Toxicology in Vitro 28 (2014) 451–456
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Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes Andrea Cˇabarkapa a,⇑, Lada Zˇivkovic´ a, Dijana Zˇukovec a, Ninoslav Djelic´ b, Vladan Bajic´ c, Dragana Dekanski c, Biljana Spremo-Potparevic´ a a
Department of Biology and Human Genetics, Institute of Physiology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Oslobodjenja Blvd 18, Belgrade, Serbia c Institute for Research and Development, Galenika a.d., Pasterova 2, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Article history: Received 27 June 2013 Accepted 23 December 2013 Available online 3 January 2014 Keywords: Dry olive leaf extract (DOLE), Adrenaline (CAS 329-63-5) Oxidative stress Comet assay
a b s t r a c t Excessive release of stress hormone adrenaline is accompanied by generation of reactive oxygen species which may cause disruption of DNA integrity leading to cancer and age-related disorders. Phenolic-rich plant product dry olive leaf extract (DOLE) is known to modulate effects of various oxidants in human cells. The aim was to evaluate the effect of commercial DOLE against adrenaline induced DNA damage in human leukocytes by using comet assay. Peripheral blood leukocytes from 6 healthy subjects were treated in vitro with three final concentrations of DOLE (0.125, 0.5, and 1 mg/mL) for 30 min at 37 °C under two different protocols, pretreatment and post-treatment. Protective effect of DOLE was assessed from its ability to attenuate formation of DNA lesions induced by adrenaline. Compared to cells exposed only to adrenaline, DOLE displayed significant reduction (P < 0.001) of DNA damage at all three concentrations and under both experimental protocols. Pearson correlation analysis revealed a significant positive association between DOLE concentration and leukocytes DNA damage (P < 0.05). Antigenotoxic effect of the extract was more pronounced at smaller concentrations. Post-treatment with 0.125 mg/mL DOLE was the most effective against adrenaline genotoxicity. Results indicate genoprotective and antioxidant properties in dry olive leaf extract, strongly supporting further explorations of its underlying mechanisms of action. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Prolonged exposure to elevated oxygen levels, exceeding normal physiological concentrations, is well known cause of oxidative DNA damage in living cells and tissues (Halliwell and Gutteridge, 1984). Oxidizing agents affect normal functioning of cells and induce structural alterations and DNA mutations that can cause or lead to cancer and age-related disorders (Khansari et al., 2009). Oxidative stress can originate from endogenous sources caused by reactive oxygen species (ROS) overproduction in cells, or from exogenous cellular substances like various environmental
Abbreviations: ROS, reactive oxygen species; DOLE, dry olive leaf extract.
⇑ Corresponding author. Address: Institute of Physiology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia. Tel.: +381 11 3951349; fax: +381 11 3974349. E-mail addresses: [email protected], [email protected] ˇ abarkapa), [email protected] (L. Zˇivkovic´), [email protected] (A. C (D. Zˇukovec), [email protected] (N. Djelic´), [email protected] (V. Bajic´), ddekan@ sezampro.rs (D. Dekanski), [email protected] (B. Spremo-Potparevic´). 0887-2333/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.12.014
genotoxic compounds (Khansari et al., 2009). Hydrogen peroxide (H2O2) is one of the well known chemicals able to produce ROS via Fenton’s reaction and cause oxidative damage to DNA and genomic instability (Anderson et al., 1994). On the other hand, pro-oxidant properties of adrenaline and catechol derivates and their effects on DNA integrity are far less examined although recent empirical evidence confirmed the ability of adrenaline to induce DNA alterations (Djelic´ et al., 2003; Djelic´ and Anderson, 2003; Dobrzynska et al., 2004). Studies have shown that DNA damage is induced during the oxidation and cyclisation of catechol derivatives, such as dopa and adrenaline in copper or iron-catalyzed Fenton reaction resulting in ROS generation (Bindoli et al., 1990; Cavalieri and Rogan, 2004; Genova et al., 2006; Miura et al., 2000). Production of free radicals during redox cycling of catecholamines is considered a key step in their genotoxic action. Since ROS generation underlies main path for inducing DNA damage in both H2O2 and adrenaline mechanism of action, we searched for compounds able to scavenge free radicals and protect the cell DNA from damage when exposed to these oxidants. Dry olive leaf extract
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(DOLE) from the Mediterranean tree Olea europaea has been reported to possess antioxidant, antimicrobial and antifungal activity which may relate to its therapeutic properties in prevention of hypertension, cancer and diabetes mellitus (Dekanski et al., 2009a,b; Fares et al., 2011; Miljkovic´ et al., 2009; Mijatovic´ et al., 2011). Phenolics and flavonoids, the main components of DOLE are widely recognized for their antioxidant activity and free radical scavenging capacity (Anter et al., 2011; Benavente-Garcia et al., 2000; Cumaoglu et al., 2011; Le Tutour and Guedon, 1992; Nousis et al., 2005). Although extensively studied for its protective action against different diseases, DOLE has been insufficiently examined for its ability to interact, either directly or indirectly, with different mutagens, and until now DOLE has not been studied in relation to oxidative DNA damage induced by hormonal stress. The main goal of this research was to evaluate the protective effect of commercial dry olive leaf extract against adrenaline and H2O2 induced oxidative DNA damage in human peripheral blood leukocytes by using the ‘‘single cell gel electrophoresis’’ (comet assay). The comet assay has been shown to be an effective, sensitive and rapid in vitro method for examining DNA damage and issues related to oxidative stress in human peripheral blood cells (Anderson et al., 1994). 2. Materials and methods 2.1. Olive leaf extract Olive leaf extract EFLAÒ 943, standardized to 18–26% of oleuropein, was purchased from Frutarom Switzerland Ltd. (Wadenswil, Switzerland). The extract was manufactured from the dried leaves of Olea europaea L., applying an ethanol (80% m/m) extraction procedure. After a filtration process, the crude extract was dried. Its total phenols content, determined by Folin–Ciocalteau assay, was 197.8 lg GAE/g of dry extract; total flavonoids and tannins content was 0.29% and 0.52%, respectively. High performance liquid chromatography (HPLC) analysis revealed a complex mixture of phenolic compounds: oleuropein (19.8%), luteolin-7-O-glucoside (0.04%), apigenine-7-O-glucoside (0.07%), quercetin (0.04%) and 0.02% of caffeic acid (Dekanski et al., 2009a). In this study, the same batch of EFLAÒ 943 was used. It was kept in sealed, previously sterilized microtubes, stored at room temperature and protected from light until use. 2.2. Subjects Peripheral blood samples were collected in heparinized containers from six healthy volunteers (4 female and 2 male subjects) aged between 19 and 35 years. All subjects were non-smokers, who abstained from alcohol, not receiving any therapy or medications, nor taking dietary supplements. 2.3. Study design For the experiment, DOLE powder was diluted in phosphatebuffered saline (PBS, Torlak Institute of Immunology and Virology, Belgrade, Serbia) to three final concentrations: 1 mg/mL, 0.5 mg/ mL and 0.125 mg/mL. We chose final concentrations of the extract according to the range of concentrations previously found to be safe and effective to use in in vitro studies (Turkez and Togar, 2011; Lee-Huang et al., 2003). Adrenaline (CAS No. 329-63-5, epinephrine injection 1 mg/mL solution, Jugoremedija A.D., Zrenjanin, Serbia) and H2O2 (CAS No. 7722-84-1, ZORKA Pharma, Sabac, Serbia) were previously examined at four doses to determine the appropriate limits of toxicity for the assay (5, 10, 50, 150 lM adrenaline and 5, 10, 25, 50 lM H2O2). According to literature data
5 lM adrenaline represents the maximal therapeutic dose for human therapy. Also, all tested concentrations correspond to those previously used in genetic toxicology studies of adrenaline (Djelic´ et al., 2003; Dobrzynska et al., 2004). We chose 10 lM adrenaline and 25 lM H2O2 for further experiments, since these were the smallest concentrations that produced consistently high level of DNA fragmentation in exposed cells as compared to the untreated controls. To determine the effect of antioxidant-rich environment in vitro on human peripheral blood leukocytes exposed to oxidative stress induced by adrenaline and H2O2, the DOLE extract was subjected to two types of interactions with the oxidants, pretreatment and post-treatment. Two independent experiments were performed: (1) In the first series (pretreatment protocol) three different concentrations of antioxidant DOLE (1, 0.5 and 0.125 mg/mL) were administered and incubated with the whole blood cell preparations on slides at 37 °C for 30 min, before washing the cells with PBS and adding two oxidizing agents separately (25 lM H2O2 for 15 min on ice, and 10 lM adrenaline for 30 min on 37 °C). (2) The second series of treatments (post-treatment protocol) was performed simultaneously on other series of cell preparations from the same subjects, first exposed to two oxidizing agents individually, then rinsed with PBS and subsequently post-treated with three mentioned concentrations of DOLE under the same conditions as in first group. Experiments were repeated six times, each carried out in duplicate. All cell preparations were incubated together with negative controls (treated only with PBS) as well as with positive controls (H2O2 and adrenaline). 2.4. The single cell gel electrophoresis assay Before processing for comet assay, cell viability for all whole blood samples were determined by using the trypan blue exclusion method (Anderson et al., 1994). Cell viability was always found to be above 90%. The comet assay was performed essentially as described by Singh et al. (1988). Briefly, the 6 ll of whole blood samples were suspended in 0.67% low-melting-point (LMP) agarose (Sigma–Aldrich, St. Louis, MO) and pipetted onto superfrosted glass microscope slides precoated with a layer of 1% of normal-meltingpoint agarose (Sigma–Aldrich, St. Louis, MO), spread using a coverslip, and maintained for 5 min on 4 °C to solidify. After gently removing the coverslips, the cell suspensions on slides were treated with DOLE and oxidants as described above in two mentioned types of interactions, pre-treatment and post-treatment. Following the treatments, all slides were covered with the third layer of 0.5% LMP agarose and again allowed to solidify on ice for 5 min. After coverslips removal, the slides were placed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X100 and 10% dimethylsulfoxide, pH 10 adjusted with NaOH) at 4 °C overnight and afterwards subjected to electrophoresis and staining with ethidium bromide (20 lg/L), performed as described by Singh et al. (1988). The comets were analyzed 15 min after staining at 100 magnification on Olympus BX 50 microscope (Olympus Optical Co., GmbH, Hamburg, Germany), equipped with a mercury lamp HBO (50 W, 516–560 nm, Zeiss). Evaluation of DNA damage was done according to Anderson et al. (1994). DNA damage in the cells was assessed by quantification of the amount of DNA released from the core of the nucleus and comets were visually scored and classified into five categories corresponding to the extent of DNA migration: (A) no damage, 95% (Fig. 1). Analysis was performed on 100 cells that were randomly selected per subject (50 cells from each of 2 replicate slides) and were always carried out by the same experienced person. DNA damage was characterized as DNA migration over 5% (B + C + D + E comet classes), and median value
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Fig. 1. Representing five classes of Comets: (A) no damage, 95%.
was calculated for all six subjects. Leukocytes undergoing apoptosis or necrosis and ‘‘hedgehog’’ cells were excluded from the analysis of the comet assay slides, and were distinguished from normal cells following the instructions given by Singh (2005). 2.5. Statistical analysis Statistical analysis was performed by using Kruskal–Wallis and Mann–Whitney U test, for comparisons of different treatments vs. the respective controls. Values are expressed as median and 25th and 75th percentile, for n = 6. Pearson’s correlation and linear regression were used to evaluate the relationship between DNA damage and DOLE concentration. All data were analyzed with The GraphPad Prism 5.0 software. A difference at P < 0.05 was considered statistically significant. 3. Results Protective ability of DOLE extract was tested using two different treatments (pretreatment and post-treatment) and the comet scores were compared to the scores obtained for the DNA damaging agents adrenaline and H2O2. The protective effect of the tested extract DOLE was evaluated for its ability to prevent hydrogen peroxide and adrenaline-induced formation of strand breaks in the nuclear DNA, represented as the median number of all cells with migrated DNA in Tables 1 and 2. The effect of pretreatment with DOLE on the level of DNA damage when cells are incubated with DOLE before the individual
exposure to two oxidants, are shown in Table 1. The score for cells from post-treatment protocol, first exposed to oxidizing agents separately and afterwards post-treated with antioxidant DOLE under same conditions as above mentioned is represented in Table 2. While an increase of DNA damage was detected in positive control cells exposed only to either 25 lM H2O2 or 10 lM adrenaline, the levels of DNA damage in leukocytes analyzed from the samples treated with different concentrations of DOLE were significantly reduced in both pretreated and post-treated samples (P < 0.001) (Tables 1 and 2). All three concentrations of DOLE strongly prevented DNA damage under both experimental conditions, seen as reduction in the number of cells with damaged DNA. As shown in Table 1, in samples pretreated with DOLE, we observed a positive association between the level of DNA damage and concentration of DOLE (0.125 mg/mL, 0.5 mg/mL, 1 mg/mL), in both adrenaline and H2O2 exposed cells (r = 0.41, P < 0.0214; r = 0.3960, P < 0.0204, respectively). Concentration–response indicated that with decline of DOLE concentration, DNA damage was less profound. Although all three concentrations of extract effectively reduced induction of DNA fragmentation by adrenaline and H2O2, treatment with smaller concentrations of extract corresponded to less DNA damage. Surprisingly, the greater level of DNA damage was observed in leukocytes pretreated with 1 mg/ mL concentration as compared to two smaller concentrations of the extract, thus the highest concentration of DOLE actually displayed the lowest protective effect and the highest variability of the results. Two smaller concentrations of DOLE (0.5 and 0.125 mg/mL) displayed approximate results in attenuation of both
Table 1 Pretreatment protocol: Number of cells with damaged DNA from six different subjects, first pretreated with 3 concentration of DOLE and subsequently exposed to two oxidants (adrenaline and H2O2 separately). Oxidants
Adrenaline 10 lM H2O2 25 lM
Positive controls
54.00 (39.50–75.75) 86.00 (71.25–96.00)
DOLE concentrations
P value
1 mg/mL
0.5 mg/mL
0.125 mg/mL
10.00* (4.50–16.75) 15.00* (8.50–28.50)
9.50* (5.25–15.25) 11.50* (5.25–13.25)
10.00* (4.75–15.00) 8.00* (5.50–12.75)
Values are expressed as median and 25th and 75th percentile for comet scores in 100 cells from 6 subjects. P < 0.05, DOLE treatment vs. oxidant; analyzed by Kruskal–Wallis test.
*
0.0045 0.0020
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Table 2 Post-treatment protocol: number of cells with damaged DNA from six different subjects, first treated with two oxidants (adrenaline and H2O2 separately) and subsequently incubated with 3 concentration of DOLE. Oxidants
Adrenaline 10 lM H2O2 25 lM
Positive controls
DOLE concentrations
54.00 (39.50–75.75) 86.00 (71.25–96.00)
P value
1 mg/mL
0.5 mg/mL
0.125 mg/mL
12.50* (4.75–16.50) 15.50* (10.25–27.25)
9.50* (5.50–11.25) 4.50* (1.50–15.00)
3.50* (1.75–6.50) 11.00* (5.50–26.25)
0.0010 0.0018
Values are expressed as median and 25th and 75th percentile for comet scores in 100 cells from 6 subjects. P < 0.05, DOLE treatment vs oxidant; analyzed by Kruskal–Wallis test.
*
80
Adrenaline 10 µM DOLE 0.125 mg/mL post-treated
60
40
20
* 0 B+C (medium damage)
4. Discussion This study demonstrates the potential of commercial dry olive leaf extract to attenuate oxidative DNA damage in human leukocytes, induced by adrenaline and hydrogen peroxide under two different experimental conditions, pretreatment and post-treatment. Our results show that all concentrations of DOLE evaluated in our study displayed DNA protective effect against adrenaline and H2O2 genotoxicity. Dry olive leaf extract so far exhibited beneficial properties in many experimental models of diseases stemming from oxidative stress, and its antioxidant properties are well documented (Anter et al., 2011; Benavente-Garcia et al., 2000; Cumaoglu et al., 2011; Le Tutour and Guedon, 1992; Nousis et al., 2005). Although the broad field of biomedical research has been exploring various protective effects of DOLE, according to our knowledge of literature, this is the first work that shows the action of DOLE in relation to a stress hormone-induced oxidative DNA damage. Since oxidative stress is being recognized as an important factor in the etiology of many chronic diseases, the examination of dietary components with antioxidant activity has received much attention (Arts and Hollman, 2005). On the other hand, the role of hormones in oxidative stress is far less examined. One of the aims of this study was to point out the fact that nonsteroidal hormones, such as adrenaline, with phenolic or catechol moieties are able to cause oxidative DNA damage (Djelic´ and Anderson, 2003; Flint et al., 2007; Miura et al., 2000). The release of catecholamines in amounts exceeding physiological concentrations has been observed to exert cytotoxic effects in neuroblasts, melanoma and myocardial cells (Behonick et al., 2001; Flint et al., 2007; Miura et al., 2000; Okamoto et al., 1996). Ambiguous assumptions that hormones can yield genetic instability are supported by novel studies that explain how biochemical events underlying hormone action might eventually lead to genetic changes (Cavalieri and Rogan, 2004; Djelic´ and Anderson, 2003; Liehr, 2001). Catabolic
Number of cells with migrated DNA
Number of cells with migrated DNA
adrenaline and H2O2 induced strand breaks, while the 1 mg/mL concentration showed to be more effective in reducing damage caused by adrenaline then H2O2. Results of the post-treated samples displayed in Table 2 follow similar concentration-dependent trend that we observed in pretreatment samples, in relation to adrenaline. The number of cells with damaged DNA was positively associated with rising concentrations of DOLE (r = 0.4602, P < 0.0047). On the other hand, in H2O2 treated samples, the correlation between level of DNA damage and DOLE concentrations was lost. The two higher concentrations of DOLE responded to previously recorded trend of efficacy, while the smallest 0.125 mg/mL concentration showed discordance with the previously mentioned results, and was much less effective against H2O2-induced oxidative damage in post-treated than pretreated samples (Table 1 vs. Table 2). Comparing the antioxidant ability of all concentrations of DOLE in both experimental conditions, it appeared that its application in post-treatment was more effective in reducing DNA damage than in pretreatment. The results also demonstrate that post-treatment with 0.125 mg/mL of DOLE was the most effective in prevention of adrenaline induced genotoxicity, while the post-treatment with 0.5 mg/mL of DOLE was most efficient in attenuating H2O2-induced DNA damage. Fig. 2 represents the antioxidant response of the most effective DOLE treatment (0.125 mg/mL post-treatment) in comparison with the genotoxic effect of adrenaline, expressed with two DNA damage distribution variables: the median number of cells with low and medium damage (B + C comet category), and the median number of cells with high and total damaged DNA (D + E comets) from all six subjects. Degree of DNA damage presented with two variables in Fig. 2 indicate that the 0.125 mg/mL DOLE post-treatment was equally effective in attenuation of low and medium oxidative DNA damage as well as in the reduction of highly damaged DNA.
20
Adrenaline 10 µM DOLE 0.125 mg/mL post-treated
15
10
5
* 0 D+E (high damage)
Fig. 2. Relationship between antioxidant activities of dole and extent of DNA damage: Degree of DNA damage in human leukocytes exposed to adrenaline compared to antioxidant response of most effective DOLE treatment (0.125 mg/mL post-treatment). *P < 0.0022 DOLE treatment vs. adrenaline, by Mann Whitney test. Data represents median comets scores from 6 subjects.
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oxidation and cyclisation of adrenaline to semiquinone noradrenochrome and adrenochrome is accompanied by the creation of byproducts such as reactive oxygen species and H2O2 (Djelic´ and Anderson, 2003; Genova et al., 2006). Similar events were also observed in relation with other catecholamines, such as dopamine and noradrenaline (Genova et al., 2006; Moldeus et al., 1983). Free radical production during redox cycling of catecholamines can eventually lead to disruption of genetic stability. Our results support the previous findings, and show that adrenaline in high concentration is able to cause DNA strand breaks. As expected, adrenaline acted as a weaker oxidant than H2O2, which caused severe strand breakage, although both agents produced DNA damage above 50% in treated cells. Under two treatments applied in this study, pretreatment and post-treatment, examined extract may act via bio-antimutagenic or desmutagenic activities (Franke et al., 2005). Deleterious effects of two oxidants were attenuated by the administration of DOLE under both experimental conditions used. The observed positive effect of DOLE treatment on the levels of oxidative DNA damage could be explained by three possible mechanisms. First, in pretreatment protocol, cells preincubated with DOLE prior to exposure to oxidants showed to be more resistant to genotoxic effects than cells of the control samples exposed only to adrenaline and H2O2. Efficiency of pretreatment can be explained with the mechanisms that some authors reported earlier, of DOLE being able to increase the cells’ antioxidant capacity by stimulating the synthesis of antioxidant enzymes and help maintain their activity during oxidative stress, thus acting at a prevention level (Abo Ghanema and Sadek, 2012). Study of El-Damrawy (2011) showed that olive leaf extract supplementation to aged males rabbits, significantly increased activity of antioxidant defence enzymes, glutathione s-transferase (GST) and superoxide dismutase (SOD) in blood plasma. In support of this view, several other studies found that polyphenolic substances, like oleuropein, the main component of DOLE, increased the gene expression of antioxidant enzymes SOD and catalase (CAT) at the transcriptional level (Masella et al., 2004). Second, the antioxidant effect of DOLE in post-treatment may be expressed directly, by acting at intervention level as free radical scavenger. Scavenging activity occurs by providing hydroxyl group, quenching free radicals and thus directly neutralizing their effect and preventing their further generation. The scavenging ability of DOLE can be attributed to its phenolic constituents (BenaventeGarcia et al., 2000; Lee and Lee, 2010). Previous phytochemical analysis of DOLE showed high oleuropein content (almost 20%), flavonoids, including luteoline-7-O-glycoside, apigenine-7-O-glycoside and quercetin, and caffeic acid (Dekanski et al., 2009a). It is important to mention that the study of Lee and Lee (2010) found that combined phenolics in Olea europaea leaf extract showed greater antioxidant potential than individual components of the extract, indicating the significance of coactions of all ingredients. The third possible mechanism by which DOLE could exert its action in post-treatment could be by stimulating the mechanisms of DNA repair, and thus lowering the level of DNA damage. This mechanism of action probably played less significant role in attenuated oxidative stress response because of the time dynamics in our study design. Namely, Chiaramonte et al. (2001) showed that significant DNA damage repair occurred within 1 h after the exposure to oxidative agent. Since leukocytes in our study were exposed to antioxidant up to 30 min after administration of oxidants, the activation of DNA repair could only partially contribute to the overall protective potential of DOLE. However, it should be noted that DOLE in post-treatment displayed higher efficiency in reducing DNA damage then in pretreatment, probably because of synergistic action of all three mechanisms, ROS scavenging as well as antioxidant enzyme activation and stimulation of DNA repair.
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Based on our results, it can be said that both interactions (preand post-treatments) of the plant extract were effective in modulating the genotoxicity of adrenaline and H2O2. The protective effect of DOLE is probably a result of synergistic activation of several molecular mechanisms such as ROS scavenging and increasing the antioxidant capacity of cells. Despite the obvious beneficial effects of DOLE, we also observed a relationship between slightly elevated comet percentage and the highest concentration of DOLE, in comparison with two lower concentrations. This indicates that the smaller concentrations of extract used in our study actually displayed stronger antioxidant activity. However, it should be mentioned that we previously evaluated genotoxic properties of the extract itself in an independent experiment, and it was shown that DOLE per se, in the absence of adrenaline or hydrogen peroxide did not cause formation of DNA strand breaks at any of the concentrations used in this study (data not shown in Results). Reviewing the literature about the mutagenic and antimutagenic effects of DOLE, we found that phenolics from olive products in some previous investigations showed that they can themselves be genotoxic and cytotoxic when applied in very high doses and can act in a prooxidant or in antioxidant way, dependant of the concentration and duration of exposure (Arantes-Rodrigues et al., 2011; Nousis et al., 2005; Prochazkova et al., 2011). Still, all three tested concentrations of DOLE used in our experiment themselves showed no genotoxicity to the cells and were efficient in attenuation of adrenaline and H2O2 induced DNA damage. Therefore, based on our results we can confirm that DOLE possesses DNA protective ability and antioxidant potential. 5. Conclusion Present findings demonstrate genoprotective and antioxidant properties of dry olive leaf extract. This data also contributes to a better understanding of genotoxic effects of adrenaline, strongly supporting the role of hormones in the process of mutagenesis induced by oxidative stress. Conflict of interest We declare that we have no financial or non-financial competing interests. Acknowledgment This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant OI 173034). Appendix A. Supplementary material Transparency Documents associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.tiv.2013.12.014. References Abo Ghanema, I.I., Sadek, K.M., 2012. Olive leaves extract restored the antioxidant perturbations in red blood cells hemolysate in streptozotocin induced diabetic rats. WASET 64, 159–165. Anderson, D., Yu, T.W., Phillips, B.J., Schmezer, P., 1994. The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the comet assay. Mutat. Res. 307, 261–271. Anter, J., Fernandez-Bedmar, Z., Villatoro-Pulido, M., Demyda-Peyras, S., MorenoMillan, M., Alonso-Moraga, A., et al., 2011. A pilot study on the DNA-protective, cytotoxic, and apoptosis-inducing properties of olive-leaf extracts. Mutat. Res. 723 (2), 165–170.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes Andrea Cˇabarkapa a,⇑, Lada Zˇivkovic´ a, Dijana Zˇukovec a, Ninoslav Djelic´ b, Vladan Bajic´ c, Dragana Dekanski c, Biljana Spremo-Potparevic´ a a
Department of Biology and Human Genetics, Institute of Physiology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia Department of Biology, Faculty of Veterinary Medicine, University of Belgrade, Oslobodjenja Blvd 18, Belgrade, Serbia c Institute for Research and Development, Galenika a.d., Pasterova 2, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Article history: Received 27 June 2013 Accepted 23 December 2013 Available online 3 January 2014 Keywords: Dry olive leaf extract (DOLE), Adrenaline (CAS 329-63-5) Oxidative stress Comet assay
a b s t r a c t Excessive release of stress hormone adrenaline is accompanied by generation of reactive oxygen species which may cause disruption of DNA integrity leading to cancer and age-related disorders. Phenolic-rich plant product dry olive leaf extract (DOLE) is known to modulate effects of various oxidants in human cells. The aim was to evaluate the effect of commercial DOLE against adrenaline induced DNA damage in human leukocytes by using comet assay. Peripheral blood leukocytes from 6 healthy subjects were treated in vitro with three final concentrations of DOLE (0.125, 0.5, and 1 mg/mL) for 30 min at 37 °C under two different protocols, pretreatment and post-treatment. Protective effect of DOLE was assessed from its ability to attenuate formation of DNA lesions induced by adrenaline. Compared to cells exposed only to adrenaline, DOLE displayed significant reduction (P < 0.001) of DNA damage at all three concentrations and under both experimental protocols. Pearson correlation analysis revealed a significant positive association between DOLE concentration and leukocytes DNA damage (P < 0.05). Antigenotoxic effect of the extract was more pronounced at smaller concentrations. Post-treatment with 0.125 mg/mL DOLE was the most effective against adrenaline genotoxicity. Results indicate genoprotective and antioxidant properties in dry olive leaf extract, strongly supporting further explorations of its underlying mechanisms of action. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Prolonged exposure to elevated oxygen levels, exceeding normal physiological concentrations, is well known cause of oxidative DNA damage in living cells and tissues (Halliwell and Gutteridge, 1984). Oxidizing agents affect normal functioning of cells and induce structural alterations and DNA mutations that can cause or lead to cancer and age-related disorders (Khansari et al., 2009). Oxidative stress can originate from endogenous sources caused by reactive oxygen species (ROS) overproduction in cells, or from exogenous cellular substances like various environmental
Abbreviations: ROS, reactive oxygen species; DOLE, dry olive leaf extract.
⇑ Corresponding author. Address: Institute of Physiology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia. Tel.: +381 11 3951349; fax: +381 11 3974349. E-mail addresses: [email protected], [email protected] ˇ abarkapa), [email protected] (L. Zˇivkovic´), [email protected] (A. C (D. Zˇukovec), [email protected] (N. Djelic´), [email protected] (V. Bajic´), ddekan@ sezampro.rs (D. Dekanski), [email protected] (B. Spremo-Potparevic´). 0887-2333/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.12.014
genotoxic compounds (Khansari et al., 2009). Hydrogen peroxide (H2O2) is one of the well known chemicals able to produce ROS via Fenton’s reaction and cause oxidative damage to DNA and genomic instability (Anderson et al., 1994). On the other hand, pro-oxidant properties of adrenaline and catechol derivates and their effects on DNA integrity are far less examined although recent empirical evidence confirmed the ability of adrenaline to induce DNA alterations (Djelic´ et al., 2003; Djelic´ and Anderson, 2003; Dobrzynska et al., 2004). Studies have shown that DNA damage is induced during the oxidation and cyclisation of catechol derivatives, such as dopa and adrenaline in copper or iron-catalyzed Fenton reaction resulting in ROS generation (Bindoli et al., 1990; Cavalieri and Rogan, 2004; Genova et al., 2006; Miura et al., 2000). Production of free radicals during redox cycling of catecholamines is considered a key step in their genotoxic action. Since ROS generation underlies main path for inducing DNA damage in both H2O2 and adrenaline mechanism of action, we searched for compounds able to scavenge free radicals and protect the cell DNA from damage when exposed to these oxidants. Dry olive leaf extract
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(DOLE) from the Mediterranean tree Olea europaea has been reported to possess antioxidant, antimicrobial and antifungal activity which may relate to its therapeutic properties in prevention of hypertension, cancer and diabetes mellitus (Dekanski et al., 2009a,b; Fares et al., 2011; Miljkovic´ et al., 2009; Mijatovic´ et al., 2011). Phenolics and flavonoids, the main components of DOLE are widely recognized for their antioxidant activity and free radical scavenging capacity (Anter et al., 2011; Benavente-Garcia et al., 2000; Cumaoglu et al., 2011; Le Tutour and Guedon, 1992; Nousis et al., 2005). Although extensively studied for its protective action against different diseases, DOLE has been insufficiently examined for its ability to interact, either directly or indirectly, with different mutagens, and until now DOLE has not been studied in relation to oxidative DNA damage induced by hormonal stress. The main goal of this research was to evaluate the protective effect of commercial dry olive leaf extract against adrenaline and H2O2 induced oxidative DNA damage in human peripheral blood leukocytes by using the ‘‘single cell gel electrophoresis’’ (comet assay). The comet assay has been shown to be an effective, sensitive and rapid in vitro method for examining DNA damage and issues related to oxidative stress in human peripheral blood cells (Anderson et al., 1994). 2. Materials and methods 2.1. Olive leaf extract Olive leaf extract EFLAÒ 943, standardized to 18–26% of oleuropein, was purchased from Frutarom Switzerland Ltd. (Wadenswil, Switzerland). The extract was manufactured from the dried leaves of Olea europaea L., applying an ethanol (80% m/m) extraction procedure. After a filtration process, the crude extract was dried. Its total phenols content, determined by Folin–Ciocalteau assay, was 197.8 lg GAE/g of dry extract; total flavonoids and tannins content was 0.29% and 0.52%, respectively. High performance liquid chromatography (HPLC) analysis revealed a complex mixture of phenolic compounds: oleuropein (19.8%), luteolin-7-O-glucoside (0.04%), apigenine-7-O-glucoside (0.07%), quercetin (0.04%) and 0.02% of caffeic acid (Dekanski et al., 2009a). In this study, the same batch of EFLAÒ 943 was used. It was kept in sealed, previously sterilized microtubes, stored at room temperature and protected from light until use. 2.2. Subjects Peripheral blood samples were collected in heparinized containers from six healthy volunteers (4 female and 2 male subjects) aged between 19 and 35 years. All subjects were non-smokers, who abstained from alcohol, not receiving any therapy or medications, nor taking dietary supplements. 2.3. Study design For the experiment, DOLE powder was diluted in phosphatebuffered saline (PBS, Torlak Institute of Immunology and Virology, Belgrade, Serbia) to three final concentrations: 1 mg/mL, 0.5 mg/ mL and 0.125 mg/mL. We chose final concentrations of the extract according to the range of concentrations previously found to be safe and effective to use in in vitro studies (Turkez and Togar, 2011; Lee-Huang et al., 2003). Adrenaline (CAS No. 329-63-5, epinephrine injection 1 mg/mL solution, Jugoremedija A.D., Zrenjanin, Serbia) and H2O2 (CAS No. 7722-84-1, ZORKA Pharma, Sabac, Serbia) were previously examined at four doses to determine the appropriate limits of toxicity for the assay (5, 10, 50, 150 lM adrenaline and 5, 10, 25, 50 lM H2O2). According to literature data
5 lM adrenaline represents the maximal therapeutic dose for human therapy. Also, all tested concentrations correspond to those previously used in genetic toxicology studies of adrenaline (Djelic´ et al., 2003; Dobrzynska et al., 2004). We chose 10 lM adrenaline and 25 lM H2O2 for further experiments, since these were the smallest concentrations that produced consistently high level of DNA fragmentation in exposed cells as compared to the untreated controls. To determine the effect of antioxidant-rich environment in vitro on human peripheral blood leukocytes exposed to oxidative stress induced by adrenaline and H2O2, the DOLE extract was subjected to two types of interactions with the oxidants, pretreatment and post-treatment. Two independent experiments were performed: (1) In the first series (pretreatment protocol) three different concentrations of antioxidant DOLE (1, 0.5 and 0.125 mg/mL) were administered and incubated with the whole blood cell preparations on slides at 37 °C for 30 min, before washing the cells with PBS and adding two oxidizing agents separately (25 lM H2O2 for 15 min on ice, and 10 lM adrenaline for 30 min on 37 °C). (2) The second series of treatments (post-treatment protocol) was performed simultaneously on other series of cell preparations from the same subjects, first exposed to two oxidizing agents individually, then rinsed with PBS and subsequently post-treated with three mentioned concentrations of DOLE under the same conditions as in first group. Experiments were repeated six times, each carried out in duplicate. All cell preparations were incubated together with negative controls (treated only with PBS) as well as with positive controls (H2O2 and adrenaline). 2.4. The single cell gel electrophoresis assay Before processing for comet assay, cell viability for all whole blood samples were determined by using the trypan blue exclusion method (Anderson et al., 1994). Cell viability was always found to be above 90%. The comet assay was performed essentially as described by Singh et al. (1988). Briefly, the 6 ll of whole blood samples were suspended in 0.67% low-melting-point (LMP) agarose (Sigma–Aldrich, St. Louis, MO) and pipetted onto superfrosted glass microscope slides precoated with a layer of 1% of normal-meltingpoint agarose (Sigma–Aldrich, St. Louis, MO), spread using a coverslip, and maintained for 5 min on 4 °C to solidify. After gently removing the coverslips, the cell suspensions on slides were treated with DOLE and oxidants as described above in two mentioned types of interactions, pre-treatment and post-treatment. Following the treatments, all slides were covered with the third layer of 0.5% LMP agarose and again allowed to solidify on ice for 5 min. After coverslips removal, the slides were placed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X100 and 10% dimethylsulfoxide, pH 10 adjusted with NaOH) at 4 °C overnight and afterwards subjected to electrophoresis and staining with ethidium bromide (20 lg/L), performed as described by Singh et al. (1988). The comets were analyzed 15 min after staining at 100 magnification on Olympus BX 50 microscope (Olympus Optical Co., GmbH, Hamburg, Germany), equipped with a mercury lamp HBO (50 W, 516–560 nm, Zeiss). Evaluation of DNA damage was done according to Anderson et al. (1994). DNA damage in the cells was assessed by quantification of the amount of DNA released from the core of the nucleus and comets were visually scored and classified into five categories corresponding to the extent of DNA migration: (A) no damage, 95% (Fig. 1). Analysis was performed on 100 cells that were randomly selected per subject (50 cells from each of 2 replicate slides) and were always carried out by the same experienced person. DNA damage was characterized as DNA migration over 5% (B + C + D + E comet classes), and median value
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Fig. 1. Representing five classes of Comets: (A) no damage, 95%.
was calculated for all six subjects. Leukocytes undergoing apoptosis or necrosis and ‘‘hedgehog’’ cells were excluded from the analysis of the comet assay slides, and were distinguished from normal cells following the instructions given by Singh (2005). 2.5. Statistical analysis Statistical analysis was performed by using Kruskal–Wallis and Mann–Whitney U test, for comparisons of different treatments vs. the respective controls. Values are expressed as median and 25th and 75th percentile, for n = 6. Pearson’s correlation and linear regression were used to evaluate the relationship between DNA damage and DOLE concentration. All data were analyzed with The GraphPad Prism 5.0 software. A difference at P < 0.05 was considered statistically significant. 3. Results Protective ability of DOLE extract was tested using two different treatments (pretreatment and post-treatment) and the comet scores were compared to the scores obtained for the DNA damaging agents adrenaline and H2O2. The protective effect of the tested extract DOLE was evaluated for its ability to prevent hydrogen peroxide and adrenaline-induced formation of strand breaks in the nuclear DNA, represented as the median number of all cells with migrated DNA in Tables 1 and 2. The effect of pretreatment with DOLE on the level of DNA damage when cells are incubated with DOLE before the individual
exposure to two oxidants, are shown in Table 1. The score for cells from post-treatment protocol, first exposed to oxidizing agents separately and afterwards post-treated with antioxidant DOLE under same conditions as above mentioned is represented in Table 2. While an increase of DNA damage was detected in positive control cells exposed only to either 25 lM H2O2 or 10 lM adrenaline, the levels of DNA damage in leukocytes analyzed from the samples treated with different concentrations of DOLE were significantly reduced in both pretreated and post-treated samples (P < 0.001) (Tables 1 and 2). All three concentrations of DOLE strongly prevented DNA damage under both experimental conditions, seen as reduction in the number of cells with damaged DNA. As shown in Table 1, in samples pretreated with DOLE, we observed a positive association between the level of DNA damage and concentration of DOLE (0.125 mg/mL, 0.5 mg/mL, 1 mg/mL), in both adrenaline and H2O2 exposed cells (r = 0.41, P < 0.0214; r = 0.3960, P < 0.0204, respectively). Concentration–response indicated that with decline of DOLE concentration, DNA damage was less profound. Although all three concentrations of extract effectively reduced induction of DNA fragmentation by adrenaline and H2O2, treatment with smaller concentrations of extract corresponded to less DNA damage. Surprisingly, the greater level of DNA damage was observed in leukocytes pretreated with 1 mg/ mL concentration as compared to two smaller concentrations of the extract, thus the highest concentration of DOLE actually displayed the lowest protective effect and the highest variability of the results. Two smaller concentrations of DOLE (0.5 and 0.125 mg/mL) displayed approximate results in attenuation of both
Table 1 Pretreatment protocol: Number of cells with damaged DNA from six different subjects, first pretreated with 3 concentration of DOLE and subsequently exposed to two oxidants (adrenaline and H2O2 separately). Oxidants
Adrenaline 10 lM H2O2 25 lM
Positive controls
54.00 (39.50–75.75) 86.00 (71.25–96.00)
DOLE concentrations
P value
1 mg/mL
0.5 mg/mL
0.125 mg/mL
10.00* (4.50–16.75) 15.00* (8.50–28.50)
9.50* (5.25–15.25) 11.50* (5.25–13.25)
10.00* (4.75–15.00) 8.00* (5.50–12.75)
Values are expressed as median and 25th and 75th percentile for comet scores in 100 cells from 6 subjects. P < 0.05, DOLE treatment vs. oxidant; analyzed by Kruskal–Wallis test.
*
0.0045 0.0020
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Table 2 Post-treatment protocol: number of cells with damaged DNA from six different subjects, first treated with two oxidants (adrenaline and H2O2 separately) and subsequently incubated with 3 concentration of DOLE. Oxidants
Adrenaline 10 lM H2O2 25 lM
Positive controls
DOLE concentrations
54.00 (39.50–75.75) 86.00 (71.25–96.00)
P value
1 mg/mL
0.5 mg/mL
0.125 mg/mL
12.50* (4.75–16.50) 15.50* (10.25–27.25)
9.50* (5.50–11.25) 4.50* (1.50–15.00)
3.50* (1.75–6.50) 11.00* (5.50–26.25)
0.0010 0.0018
Values are expressed as median and 25th and 75th percentile for comet scores in 100 cells from 6 subjects. P < 0.05, DOLE treatment vs oxidant; analyzed by Kruskal–Wallis test.
*
80
Adrenaline 10 µM DOLE 0.125 mg/mL post-treated
60
40
20
* 0 B+C (medium damage)
4. Discussion This study demonstrates the potential of commercial dry olive leaf extract to attenuate oxidative DNA damage in human leukocytes, induced by adrenaline and hydrogen peroxide under two different experimental conditions, pretreatment and post-treatment. Our results show that all concentrations of DOLE evaluated in our study displayed DNA protective effect against adrenaline and H2O2 genotoxicity. Dry olive leaf extract so far exhibited beneficial properties in many experimental models of diseases stemming from oxidative stress, and its antioxidant properties are well documented (Anter et al., 2011; Benavente-Garcia et al., 2000; Cumaoglu et al., 2011; Le Tutour and Guedon, 1992; Nousis et al., 2005). Although the broad field of biomedical research has been exploring various protective effects of DOLE, according to our knowledge of literature, this is the first work that shows the action of DOLE in relation to a stress hormone-induced oxidative DNA damage. Since oxidative stress is being recognized as an important factor in the etiology of many chronic diseases, the examination of dietary components with antioxidant activity has received much attention (Arts and Hollman, 2005). On the other hand, the role of hormones in oxidative stress is far less examined. One of the aims of this study was to point out the fact that nonsteroidal hormones, such as adrenaline, with phenolic or catechol moieties are able to cause oxidative DNA damage (Djelic´ and Anderson, 2003; Flint et al., 2007; Miura et al., 2000). The release of catecholamines in amounts exceeding physiological concentrations has been observed to exert cytotoxic effects in neuroblasts, melanoma and myocardial cells (Behonick et al., 2001; Flint et al., 2007; Miura et al., 2000; Okamoto et al., 1996). Ambiguous assumptions that hormones can yield genetic instability are supported by novel studies that explain how biochemical events underlying hormone action might eventually lead to genetic changes (Cavalieri and Rogan, 2004; Djelic´ and Anderson, 2003; Liehr, 2001). Catabolic
Number of cells with migrated DNA
Number of cells with migrated DNA
adrenaline and H2O2 induced strand breaks, while the 1 mg/mL concentration showed to be more effective in reducing damage caused by adrenaline then H2O2. Results of the post-treated samples displayed in Table 2 follow similar concentration-dependent trend that we observed in pretreatment samples, in relation to adrenaline. The number of cells with damaged DNA was positively associated with rising concentrations of DOLE (r = 0.4602, P < 0.0047). On the other hand, in H2O2 treated samples, the correlation between level of DNA damage and DOLE concentrations was lost. The two higher concentrations of DOLE responded to previously recorded trend of efficacy, while the smallest 0.125 mg/mL concentration showed discordance with the previously mentioned results, and was much less effective against H2O2-induced oxidative damage in post-treated than pretreated samples (Table 1 vs. Table 2). Comparing the antioxidant ability of all concentrations of DOLE in both experimental conditions, it appeared that its application in post-treatment was more effective in reducing DNA damage than in pretreatment. The results also demonstrate that post-treatment with 0.125 mg/mL of DOLE was the most effective in prevention of adrenaline induced genotoxicity, while the post-treatment with 0.5 mg/mL of DOLE was most efficient in attenuating H2O2-induced DNA damage. Fig. 2 represents the antioxidant response of the most effective DOLE treatment (0.125 mg/mL post-treatment) in comparison with the genotoxic effect of adrenaline, expressed with two DNA damage distribution variables: the median number of cells with low and medium damage (B + C comet category), and the median number of cells with high and total damaged DNA (D + E comets) from all six subjects. Degree of DNA damage presented with two variables in Fig. 2 indicate that the 0.125 mg/mL DOLE post-treatment was equally effective in attenuation of low and medium oxidative DNA damage as well as in the reduction of highly damaged DNA.
20
Adrenaline 10 µM DOLE 0.125 mg/mL post-treated
15
10
5
* 0 D+E (high damage)
Fig. 2. Relationship between antioxidant activities of dole and extent of DNA damage: Degree of DNA damage in human leukocytes exposed to adrenaline compared to antioxidant response of most effective DOLE treatment (0.125 mg/mL post-treatment). *P < 0.0022 DOLE treatment vs. adrenaline, by Mann Whitney test. Data represents median comets scores from 6 subjects.
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oxidation and cyclisation of adrenaline to semiquinone noradrenochrome and adrenochrome is accompanied by the creation of byproducts such as reactive oxygen species and H2O2 (Djelic´ and Anderson, 2003; Genova et al., 2006). Similar events were also observed in relation with other catecholamines, such as dopamine and noradrenaline (Genova et al., 2006; Moldeus et al., 1983). Free radical production during redox cycling of catecholamines can eventually lead to disruption of genetic stability. Our results support the previous findings, and show that adrenaline in high concentration is able to cause DNA strand breaks. As expected, adrenaline acted as a weaker oxidant than H2O2, which caused severe strand breakage, although both agents produced DNA damage above 50% in treated cells. Under two treatments applied in this study, pretreatment and post-treatment, examined extract may act via bio-antimutagenic or desmutagenic activities (Franke et al., 2005). Deleterious effects of two oxidants were attenuated by the administration of DOLE under both experimental conditions used. The observed positive effect of DOLE treatment on the levels of oxidative DNA damage could be explained by three possible mechanisms. First, in pretreatment protocol, cells preincubated with DOLE prior to exposure to oxidants showed to be more resistant to genotoxic effects than cells of the control samples exposed only to adrenaline and H2O2. Efficiency of pretreatment can be explained with the mechanisms that some authors reported earlier, of DOLE being able to increase the cells’ antioxidant capacity by stimulating the synthesis of antioxidant enzymes and help maintain their activity during oxidative stress, thus acting at a prevention level (Abo Ghanema and Sadek, 2012). Study of El-Damrawy (2011) showed that olive leaf extract supplementation to aged males rabbits, significantly increased activity of antioxidant defence enzymes, glutathione s-transferase (GST) and superoxide dismutase (SOD) in blood plasma. In support of this view, several other studies found that polyphenolic substances, like oleuropein, the main component of DOLE, increased the gene expression of antioxidant enzymes SOD and catalase (CAT) at the transcriptional level (Masella et al., 2004). Second, the antioxidant effect of DOLE in post-treatment may be expressed directly, by acting at intervention level as free radical scavenger. Scavenging activity occurs by providing hydroxyl group, quenching free radicals and thus directly neutralizing their effect and preventing their further generation. The scavenging ability of DOLE can be attributed to its phenolic constituents (BenaventeGarcia et al., 2000; Lee and Lee, 2010). Previous phytochemical analysis of DOLE showed high oleuropein content (almost 20%), flavonoids, including luteoline-7-O-glycoside, apigenine-7-O-glycoside and quercetin, and caffeic acid (Dekanski et al., 2009a). It is important to mention that the study of Lee and Lee (2010) found that combined phenolics in Olea europaea leaf extract showed greater antioxidant potential than individual components of the extract, indicating the significance of coactions of all ingredients. The third possible mechanism by which DOLE could exert its action in post-treatment could be by stimulating the mechanisms of DNA repair, and thus lowering the level of DNA damage. This mechanism of action probably played less significant role in attenuated oxidative stress response because of the time dynamics in our study design. Namely, Chiaramonte et al. (2001) showed that significant DNA damage repair occurred within 1 h after the exposure to oxidative agent. Since leukocytes in our study were exposed to antioxidant up to 30 min after administration of oxidants, the activation of DNA repair could only partially contribute to the overall protective potential of DOLE. However, it should be noted that DOLE in post-treatment displayed higher efficiency in reducing DNA damage then in pretreatment, probably because of synergistic action of all three mechanisms, ROS scavenging as well as antioxidant enzyme activation and stimulation of DNA repair.
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Based on our results, it can be said that both interactions (preand post-treatments) of the plant extract were effective in modulating the genotoxicity of adrenaline and H2O2. The protective effect of DOLE is probably a result of synergistic activation of several molecular mechanisms such as ROS scavenging and increasing the antioxidant capacity of cells. Despite the obvious beneficial effects of DOLE, we also observed a relationship between slightly elevated comet percentage and the highest concentration of DOLE, in comparison with two lower concentrations. This indicates that the smaller concentrations of extract used in our study actually displayed stronger antioxidant activity. However, it should be mentioned that we previously evaluated genotoxic properties of the extract itself in an independent experiment, and it was shown that DOLE per se, in the absence of adrenaline or hydrogen peroxide did not cause formation of DNA strand breaks at any of the concentrations used in this study (data not shown in Results). Reviewing the literature about the mutagenic and antimutagenic effects of DOLE, we found that phenolics from olive products in some previous investigations showed that they can themselves be genotoxic and cytotoxic when applied in very high doses and can act in a prooxidant or in antioxidant way, dependant of the concentration and duration of exposure (Arantes-Rodrigues et al., 2011; Nousis et al., 2005; Prochazkova et al., 2011). Still, all three tested concentrations of DOLE used in our experiment themselves showed no genotoxicity to the cells and were efficient in attenuation of adrenaline and H2O2 induced DNA damage. Therefore, based on our results we can confirm that DOLE possesses DNA protective ability and antioxidant potential. 5. Conclusion Present findings demonstrate genoprotective and antioxidant properties of dry olive leaf extract. This data also contributes to a better understanding of genotoxic effects of adrenaline, strongly supporting the role of hormones in the process of mutagenesis induced by oxidative stress. Conflict of interest We declare that we have no financial or non-financial competing interests. Acknowledgment This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant OI 173034). Appendix A. Supplementary material Transparency Documents associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.tiv.2013.12.014. References Abo Ghanema, I.I., Sadek, K.M., 2012. Olive leaves extract restored the antioxidant perturbations in red blood cells hemolysate in streptozotocin induced diabetic rats. WASET 64, 159–165. Anderson, D., Yu, T.W., Phillips, B.J., Schmezer, P., 1994. The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the comet assay. Mutat. Res. 307, 261–271. Anter, J., Fernandez-Bedmar, Z., Villatoro-Pulido, M., Demyda-Peyras, S., MorenoMillan, M., Alonso-Moraga, A., et al., 2011. A pilot study on the DNA-protective, cytotoxic, and apoptosis-inducing properties of olive-leaf extracts. Mutat. Res. 723 (2), 165–170.
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