In vitro genotoxic and antigenotoxic effects of cynarin Esra Erikel, Deniz Yuzbasioglu, Fatma Unal
Reference: JEP 11809
To appear in: Journal of Ethnopharmacology
Received Date: 29 November 2018 Revised Date: 12 March 2019 Accepted Date: 13 March 2019
Please cite this article as: Erikel, E., Yuzbasioglu, D., Unal, F., In vitro genotoxic and antigenotoxic effects of cynarin, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.03.036.
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Chromosome aberrations (CAs) test
Sister Chromatid Exchanges (SCEs) test
Micronucleus (MN) test
•Cynarin (12-194 µM) alone and simultaneously with MMC or H2O2 (only for comet assay)
An artichoke phytochemical
ti Not genotoxic effect
ti Strong anti-genotoxic effect
•Reduced DNA damage caused by H2O2,
•Decreased frequency of CAs, SCEs and MN induced by MMC at some concentration
In Vitro Genotoxic and Antigenotoxic Effects of Cynarin
Esra Erikel, Deniz Yuzbasioglu*, Fatma Unal
Genetic Toxicology Laboratory, Department of Biology, Science Faculty, Gazi University,
06500, Teknikokullar, Ankara, Turkey
CAs, chromosome aberrations; SCEs, sister chromatid exchanges; MN, micronucleus; SCGE, single cell gel electrophoresis; MI, mitotic index; RI, replication index; NDI, nuclear division index; BN, binucleate; MMC, mitomycin-C; H2O2, Hydrogen peroxide.
Prof. Dr. Deniz YUZBASIOGLU Tel. +90 312 202 1205
Fax. +90 312 212 2279
E-mail address: [email protected]
Ethnopharmacological relevance: Cynarin is an artichoke phytochemical that possesses a variety of pharmalogical features including free-radical scavenging and antioxidant activity. The origin of artichoke species appears to be Mediterranean region. Two of these species, globe artichoke (Cynara cardunculus var. scolymus L.) and cardoon (Cynara cardunculus var. altilis DC), are widely cultivated and consumed. This vegetable, as the basis of the mediterranean diet, has been used as herbal medicine for its therapeutic effects since ancient times. Therefore, this study was performed to determine genotoxic and antigenotoxic effects of cynarin against MMC (mitomycin C) and H2O2 (hydrogen peroxide) induced genomic instability using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN), and comet assays in human lymphocytes.
Materials and methods: Lymphocytes obtained from two healthy volunteers (1 male and 1 female) were exposed to different concentrations of cynarin (12-194 µM) alone and the combination of cynarin and MMC (0.60 µM) or cynarin and H2O2 (100 µM, only for comet assay).
Results: Cynarin alone did not induce significant genotoxic effect in the CA, SCE (except 194 µM), MN, and comet assays. The combination of some concentrations of cynarin and MMC decreased the frequency of CAs, SCEs and MN induced by MMC. Furthermore, the combination of cynarin and H2O2 reduced all comet parameters at all the concentrations compared to H2O2 alone. While the highest concentrations of cynarin significantly decreased mitotic index (MI), the combination of cynarin and MMC increased the reduction of MI induced by MMC alone.
Conclusion: All the results obtained in this study demonstrated that cynarin exhibited antigenotoxic effects rather than genotoxic effects. It is believed that cynarin can act as a potential chemo-preventive against genotoxic agents.
Key Words: Cynarin, Phytochemical, Genotoxicity, Antigenotoxicity, Human Lymphocytes
Recently, the use of phytochemicals derived from nutritional plants has gained interest as chemo-preventive agent because these agents are known to prevent or reverse the multi-step processes of carcinogenesis (Vlaykova et al., 2013; Srivastava et al., 2016). Artichoke species belonging to Cynara that is relatively a small genus and originating from the Mediterranean region are cultivated for their heads and leaves. Two of these crops are globe artichoke (Cynara cardunculus var. scolymus L.) and cardoon (Cynara cardunculus var. altilis DC). Traditionally they are used as food (vegetable or tea) and medicine (Foti et al., 1999; Portis et al., 2005; Ierna and Mauromicale, 2010; Christaki et al., 2012). They have been generally used for treatment of hepatic insufficiency as a choleretic, diuretic and hypocholesterolemic activities since ancient times (Wang et al., 2003; Speroni et al., 2003; Da Silva et al., 2017; Guven et al., 2018). Numerous studies on artichokes showed that these species have health- protective effect as hepatoprotection, hypocholesterolemic, anti-oxidative, anti-inflammatory, anti-microbial, anti-HIV, and anti-carcinogenic activities (Adzet et al., 1987; Clifford, 2000; Lupattelli et al., 2004; Zhu et al., 2004; Lattanzio et al., 2009; Salama and El Baz, 2013, Martínez-Esplá et al., 2017; Souza et al., 2018). In addition, artichoke extracts exhibited antigenotoxic effect in different test sytems (Table 1). These properties of artichoke depend on its phytochemical profile including flavones (mainly apigenin and luteolin), glycosides, and hydroxycinnamic derivates as mono- and di-caffeoylquinic acid (e.g., cynarin and chlorogenic acid) (Lattanzio and Van Sumere, 1987; Miadokova at el., 2008; Martínez-Esplá et al., 2017; Rezazadeh et al., 2018).
Cynarin is a phenolic compound that is derivative of di-caffeoylquinic acid in artichoke heads and leaves (Jiménez-Escrig et al., 2003; Wang et al., 2003; Mulinacci et al., 2004) and possesses strong antioxidant activity (Adzet et al., 1987; Wang et al., 2003; Jun et al., 2007). Pharmacological studies demonstrated that extracts of artichoke (Cynara scolymus) and its main active component cynarin have choleretic and hypocholesterolemic efficiency (Alonso et al., 2006; Souza et al., 2018). Moreover, cynarin and caffeic acid in artichoke has hepatoprotective activity against carbon tetrachloride (CCl4) toxicity in isolated rat hepatocytes (Adzet et al., 1987; Lattanzio et al., 2009). To the best of our knowledge, there is no genotoxicity or antigenotoxicity study on cynarin.
The aim of the present study was to investigate the potential genotoxic and also antigenotoxic effects of cynarin (1.5-dicaffeoylquinic acid) using four different genotoxicity assays as chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN), and comet assay (single cell gel electrophoresis-SCGE) in human lymphocytes in vitro. These assays (except SCE) are OECD (The Organisation for Economic Co-operation and Development) guidelines that are frequently used as sensitive assays to evaluate potential mutagenic, clastogenic and carcinogenic effects of physical and chemical agents (Yuzbasıoglu et al., 2008; Yilmaz et al., 2009; Siddique et al., 2010; Jacociunas et al., 2012; Unal et al., 2013; Ataseven et al., 2016; Avuloglu-Yilmaz et al., 2017a; Yuzbasioglu et al., 2018). Despite the in vitro sister chromatid exchange test in mammalian cells was excluded from the OECD test guidelines on 2nd April 2014, it is still being used in different genetic and genotoxic evaluations (Engen et al., 2015; Medves et al., 2016; Lima et al., 2016; Unal et al., 2016; Avuloglu-Yilmaz et al., 2017a).
In vitro genotoxicity assays as biological marker are used to determine mutagenic and carcinogenic effects of physical and chemical agents in cells or organisms (Phillips and Arlt, 2009). Similarly, these tests can also be used to identify anti-genotoxic, anti-mutagenic and/or anti-carcinogenic effects of plant extracts and phytochemicals against some chemicals (Siddique et al., 2010; Unal et al., 2013; Słoczyńska et al., 2014; Sponchiado etal., 2016; Lemes et al., 2017; Makhuvele et al., 2018). Mutations that identified as damage in genetic material have been considered as a cancer-initiating mechanism. Particularly, there is a significant correlation between the increased frequency of CA and MN in human peripheral lymphocytes and the incidence of cancer (Bonassi et al., 2008; Ginzkey et al., 2014; Huerta et al., 2014; Minina et al., 2018; Russo and Degrassi, 2018). There is no association between increased SCE frequency and cancer formation. However, this increase has been considered as a biological indicator of exposure to genotoxic agents and appears to indicate DNA damaging effects and/or subsequent repair by homologous recombination (Norppa et al., 2006; Sebastià et al., 2014; Mourelatos, 2016). Additionally, many studies investigating specific antioxidant compounds have approved that the reduction observed in the frequency of CA, SCE, MN and other nuclear abnormalities is indicative of the antigenotoxic activity of the compounds (Stanimirovic et al., 2005; Siddique et al., 2008; Unal et al., 2013; Słoczyńska et al., 2014; Izquierdo‐Vega et al., 2017). The comet assay is a relatively sensitive, simple, and reliable method for measuring DNA damage and repair (Singh et al., 1988; Bausinger and Speit, 2016; Møller, 2018). Furthermore, it has been prevalently applied to evaluate protective
effects of dietary antioxidants against certain genotoxic carcinogens and oxidative agents (Møller and Loft, 2006; Collins, 2014; Cheng et al., 2017). In this paper, we investigated whether cynarin is genotoxic or not and has the capacity to protect DNA damage induced by mitomycin C (MMC) and hydrogen peroxide (H2O2) in human lymphocytes.
2.Materials and Methods
Cytochalasin-B (CAS. No: 14930-96-2), bromodeoxyuridine (CAS. No: 59-14-3), mitomycin C (CAS. No: 50-07-7), NaCl (CAS. No: 7647-14-5), and colchicine (CAS. No: 64-86-8) were obtained from Sigma. DMSO (CAS. No: 67-68-5), EDTA (CAS. No: 6381-92-6), NaOH (CAS. No: 1310-73-2), tris (CAS. No: 77-86-1), triton X-100 (CAS. No: 9002-93-1), low melting agarose (CAS. No: 9012-36-6), normal melting agarose (CAS. No: 9012-36-6), EtBr (CAS. No: 1239-45-8), and H2O2 (CAS. No: 7722-84-1) were obtained from Applichem. PBS (CAS. No: L1825), Biocoll (CAS. No: L 6115), and Chromosome Medium B (CAS. No: F5023) were obtained from Biochrom AG. Test substance cynarin (≥98% – HPLC) was purchased from Sigma (CAS No: 30964-13-7) (product code: D8196, 1.5-dicaffeoylquinic acid) and dissolved in %50 methanol. In Fig. 1 (Sigma-Aldrich, 2014), the chemical structure, molecular formula, and molecular weight of cynarin are shown.
Molecular Formula: C25H24O12 Molecular Weight: 516.45 g/mol
Figure 1. Structure of cynarin (Sigma-Aldrich, 2014)
2.2.Cell cultures and concentration selection
The present research was carried out with the permission of ethical committee of the Faculty of Medicine, Gazi University (13.02.2013-29) and performed in accordance with Declaration of Helsinki. To investigate the genotoxic and antigenotoxic effects of cynarin, two protocols were performed; peripheral lymphocytes were treated with cynarin alone and simultaneously
treated with a mutagen or oxidant. Mitomycin-C (MMC), an antineoplastic agent, was used in CA, SCE and MN assays, and hydrogen peroxide (H2O2), an oxidant, was used in comet assay to induce chromosome and DNA damage. Peripheral blood samples were collected from two healthy (one female and one male, aged 20-25 years) non-smoker donors.
To determine the used concentrations of cynarin, preliminary studies were carried out in human lymphocytes in vitro in six different concentrations (6, 12, 24, 48, 97, and 194 µM). As a result, 12, 24, 48, 97, and 194 µM were found to be the best suitable concentrations depending on cell-proliferating activity and mitotic index (to analyse sufficient number of metaphases). Heparinized blood (0.2 mL) was added to 2.5 mL Chromosome Medium B (containing fetal bovine serum, heparin, antibiotics, and phytohemagglutinin) and incubated with six different concentrations (6, 12, 24, 48, 97, and 194 µM) of cynarin alone and the combination of cynarin and MMC (for 24h and 48h) at 37°C.
2.3.Chromosome Aberration and Sister Chromatid Exchange assays
Whole heparinized bloods were incubated in chromosome medium supplemented with bromodeoxyuridine (10 µg/mL), a thymine analogue, at 37ºC for 72 h. After the beginning of culture, at 24h and 48h, the cells were treated with five different concentrations of cynarin (12, 24, 48, 97, and 194 µM) alone and the combination of cynarin and MMC (0.60 µM). In addition, a negative (distilled water), a solvent (50% methanol stock solution, 0.36% in culture) and a positive control (MMC, 0.60 µM) were also used in all assays. CA and SCE assays were applied with some modifications of Evans (1984) and Perry and Thompson’s study (1984), respectively (Yuzbasioglu et al., 2006; Erikel et al., 2017).
To assess chromosomal aberrations, 100 well-spread metaphases were evaluated blindly per donor and treatment (totally 200 metaphases). The mean frequency of abnormal cells and the number of CAs per cell (CAs/cell) were determined. The mitotic index (MI) was detected by scoring 1000 cells per treatment and donor.
For the SCE test, the method of Speit and Houpter (1985) was followed with some minor modifications and the preparations were stained with Giemsa (Yuzbasioglu et al., 2006). For scoring SCEs, 25 cells (totally 50 cells) undergoing the second mitotic division and having well distributed chromosomes were examined blindly for each donor.
The replication index (RI) was determined by scoring a total of 200 cells (100 cells from each donor). RI was determined by the formula: RI= [(1×M1) + (2×M2) + (3×M3)]/N (Schneider and Lewis, 1981) where M1, M2, and M3 represent the number of cells undergoing first, second, and third mitosis, respectively, and N represents the total number of metaphases scored.
Micronucleus assay was carried out according to the procedures of Fenech (2000) and Palus et al. (2003). The human lymphocytes in cultures were treated with five different concentrations of cynarin (12, 24, 48, 97, and 194 µM) alone and also cynarin and Mitomycin-C (MMC, 0.60 µM) simultaneously, at 37°C for 72 h. A negative (sterile distilled water), a solvent (50% methanol stock solution, 0.36% in culture) and a positive control (MMC, 0.60 µM) were also maintained. To block cytokinesis, cytochalasin B (5.2 µg/mL) was added at the 44th hour of culture. Micronuclei were analysed blindly in 1000 binucleated cells per donor (totally 2000 binucleated cells). Additionally, nuclear division index (NDI) which indicates the average number of cell cycles was utilized to determine cell proliferation. 500 cells per donor (totally 1000 cells) were scored to determine the percentage of cells with 1-4 nuclei. NDI was calculated according to Surrales et al. (1995), as follows; [(1×N1) + (2×N2) +(3×(N3 + N4))]/N, where N1-N4 represent the number of cells with 1-4 nuclei, respectively, and N is the total number of cells scored.
2.3.3.Comet Assay (Alkaline Single Cell Gel Electrophoresis-SCGE)
Comet assay was performed according to the procedure of Singh et al. (1988) with some modifications. The lymphocytes were isolated using Biocoll separating solution from blood that was taken from healthy donors. The viability of cells was found to be >96% using Trypan Blue Exclusion Test. The isolated human lymphocytes were treated with five different concentrations of cynarin (12, 24, 48, 97, and 194 µM) alone and cynarin and H2O2 (100 µM) simultaneously, and incubated at 37°C for 1h. A negative (sterile distilled water), a solvent (50% methanol stock solution, 0.36% in culture) and a positive control (H2O2, 100 µM) were also run.
Following the time off incubation, the lymphocytes were centrifuged (at 1348g, 5 min). After removing supernatant, they were re-suspended in PBS and the cells were suspended in low- melting point agarose (0.65%). Afterwards, the suspension was fast layered onto slides pre-
coated with normal-melting point agarose (0.65%), and immediately covered with a cover slip. Preparations were incubated into the lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris pH = 10, in which 10% DMSO and 1% Triton X-100 were added) for 1 hour at 4°C. The slides were incubated in ice-cold (20 min) electrophoresis solution (0.3 M NaOH, 1 mM EDTA, pH > 13). After electrophoresis at 25 V, 300 mA for 20 min, the slides were rinsed in neutralization buffer (0.4 M Tris, pH = 7.5). At last, each slide was stained with 20 mg/mL of ethidium bromide.
The fluorescent microscope (Olympus BX51) having an excitation filter of 546 nm and a barrier filter of 590 nm was used at 400× magnification for image analysis and comet scoring. To determine the DNA damage,100 cells for each experiment were analysed with regards to comet parameters that are the tail length (µm), tail intensity (%) and tail moment (200 comets per concentration), using specialized Image Analysis System (Comet Assay IV, Perceptive Instruments Ltd., UK).
z test was applied for the percentage of abnormal cells, CA/cell, RI, MI, the frequency of MN, and NDI. T-test was applied for the SCEs and the comet assays. Regression analysis was performed in order to reveal dose-response relationships for the percentage of abnormal cells, CA/cell, SCE, MN, MI, and comet parameters.
3.1.Chromosomal aberrations assay
Cynarin alone did not significantly increase the frequency of CAs at all the concentrations compared to solvent controls at both 24 h and 48 h (Table 2). However, it induced four types of structural aberrations (chromatid and chromosome breaks, fragments and sister chromatid unions) in both treatment periods and, a numerical aberration (polyploidy) in only 48h treatment.
In lymphocytes treated with the combination of Cynarin+MMC at 24 h, the frequency of abnormal cells significantly reduced compared to positive control (MMC) at only the two highest concentrations (97 and194 µM). Similarly, CA/cell significantly reduced at all concentrations (except 12 µM) compared to positive control. In addition, the frequency of abnormal cells and the number of CA/cell reduced significantly in all the concentrations of
Cynarin+MMC compared to positive control at 48 h treatment (Table 2). These effects were concentration independent in both 24 h and 48 h treatments (r=0.38 and r=0.36 for the frequency of abnormal cell; r=0.39 and r=0.35 for CAs/cell, for 24h and 48 h, respectively). In this study, while there are four different types of structural aberrations (chromatid breaks, fragments, chromosome breaks and chromatid exchanges) at 24 h, there are six different types of structural aberrations (chromatid breaks, fragments, chromatid exchanges, chromosome breaks, dicentric chromosomes and sister chromatid unions) and a numerical aberration (polyploidy) at 48 h treatment. Moreover, it was determined that most protective concentrations of cynarin were 97 and 48 µM against MMC induced chromosomal aberrations at 24 h and 48 h, respectively.
3.2.Sister-chromatid exchanges, mitotic and replication indices
The results of the SCEs analysis, mitotic and replication indices are shown in Table 3. Cynarin alone did not significantly increase the frequency of SCEs/cell at all the concentrations at 24 h. However, it significantly increased the number of SCEs/cell at only 194 µM at 48 h (r=0.81), compared to solvent control. Treatment of Cynarin+MMC significantly reduced the number of SCEs/cell in the lower three concentrations (24, 48 and 97 µM) in all the treatment times, compared to positive control (MMC). This reduction in SCEs was slightly concentration-dependent in both application periods (r= 0.61 at 24 h, r=0.57 at 48 h). It was found that the most effective concentrations to reduce the frequency of SCEs were 97 µM and 48 µM, at 24 h and 48 h, respectively.
Treatment of cynarin alone significantly reduced MI at the highest concentration (194 µM at 24 h, 97 and 194 µM at 48 h) compared to solvent control. The decline in MI was slightly concentration-dependent (r=-0.58 and, r=-0.42, at 24 and 48 h, respectively). In the simultaneous treatment of Cynarin+MMC, MI increased concentration-dependently in all the concentrations and treatment periods compared to positive control (r=0.76 at 24 h, r=0.45, weak rate, at 48 h). Both Cynarin alone and the combination of Cynarin and MMC treatments did not considerably affect the RI.
3.3.Micronucleus assay and nuclear division index
Table 4 shows that cynarin alone did not significantly increase the frequency of lymphocytes with micronucleus compared to solvent control. On the other hand, simultaneous treatment of cynarin and MMC diminished the frequency of micronuclei compared to positive control in
all the concentrations (except 12 µM). However, this decline was significant only at 24 µM and concentration-independent (r=0.10). Furthermore, NDI were unaffected in all the treatments.
3.4.Comet Assay (Single Cell Gel Electrophoresis)
Table 5 presents three comet parameters (tail intensity, tail length and tail moment) following the treatments of lymphocytes with cynarin alone and simultaneous treatment with Cynarin+H2O2. Treatment of cynarin alone did not significantly increase the comet parameters compared to solvent control. On the other hand, simultaneous treatment of cynarin+H2O2 reduced all the comet parameters at all the concentrations compared to H2O2 alone (positive control). This reduction was significant and concentration-dependent for tail intensity (r=0.76), tail length (r=0.64) and tail moment (r=0.73).
Medical and nutritional plants are widely used in folk medicine and to develop new drugs. Therefore, to investigate mutagenic potentials of plant extracts and plant chemicals during pre-clinical assessment is extremely important due to both safety and economic concerns (Melo-Reis et al., 2011; Sponchiado et al., 2016). At present, plant metabolites derived from natural sources rather than synthetic ones are preferred to treat diseases in humans and animals. Any compound which is non-mutagenic as a good candidate for therapeutic use is highly important. Moreover, regulation of the compounds which has antigenotoxic activity plays an important role in the development of chemo-preventive strategies (Vlaykova et al., 2013; Avato and Argentieri, 2018).
The results of this study showed that cynarin alone did not significantly induce the frequency of CAs and CA/cell at all the concentrations and exposure times. On the other hand, simultaneous application of Cynarin and MMC significantly reduced the frequency of chromosomal abnormalities in comparison to MMC treated cells. It is widely accepted that as the number of chromosomal abnormalities increases, cellular toxicity increases as well.The frequency of chromosomal abnormalities in peripheral blood lymphocytes reflects a biological indicator of cancer risk in humans (Hagmar et al., 1998; Norppa et al., 2006; Bonassi et al., 2008; Doak et al., 2012; Santovito et al., 2014; Minina et al., 2018).
According to the results of the SCEs analysis, cynarin significantly increased the number of SCEs/cell at only 194 µM (the highest) concentration at 48h treatment. On the other hand, simultaneous treatment of Cynarin+MMC considerably decreased the number SCEs/cell in comparison to MMC alone at the three concentrations (24, 48, and 97 µM) at both treatment periods. Although it is known that the SCE assay is not as good as CA assay as a biomarker in cancer risk assessment, it is widely used in the evaluation of genomic damage (Preston et al., 2010; Santovito et al., 2014). S-phase dependent agents such as mitomycin-C are among the most prominent SCE inducing agents. SCEs may potently occur due to the conditions that gather single strand breaks or subsequent double strand breaks generation during replication at cellular level (Zhang, 2013; Mourelatos, 2016; Salawu et al., 2018).
In this study, the MI is one of the important parameters used to determine cell proliferation and cytotoxicity (Eroglu et al., 2006; Ping et al., 2012). Treatment of MMC (0.60 µM) alone reduced MI compared to control groups at both treatments periods. Cynarin alone significantly decreased MI only at the highest concentration (194 µM, at 24 h) compared to solvent controls. Simultaneous treatment of cynarin+MMC increased MI at both treatment periods and in all the concentrations compared to MMC itself. The decrease in MI can be due to the inhibition of G2 phases of the cell cycle which allows cells to progress mitosis as well as a drop in ATP level or a defect in the energy production site (Epel, 1963; Jain and Andsorbhoy; 1988; Avuloglu-Yılmaz et al., 2017b). No significant differences were observed between cynarin treated and the combination of cynarin and MMC treated cells in NDI and RI.
Micronucleus assay in human lymphocytes is an in vitro technique that is often used to detect and measure chromosomal damage resulting from exposure to chemical and radiological genotoxins (Kirsch-Volders, 2014; Fenech et al., 2016; Rodrigues et al., 2018). Micronucleus derives from whole chromosomes which are not included in the main daughter nuclei (clastogenicity) or from chromosome fragments (aneugenicity) that are lost during nuclear division (Fenech, 2007; Pardini et al., 2017; Kirsch-Volders et al., 2018). Cynarin alone did not affect the frequency of MN formation compared to control groups in all the treatments. Simultaneous administration of cynarin and MMC reduced the frequency of MN against MMC itself in all the concentrations (except 12 µM). However, this decline was statistically significant at only 24 µM concentration. MN frequency has been prevalently considered as a biomarker of chromosomal damage. Furthermore, it has been shown that there is a significant
correlation between increased MN frequency in human peripheral lymphocytes and cancer incidence (Bonassi et al., 2011; Chandirasekar et al., 2014; Pardini et al., 2017; Russo and Degrassi, 2018).
The comet assay is one of the most preferred methods for the analysis of genotoxic and geno- protective effects in single eukaryotic cell since it is very sensitive, simple, and reliable. Alkaline comet assay used in this study allows single and double strand breaks, alkali labile sites, and delayed or incomplete excision repair sites in the DNA (Rojas et al., 1999; Gyori et al., 2014; Collins, 2014; Pérez-Iglesias et al., 2017; Møller, 2018). Cynarin alone did not significantly increase comet parameters (tail intensity, tail length, and tail moment) in isolated lymphocytes. On the other hand, simultaneous administration of Cynarin and H2O2 reduced comet parameters in a concentration-dependent manner in all the concentrations compared to H2O2 alone.
Our results indicated that cynarin alone did not possess genotoxic effect. Furthermore, it has antigenotoxic effects against genomic damage induced by MMC and H2O2. The present investigation is the first study which assesses the genotoxic and antigenotoxic effects of cynarin by using CA, SCE, MN, and comet assays in human lymphocytes. However, the genotoxic, antigenotoxic, and cytotoxic effects of the extracts from artichoke were evaluated in various in vivo and/or in vitro studies (Table 1). While some of these studies are on the leaf extracts of Cynara scolymus L. (global artichoke), only one is on the leaf extract of Cynara carcundulus L. Cynara cardunculus L. leaf extract (CCE) did not exhibit genotoxic potential in gene mutation tests in S. cerevisia and S. typhimurium, and chromosomal aberration test in Vicia faba L. Researchers demonstrated that CCE has antigenotoxic potential in S. cerevisia and Vicia faba L. but not in S. typhimurium. The mutagenic agents used in that study were 4- NQO, MNU, and 2-AF. Interestingly, antigenotoxic effect of CCE was observed at only simultaneous treatments. Protective effect induced by simultaneous treatment of CCE on yeast cells might have resulted from its desmutagenic activities via direct inactivation of the genotoxic agent and/or free radical reactive oxygen species scavenging (Miadokova et al. 2008).
C. scolymus leaf extract (CSE) has non-genotoxic effects in some test systems. In an in vivo study, CSE (500, 1000, and 2000 mg/kg, 3 days) did not increase the frequency of micronucleus in peripheral blood cells in mice. While SCE did not significantly increase the
comet parameters in peripheral blood cells compared to the control, a significant increase in the frequency of damage was detected only at the highest concentration (2000 mg/ kg) in bone marrow cells (Zan et al., 2013). In another in vivo investigation, CSE (250 mg/kg, 28 days) did not increase the frequency of CAs in rat bone marrow cells (Donya and Ibrahim, 2012). Bloom head (BHE) and leaf (CSE) extracts obtained from C. scolymus L. were not genotoxic in somatic mutation and recombination test (SMART) in Drosophila melanogaster (Jacociunas et al., 2014). On the contrary, CSE increased DNA damage in CHO and HepG2 cells and MN frequency in CHO cells (Jacociunas et al. 2012, 2013a, Da Silva et al. 2017). Jacociunas et al. (2012) reported that CSE induced DNA damage at all the concentrations (0.62, 1.25, 2.5, and 5.0 mg/mL) and treatment periods (1- and 24-h) in Chinese hamster ovary cells. Jacociunas et al. (2013a) showed that while the same concentrations and treatment period of CSE enhanced micronucleus frequency; it did not induce nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in CHO cells. Nuclear division index (NDI) was also not affected. They explained that CSE induced chromosomal mutations was due to pro- oxidant activity of phenolic compounds, especially flavonoids. In another study demonstrating genotoxic effect of CSE, comet test was performed in HepG2 cells (Da Silva et al. 2017). Researchers have chosen the same concentrations and durations with Jacociunas et al. (2012) and (2013a, b). In these studies, genotoxic potential of CSE was attributed to the high concentrations selected. Antigenotoxic potential of CSE has also been examined and it has been demonstrated to be antigenotoxic in all except one study. Jacociunas et al. (2014) reported that CSE (0.0435 and 0.0875 g/mL) and BHE (0.0276 and 0.0552 g/mL) did not have an antigenotoxic effect against bleomycin (BLM, 0.01 mM) or mitomycin C (MMC, 0.5 mM) induced mutagenicity in the co- or post-treatment in Drosophila melanogaster. On the other hand, they observed that BHE significantly increased the genotoxicity induced by ethyl methane sulfonate (EMS, 12.5 mM). CSE did not affect the genotoxicity triggered by EMS in the co-treatment period whereas it significantly increased the genotoxicity in the post- treatment period. Researchers stressed that artichoke extracts promoted the repair processes such as homologous recombination in proliferative cells in D. melanogaster (Jacociunas et al., 2014). CSE showed to be antigenotoxic particularly in low concentrations in different test systems. Simultaneous treatment of this extract (0.62, 1.25, and 2.5 mg/mL concentrations) exhibited antigenotoxic effect against alkylating agent ethyl methanesulfonate (EMS-350 µM) in CHO cells in comet assay (Jacociunas et al. 2012). In another study, antigenotoxic potantial of CSE (0.62, 1.25, 2.5 and 5.0 mg/mL) was determined against genomic alterations induced by EMS (350 µM) in Chinese hamster ovary cells (CHO). In another study, cytokinesis block
micronucleus (CBMN) cytome assay was performed with three protocols; pre-, simultaneous and post-treatment. While CSE decreased the frequency of MNi and NBUDs at only 0.62 mg/mL, it raised the MNi frequency at 5 mg/mL, which is the highest concentration. Simultaneous treatment of CSE significantly supressed the induction of MNi (Jacociunas et al., 2013b). Similarly, Da Silva et al. (2017) demonstrated CSE induced antigenotoxic effects on human hepatocyte cultures (HepG2 cells) using comet assay. In pre-treatment, artichoke extracts (0.62, 1.25, and 2.5 mg/mL) displayed a protective effect against hydrogen peroxide (H2O2) whereas in simultaneous and post-treatment protocols, it decreased H2O2-promoted DNA damage only at the low concentration (0.62 mg/mL). However, the highest concentration of artichoke extract enhanced H2O2 genotoxicity in the simultaneous treatment protocol. Da Silva et al. (2017) explained that artichoke can be genotoxic to HepG2 via pro- oxidant activity by the formation of reactive oxygen species and modulating DNA damage induced by H2O2 by inhibiting the activation of intermediate reactive species and/or inducing detoxifying mechanisms. Another possibility could be the blockage of reactive molecules by chemical or enzymatic reactions with electrophiles or scavenging reactive oxygen species. In another study it was demonstrated that hydroethanolic leaves extract of Cynara scolymus L. (250mg/kg, for 4 weeks, pre-treatment) exhibited antigenotoxic effect against paracetamol (4 g.kg-1b.wt, p.o.) by reducing DNA damage and percentage of chromosomal aberrations in rat bone marrow cells. Researchers suggested that this effect was probably originated from antioxidant phytochemicals (phenols, flavonoids, etc.) found in leave extracts (Donya and Ibrahim, 2012).
According to these studies, artichoke extracts inhibited antigenotoxic effects against mutagens induced DNA damage generally at low concentration in pre- and simultaneous treatment protocols. The authors explained that these effects may be related to phytochemicals (caffeic acid and ferulic acid, cynarin, and chlorogenic acid etc.) of artichoke extracts which can have desmutagenic potential. They are responsible for genotoxic and antigenotoxic effects due to either prooxidant or free radical scavenger properties.
The phytochemical analysis of global artichoke identified hydroxycinnamic acids and flavones such as chlorogenic acid, cynarin (1,5-di-caffeoylquinic acid), luteolin and luteolin glycosides (for example scolymoside and cyanoroside) that are regarded as essential compounds of artichokes (Fritsche et al., 2002; Goñi et al., 2005; Martínez-Esplá et al., 2017; Rezazadeh et al., 2018). The results of our study demonstrated that cynarin, a derivative of
caffeic and quinic acid like chlorogenic acid, decreased genetic toxicity induced by MMC and oxidative stress caused by H2O2 in human lymphocytes. This effect of cynarin which can act as a powerful antimutagenic may be associated with high antioxidant capacity due to two adjacent hydroxyl groups on each of its phenolic rings (Wang et al., 2003; Jun et al., 2007), inhibiting free radical attacks formed by MMC or H2O2. These results correspond to those obtained by Cinkilic et al. (2013) and Devipriya et al. (2008) who studiesquinic acid (0.5-4 µg/mL), chlorogenic acid (0.5-4 µg/mL) and caffeic acid (5.5-88 µM) by using pre-treatment in human lymphocytes. Both these studies suggested radioprotective effects against gamma radiations. and concluded that these phenolic acids have two adjacent hydroxyl groups on an aromatic residue which scavenge reactive oxygen species, thus having antioxidant, antimutagenic and, anticarcinogenic activities in vitro.
Kada et al. (1982) classified antimutagenic agents according to their movement mechanisms as desmutagens or bioantimutagens. Desmutagens, extracellularly interacting directly with mutagens, might be chemically inactivate mutagenic agents prior to the arrival to DNA. Unlike desmutagens, bioantimutagens that act within the cell suppress genom damage by reversing mutagenic effects and are able to interfere with DNA repair and replication (Słoczyńska et al., 2014; Al-dualimi et al., 2018). On the other hand, anti-mutagenic agents that act in pre- or simultaneous treatment incorporate into desmutagens, while those acting in the post-treatment are bio-antimutagens (De Flora and Ramel, 1988; Srivastava et al., 2016). As a result of all, it can be estimated that cynarin follows a chemoprevention pathway involving the desmutagenic mechanism of action and exhibits antioxidant activity by scavenging free radicals in the simultaneous treatment.
In recent years, phytochemicals in dietary plants have been of great interest as chemo- preventive agents due to their ability to prevent or suppress the multiple steps of carcinogenesis, especially induced by the genotoxic action. The results of the present study indicate that antioxidant phytochemicals such as cynarin are able to protect DNA against chemically induced lesions. Thus, it can be considered that cynarin exhibits strong chemo- preventive activity because of its antioxidant capacity. However, these findings are insufficient to establish chemo-preventive strategies using phytochemicals such as cynarin. Mechanisms of chemo-prevention of cynarin are not yet very clear. Therefore, more in vitro and/or in vivo studies in different test systems are needed to establish cynarin as chemopreventive against genetic instability and health promoter.
E. Erikel and D. Yuzbasioglu designed the research and carried out the experimental stages and obtained the data. F.Unal contributed the comet assay and the statistical analyzes. The authors read and approved the manuscript. This study is a part of the master thesis of E. Erikel.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Table 1. Literature review of genotoxic and antigenotoxic effects of different artichoke species extracts in various assay systemsa
Positive chemicals Extracts treatment and
End point Cell/animal Referance
(mutagen) concentration Antigenotoxicity
Gene mutation S. cerevisiae 4-NQO, 3×10-6 M CCE; 0.0025% and 0.005%; – + Miadokova et al. (2008)
(D7 strain) simultaneous treatment
CA Vicia sativa L. MNU, 0.5–2.0 mM CCE; 0.0025%; simultaneous – +
Gene mutation S. typhimurium 2-AF, 100 µg/plate CCE; 600, 300, 200, 100 µL/plate; – –
(TA 98, S9 mix) preincubation (enhanced mutagenicity)
Comet assay CHO EMS, 350 µM CSE; 0.62, 1.25, 2.5, and + + (only simultaneous, 0.62- Jacociunas et al. (2012)
5.0 mg/mL (1 and 24h); pre-, 2.5 mg/mL) simultaneous and post-treatment
CA (in bone marrow) Rat Paracetamol, 4 g.kg-1 CSE; 250 mg/kg; pre-treatment; – + Donya and Ibrahim (2012)
b.wt, p.o; 24h 28 days
CBMN CHO CSE; 0.62, 1.25, 2.5 and 5.0 + (only MN Jacociunas et al. (2013a),
mg/mL (1 h and 24 h) frequencies)
CBMN CHO EMS, 350 µM CSE; 0.62, 1.25, 2.5 and 5.0 + (pre-, simultaneous Jacociunas et al. (2013b)
mg/mL (1 h and 24 h); pre-, treatment) simultaneous and post-treatment
MN and Comet assays Mice CSE; 500, 1000, and 2000 mg/kg -/+ (2000 mg/kg, in Zan et al. (2013)
(peripheral blood (3 days) bone marrow) lymphocytes and bone
SMART test D. melanogaster BLM, 0.01 mM; C. Scolymus; BHE (0.0276 and – – Jacociunas et al. (2014)
MMC, 0.5 mM; 0.0552 g/mL); CSE (0.0435 and
EMS, 12.5 mM 0.0875 g/mL); simultaneous and
Comet assay HepG2 H2O2, 1.4 mM CSE; 0.62, 1.25, 2.5 and + + (except 5.0 mg/mL in the Da Silva et al. (2017)
5.0 mg/mL (1 and 24h); pre-, pre-treatment; only 0.62
simultaneous and post-treatment mg/mL in the simultaneous
and post treatment)
a Notes: Positive: +, negative: -, CCE: Cynara carcundulus L. leaf extract, CSE: Cynara scolymus L. leaf extract, BHE: Bloom head extract, 4-NQO: 4-nitroquinoline-N-oxide, MNU:N-nitroso- N′-methylurea, 2-AF: 2-aminofluorene, EMS: Ethyl methane sulfonate, BLM: Bleomycin, MMC: Mitomycin-C, H2O2: Hydrogen peroxide
Table 2. Frequencies of chromosomal aberrations and abnormal cells in cultured human peripheral lymphocytes after treatment with different concentration of Cynarin and Cynarin+MMC
Test substance Numerical Abnormal cell CA/Cell
Period Concentration Structure abnormalities Abnormalies ± SE (%) ± SE
ctb csb f scu cte dic p
Negative Control 24 0.00 7 1 2 – – – – 4.50±1.46 0.05±0.02
Solvent Control 24 0.36% 8 – 4 1 – – – 6.00±1.67 0.06±0.02
Positive Control (MMC) 24 0.60 32 6 13 – – – – 22.50±2.95●a 0.25±0.03●a
Cynarin 24 12 8 – 1 – – – – 4.50±1.46 0.04±0.01
24 10 – 2 – – – – 5.00±1.54 0.06±0.02
48 6 2 – 1 – – – 4.50±1.46 0.04±0.01
97 8 – 2 – – – – 5.00±1.54 0.05±0.02
194 11 – 1 – – – – 6.00±1.67 0.06±0.02
Cynarin+MMC 24 12 24 7 4 – 1 – – 16.50±2.62 0.18±0.03
24 26 1 6 – – – – 16.50±2.62 0.16±0.03*
48 26 4 4 – 1 – – 16.00±2.59 0.17±0.03*
97 18 2 4 – 1 – – 11.00±2.21** 0.12±0.02**
194 19 4 4 – – – – 12.50±2.33* 0.13±0.02 **
Negative Control 48 0.00 5 1 1 – – – 1 4.00±1.39 0.04±0.01
Solvent Control 48 0.36% 6 1 1 2 – 1 – 5.50±1.16 0.05±0.02
Positive Control (MMC) 48 0.60 33 16 3 5 4 1 – 27.50±3.16●a 0.31±0.03●a
Cynarin 48 12 6 1 2 1 – – 1 5.50±1.16 0.05±0.02
24 6 2 – 1 – – – 4.50±1.47 0.04±0.01
48 5 1 – 1 – – – 3.50±1.30 0.03±0.01
97 8 1 – 1 – – – 5.00±1.54 0.05±0.02
194 10 1 1 1 – – – 6.50±1.74 0.06±0.02
Cynarin+MMC 48 12 21 5 7 1 – 1 – 16.50±2.62** 0.17±0.03**
24 16 3 6 1 2 – – 13.00±2.38*** 0.14±0.02***
48 10 – 1 – 7 1 – 9.50±2.07*** 0.09±0.02***
97 13 2 6 1 2 2 1 12.00±2.30*** 0.13±0.02***
194 12 1 4 1 3 1 – 11.00±2.21*** 0.11±0.02***
ctb: chromatid break, csb: chromosome break, f: fragment, cte: chromatid exchange, dic: dicentric chromosome, scu: sister chromatid union, p: polyploidy (200 metaphases were scored for each treatment)
* Significantly different from the positive control p< 0.05 (z test) ** Significantly different from the positive control p< 0.01 (z test) *** Significantly different from the positive control p< 0.001 (z test) ● Significantly different from the negative control p< 0.001 (z test) a Significantly different from the solvent control p< 0.001 (z test) Table 3. Frequencies of SCE, RI and the MI in cultured human peripheral lymphocytes after treatment with different concentration of Cynarin and Cynarin+MMC Treatment Min-max SCE/cell ± SE M1 M2 M3 RI ± SE MI ± SE Period Concentration Test substance SCE (hour) (µM) Negative Control 24 0.00 1-12 4.60±0.36 24 50 126 2.51±0.05 8.75±0.63 Solvent Control 24 0.36% 2-10 4.22±0.24 22 50 128 2.53±0.05 8.40±0.62 Positive Control (MMC) 24 0.60 12-38 22.42±0.94 c1c2 37 56 107 2.37±0.06 4.45±0.46 a2 b2 Cynarin 24 12 1-10 4.58±0.28 22 50 128 2.53±0.05 7.55±0.59 24 0-10 4.30±0.35 16 50 134 2.59±0.05 7.95±0.60 48 0-11 4.80±0.35 12 50 138 2.63±0.05 8.05±0.61 97 0-10 4.08±0.27 17 50 133 2.58±0.05 7.70±0.60 194 2-10 4.60±0.30 19 50 131 2.56±0.05 6.70±0.56 a1 b1 Cynarin+MMC 24 12 12-33 21.56±0.86 33 60 107 2.37±0.06 5.90±0.53 * 24 11-39 18.88±0.92 c3 32 53 115 2.41±0.06 7.10±0.57 *** 48 9-29 19.00±0.77 c3 39 72 89 2.25±0.06 7.90±0.60 *** 97 5-28 17.46±0.89 c3 29 74 97 2.34±0.06 8.40±0.62 *** 194 11-36 21.16±0.80 39 72 89 2.25±0.06 7.10±0.57 *** Negative Control 48 0.00 0-9 4.22±0.29 60 62 80 2.44±0.06 6.95±0.57 Solvent Control 48 0.36% 0-12 4.80±0.38 45 63 92 2.04±0.06 5.70±0.52 Positive Control (MMC) 48 0.60 12-96 41.12±3.41 c1c2 61 75 68 2.08±0.06 2.45±0.35 a2 b2 Cynarin 48 12 2-7 4.44±0.19 54 51 95 2.21±0.06 6.90±0.57 24 0-7 4.38±0.27 35 72 93 2.29±0.06 6.30±0.54 48 1-7 4.64±0.24 47 57 99 2.25±0.06 5.90±0.53 97 1-8 4.78±0.21 51 66 83 2.16±0.06 5.15±0.49 a1 194 3-10 6.04±0.25 c1c2 42 59 99 2.29±0.06 5.05±0.49 a1 Cynarin+MMC 48 12 10-96 42.78±3.61 84 85 46 1.81±0.06 5.15±0.49 *** 24 7-77 36.34±3.41 c3 62 71 67 2.03±0.06 5.85±0.52 *** 48 5-71 29.44±2.89 c3 51 97 52 2.01±0.05 6.75±0.56 *** 97 9-76 35.80±3.27 c3 83 80 39 1.78±0.06 4.40±0.46 *** 194 9-87 38.42±3.66 72 87 41 2.35±0.06 4.25±0.45 ** * Significantly different from the positive control p< 0.05 (z test) b1 Significantly different from the solvent control p< 0.05 (z test) ** Significantly different from the positive control p< 0.01 (z test) b2 Significantly different from the solvent control p< 0.001 (z test) *** Significantly different from the positive control p< 0.001 (z test) c1 Significantly different from the negative control p<0.05 (t test) a1 Significantly different from the negative control p< 0.05 (z test) c2 Significantly different from the solvent control p<0.05 (t test) a2 Significantly different from the negative control p< 0.001 (z test) c3 Significantly different from the positive control p<0.05 (t test) Table 4. Frequencies of micronuclei and nuclear division index (NDI) in cultured human peripheral lymphocytes after treatment with different concentration of Cynarin and Cynarin+MMC Treatment Distribution of BN MN ± SE Nuclear division cells according to the BN cells scored (%) index Test substance no. of MN Period Concentration (NDI) (hour) (µM) (1) (2) Negative Control 48 0.00 2000 5 - 0.25±0.11 1.72±0.41 Solvent Control 48 0.36% 2000 6 - 0.30±0.12 1.64±0.40 Positive Control (MMC) 48 0.60 2000 19 - 0.95±0.22 a b 1.63±0.40 Cynarin 48 12 2000 4 2 0.40±0.14 1.35±0.36 24 2000 4 - 0.20±0.09 1.40±0.37 48 2000 5 - 0.25±0.11 1.72±0.41 97 2000 4 - 0.20±0.09 1.75±0.41 194 2000 3 - 0.15±0.08 1.70±0.40 MMC+Cynarin 48 12 2000 22 - 1.10±0.23 1.65±0.40 24 2000 8 - 0.40±0.14* 1.69±0.40 48 2000 9 - 0.45±0.15 1.68±0.40 97 2000 11 - 0.55±0.17 1.60±0.39 194 2000 9 - 0.45±0.15 1.61±0.39 * Significantly different from the positive control p< 0.05 (z test) a Significantly different from the negative control p< 0.01 (z test) b Significantly different from the solvent control p< 0.05 (z test) Table 5. Mean of DNA damage in isolated human peripheral lymphocytes after treatment with different concentration of Cynarin and Cynarin+H2O2 Period Concentration Tail intensity Tail Lenght Tail Moment Test substance (hour) (µM) (%) (µm) Negative Control 1 0.00 8.30±1.02 54.45±0.75 2.11±0.39 Solvent Control 1 0.36% 6.24±0.56 54.95±0.74 1.32±0.12 Positive Control (H2O2) 1 100 44.55±2.58 α β 214.15±9.57 α β 40.97±3.19 α β Cynarin 1 12 7.86±0.84 56.51±0.79 1.87±0.29 24 6.59±0.75 54.23±0.75 1.50±0.23 48 6.28±0.71 52.71±0.78 1.48±0.27 97 7.42±0.75 54.32±0.81 1.77±0.30 194 7.74±0.75 55.49±0.73 1.66±0.15 Cynarin+ H2O2 1 12 7.28±0.89* 57.97±1.23* 1.80±0.33* 24 8.04±0.84* 61.21±1.49* 1.86±0.33* 48 97 194 7.78±0.82* 6.99±0.64* 9.97±1.12* 63.18±1.62* 57.38±1.10* 58.48±1.19* 1.80±0.25* 1.44±0.13* 2.55±0.46* α Significantly different from the negative control p< 0.05 (t test) * Significantly different from the positive control p< 0.05 (t test) β Significantly different from the solvent control p< 0.05 (t test) References Adzet, T., Camarasa, J., and Laguna, J.C., 1987. 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