bee Propolis, Food and Chemical Toxicology. 36 (1998) [3] Castaldo, S., et al. Propolis, an old remedy used in modern medicine, Fitoterapia. 73 (2002) S1-6. [4] Montoro, A., et al. Assessment by cytogenetic analysis of the radioprotection properties of propolis extract. Radiat Prot Dosimetry. 115(1-4) (2005) [5] Montoro, A., et al. Radioprotección (ISSN ), 14 (2007) [6] Norppa, H., et al. Chromosomal aberrations and SCEs as biomarkers of cancer risk. Mutat Res. 30 (2006) [7] Rojas, E., et al. Mitotic index and cell proliferation kinetics for identification of antineoplastic activity. Anticancer Drugs. 4 (1993) [8] Ozkul, Y., Silici S, Eroğlu E. The anticarcinogenic effect of propolis in human lymphocytes culture.phytomedicine. 12 (2005) [9] Ozkul, Y., Eroglu HE, Ok E.Genotoxic potential of Turkish propolis in peripheral blood lymphocytes.pharmazie. 61 (2006) [10] Tavares, D.C., et al. Propolis-induced genotoxicity and antigenotoxicity in Chinese hamster ovary cells. Toxicol In Vitro. 20 (2006)

8 [11 a ] Benkovic, V., et al. Radioprotective effects of propolis and quercetin in gamma-irradiated mice evaluated by the alkaline comet assay. Phytomedicine. (2008) Epub ahead of print [11 b ] Benkovic, V., et al. Evaluation of the radioprotective effects of propolis and flavonoids in gamma-irradiated mice: the alkaline comet assay study. Biol Pharm Bull. 31 (2008) [12] Perry, P. and Wolff, S.. New Giemsa Method for differential staining of sister chromatids. Nature. 261 (1974) [13] Farré, R. et al.. El própolis y la salud. Ars. Pharmaceutica, 45 (2004) [17] Montoro, A. Evaluación citogenética del efecto radioprotector del extracto etanólico de propóleos, PhD Thesis, Univ. Politécnica de Valencia, Spain (2006) [14] Weng, M.S., et al.. Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21waf1/cip1 expression: involvement of p38 mitogen-activated protein kinase. Biochem. Pharmacol. 15 (2005) [15] Vijayalaxmi. et al. Melatonin protects human blood lymphocytes from radiation-induced chromosome damage. Mutat. Res. 346 (1995) [16] Gadano, A.B. et al. Argentine folk medicine: genotoxic effects of Chenopodiaceae family.j Ethnopharmacol. 16 (2006)