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Evaluation of in vitro and in vivo genotoxic and
antigenotoxic effects of Rhus coriaria
Taygun Timocin, Mehmet Arslan & Hasan Basri Ila
To cite this article: Taygun Timocin, Mehmet Arslan & Hasan Basri Ila (2019): Evaluation of in�vitro and in�vivo genotoxic and antigenotoxic effects of Rhus�coriaria, Drug and Chemical Toxicology, DOI: 10.1080/01480545.2019.1593433
To link to this article: https://doi.org/10.1080/01480545.2019.1593433
Published online: 04 Apr 2019.
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RESEARCH ARTICLE
Evaluation of in vitro and in vivo genotoxic and antigenotoxic effects of
Rhus coriaria
Taygun Timocina , Mehmet Arslanband Hasan Basri Ilaa a
Faculty of Science and Letters, Department of Biology, Cukurova University, Adana, Turkey;bDepartment of Nursing, School of Health Sciences, Ardahan University, Ardahan, Turkey
ABSTRACT
Rhus coriaria has been important in the treatment of many diseases in traditional use. In this content, the genotoxic, antigenotoxic, and oxidative stress effects of methanol extract of R. coriaria (RCE) were investigated in this study. Two hundred fifty, 500, or 750mg/mL concentrations of RCE were not found to have DNA damaging effect on pET22-b(þ) plasmid and were unable to induce micronuclei in human lymphocytes (24 or 48 h treatment period). However, it did not inhibit the genotoxic effect of mitomycin-c (0.25mg/mL). Cytotoxic effects of RCE were investigated using mitotic index (MI) and nuclear division index (NDI). Five hundred, 1000, and 2000 mg/kg concentrations of RCE did not induce chromosome aberrations in rat bone marrow cells for 12 or 24 h treatment period. In addition, 2000 mg/kg concentration of RCE showed an antigenotoxic effect by decreasing to genotoxic effect of 400 mg/kg urethane at 12 and 24 h treatment periods. RCE showed cytotoxic effects by significantly decreasing NDI. Moreover, RCE increased cytotoxic effect of Mitomycin C (MMC). However, RCE did not induce cytotoxicity in rat bone marrow cells. The highest concentration of RCE reduced total oxidant level in 12 h treatment. Interestingly, the lowest total oxidant level was found in rats blood treated with the lowest concentration RCE and urethane together. Thousand and 2000 mg/kg concentrations of RCE decreased total antioxidant levels of rat blood at 24 h treatment period. Our results showed that RCE possess cytotoxic effect in short-term treatments in vitro. However, it does not demonstrate genotoxic or cytotoxic effects in vivo.
ARTICLE HISTORY
Received 30 November 2018 Revised 21 February 2019 Accepted 2 March 2019
KEYWORDS
Rhus coriaria; genotoxicity; antigenotoxicity; cytotox-icity; plasmid;
oxidative stress
1. Introduction
Rhus coriaria (RC) is a species of the genus Rhus belonging to the family Anacardiaceae. It is commonly known as sumac (Tanner’s Sumac) (El Hasasna et al. 2016) and grows as a small tree with a height range of 1–4 m in the wild (Bursal and K€oksal 2011). Its growth area includes the region from the Canary Islands through the Mediterranean region to Iran and Afghanistan. It is a native plant for Mediterranean and Southeastern Anatolian Region of Turkey (Nasar-Abbas and Halkman 2004). The name ‘sumac’ is derived from ‘sumaga’ and it means red in Syriac (Wetherilt and Pala 1994). RC is commonly used as a spice and a medicinal herb in the Middle East and The Mediterranean, especially for wound healing (Bursal and K€oksal 2011). Dried fruit of sumac is ground with salt and used as a condiment over grilled meat, kebabs, and salads (Nasar-Abbas and Halkman 2004). Also, sumac has been used in the treatment of many diseases such as cancer, stroke, diarrhea, hypertension, and more (Shafiei et al. 2011, Ali-Shtayeh et al. 2013, Abu-Reidah et al.2015).
Previous studies have indicated that RC has anti-fungal (McCutcheon et al. 1994), anti-inflammatory (Fourie and Snyckers 1984) and anti-microbial (McCutcheon et al. 1992) effects. These effects have been shown to be mediated by
the phenolic acids, flavonoids, and tannins that it contains (Zargham and Zargham 2008, Pourahmad et al. 2010, Shabana et al. 2011, Capcarova et al.2012). Al-Bataina et al. (2003) showed that RC did not demonstrate mutagenic effects in the Ames Test. In fact, it showed anti-mutagenic effect (Park et al. 2004). In addition, RCE treatment did not cause DNA damage in cells according to Chakraborty et al. (2009).
Traditional use of RC fruit for many diseases and its prop-erties were the inspiration for this work. In addition, there has been no previous research on the genotoxic and antige-notoxic properties of RC in the literature. The effects of a compound, product, and environmental factors on the gen-etic material of cells or organisms are determined by geno-toxicity tests (Hagmar et al. 1994, Albertini et al. 2000, Liou et al. 2002, Bonassi et al. 2007). Increases in CA and MN fre-quencies are considered biomarkers for cancer risk in humans (Hagmar et al. 1998, Ginzkey et al. 2014, Huerta et al. 2014, Topaktas et al.2017). Although they vary among species, fac-tors of in vivo metabolism, pharmacokinetics, and DNA repair processes are active and contribute to the response to geno-toxic chemicals (OECD 2016). Therefore, an in vivo chromo-some aberration test was used to detect structural chromosome aberrations. The mechanism of micronuclei is such that acentric fragments or whole chromosomes, which
CONTACTTaygun Timocin ttimocin@hotmail.com Faculty of Science and Letters, Department of Biology, Cukurova University, Adana 01330, Turkey
ß 2019 Informa UK Limited, trading as Taylor & Francis Group https://doi.org/10.1080/01480545.2019.1593433
are unable to migrate to the cell poles with the rest of the chromosomes, appear as small nuclei in interphase cells. Thus, the presence of micronuclei is accepted as a sign for clastogenic and/or aneugenic effects of chemical substances (Giri et al. 2002, Topaktas et al. 2017, Ribeiro et al.2018). In addition, DNA strand breaks induced by oxidants are related to the prevention of cancer (Hiramoto et al.1993).
Therefore in this study, we evaluated the genotoxic, anti-genotoxic, DNA damage-protection, and oxidant-antioxidant effects of RC extract (RCE) by the in vivo and in vitro test sys-tems using pET22-b(þ) plasmid, human peripheral lympho-cytes, rat bone marrow cells, and blood.
2. Materials and methods 2.1. Rhus coriaria extract (RCE)
RC was collected from Saribasak Village, Sahinbey, Gaziantep, Turkey (3706’09.2"N 3714’56.5"E). RC fruits were harvested on July 29 2016. Professor Necattin Turkmen, a botany pro-fessor at Cukurova University, identified RC fruits. To obtain the plant extract, RC fruits were freeze-dried, after which 15 g of dried fruit were weighed, stirred with pure methanol, and milled with a laboratory-type blender. Next, the resulting substance was heated in an ultrasonic water bath at 30C for 45 min, centrifuged at 3500 g for 10 min, and filtered with blotting paper. The methanol was evaporated at 40C using a vacuum evaporator, and finally, the extract remaining at the bottom was dissolved in purified water.
2.2. Chemicals
Colchicine (CAS No: 64–86–8), cytochalasin B (CAS No: 14930–96–2), Mitomycin C (MMC) (CAS No: 50–07–7) and urethane (CAS No: 51–79–6) were purchased from Sigma-Aldrich company (Steinheim, Germany).
2.3. Animals
Permission to work with rats was obtained from the Cukurova University Local Ethical Committee. Eight- to twelve-week-old Sprague-Dawley albino rats were used in the study, their weights ranging from 200 to 250 g. Female and male rats were caged separately at an acclimatized (24 ± 2C) room temperature within 12 h light/12 h dark photoperiod. There was no restriction on water or food consumption.
2.4. Concentration selection
For the in vitro MN test, LD50 of RCE in the mitotic index of human lymphocytes was 750mg/mL, which was selected as the highest concentration. The decreasing concentrations were 500 and 250mg/mL. For in vivo CA test, the highest concentration (2000 mg/kg) allowed by OECD was used because it has been found to be non-cytotoxic (OECD Test No 475 2016). The other two concentrations were 1000 and 500 mg/kg.
2.5. DNA damage and protection activity assay
In this research, pET22-b(þ) plasmid DNA was used to evalu-ate DNA damaging and/or protective activity of RCE. Two hundred fifty, 500, and 750mg/mL concentrations of RCE were applied to plasmid with/without FeSO4þH2O2 (positive
controls, Kanwal et al. 2011). The experiments were per-formed in a microfuge tube included 3mL pET22-b(þ) plas-mid DNA (100 ng/mL) and 3mL RCE and it was completed with dH2O to 10mL. In addition, untreated pET22-b(þ) plas-mid DNA was used as negative control. Plasplas-mids were treated with extracts for 30 min to evaluate DNA damaging activity. To determine the protective activity of the extract, the plasmid was treated with extract concentrations (250, 500, and 750mg/mL) and 3 ml FeSO4þ1 mL H2O2 together for
30 min. After the incubation time, the plasmid was stained with loading dye and loaded to 0.8% agarose gel. The elec-trophoresis was performed at 120 V for 80 min. The gels were stained with ethidium bromide and photographed using Vilber Lourmat gel imaging system.
2.6. In vitro cytokinesis-block micronucleus test
The cytokinesis-block micronucleus test has been frequently used to determine genotoxicity (Fenech et al. 2003, Fenech
2006, OECD2010). The cytokinesis-block micronucleus (CBMN) test was performed as described by Timocin et al. (2016) and Celik and Topaktas (2018). Peripheral blood from four healthy, nonsmoking volunteers (no known illness or recent exposures to genotoxic agents; two males and two females, ages: 23–27) was used with the permission of the Cukurova University Clinical Research Ethics Committee (Meeting No: 53; May 13 2016). A total of 0.2 mL peripheral blood was added to 2.5 mL chromosome medium and the cells were incubated at 37C for 68 h. Concentrations of 250, 500, and 750mg/mL were used in this test. Because the human cell cycle is 24 h, treatment periods were determined as 24 and 48 h. Because, the dur-ation of a human cell cycle is 24 h. The blood cells were treated with 250, 500, and 750mg/mL concentrations of RCE alone for genotoxicity testing and also in combination with 0.25mg/mL MMC for the antigenotoxicity assessment. A negative control (untreated cultures) and positive control (0.25mg/mL MMC) were also established simultaneously. The concentra-tions of RCE were added 20 h after initiating the culture (48 h treatment period) and 44 h after initiating the culture (24 h treatment period). Cytochalasin B (6mg/mL) was added to all tubes to block cytokinesis 44 h after the start of incubation. At the end of 68 h incubation time, all tubes were centrifuged at 805 g for 5 min and treated with 37C; 0.4% KCl (hypotonic solution). The tubes were centrifuged at 290 g for 10 min and then fixed once with cold fixative (1/5/6 glacial acetic acid/ methanol/NaCl isotonic solution) for 20 min. After that, the cells were fixed twice with another cold fixative (1/5 glacial acetic acid/methanol) for 15 min. After every fixation process, cells were centrifuged at 290 g for 10 min. Then the cells were spread on cold glass slides, dried and stained with 5% Giemsa. For each donor, 1000 binucleated lymphocytes were examined (in total 4000 binucleated cells) to determine the percentage of MN and the percentage of micronucleated binuclear
(MNBN) cell frequency, as the indicators of genotoxic effect. Cytotoxicity was evaluated using the nuclear division index (NDI), which indicates the average number of cell cycles. To evaluate the cytotoxic effect, 1000 cells for each donor (4000 cells for each group) were scored as 1, 2, 3, or 4 nuclei and the following formula was used to calculate nuclear division index (NDI): [(1 M1)þ(2 M2)þ(3 M3)þ(4 M4)]/N; where M1–M4 is the number of cells with one to four nuclei and N is the total number of cells scored (Fenech2000). Micros Austria light microscope was used to examine slides at 400 magnifi-cation. All microscopy examinations were performed by one individual (T.T.)
2.7. In vivo chromosome aberration test
In this study, Mammalian Bone Marrow Chromosomal Aberration Test Protocol of OECD was followed (OECD Test No 475 2016). Five hundred, 1000, and 2000 mg/kg concentrations of RCE were used. Since the cell cycle of the rats is 12 h, 12 and 24 h were chosen as the RCE treatment periods. According to Kayraldiz and Topaktas¸ (2007), a concentration of Urethane equal to 200 mg/kg of body weight had a genotoxic effect in rat bone marrow cells. Therefore, urethane was used as a posi-tive control. To investigate the antigenotoxic effects of RCE, the rats were administered with 400 mg/kg (b.w.) of urethane along with RCE for 12 or 24 h treatment periods. In order to arrest metaphase, colchicine (3 mg/kg b.w.) was injected intraperito-neally two hours before the rats were sacrificed by cervical dis-location. Experiments were started 12 h after treatment with the RCE. At intervals of 12 and 24 h after treatment, the harvesting experiments commenced. Bone marrow was extracted from the femurs and put into a 0.9% NaCl (37C) solution. The cells were centrifuged at 805 g for 5 min. The supernatant was removed and hypotonic solution (0.4% KCl, 37C) was added to all tubes. The cells were incubated at 37C for 20 min and then centrifuged at 290 g for 10 min. The supernatant was dis-carded again and cold fixative (1/3, glacial acetic acid/methanol) was added to the tubes, after which the cells were incubated for 20 min in cold fixative at room temperature. This fixative process was repeated three times in total. The fixed cells were dropped onto glass slides and dried, and the dried slides were stained with 5% Giemsa. To determine different types of struc-tural and numerical CA, 200 metaphases for each rat (1200 metaphases per each treatment group) were examined to cal-culate the percentage of chromosome aberrations and CA/cell ratio. Also, the mitotic index, which is related to cell division (Yuet Ping et al.2012, Topaktas et al.2017), was determined by scoring 3000 cells from each animal. Micros Austria light micro-scope was used to examine chromosome aberrations at 1000 magnification. Mitotic index examinations were performed at 400 magnification. All microscopy examinations were per-formed by one individual (T.T.)
2.8. Total oxidant and antioxidant status
Total oxidant and antioxidant status were measured in peripheral blood of rats. The plasma of blood samples was separated by centrifugation at 1810 g for 10 min. The supernatant was stored at 80C until the spectrophotometric analysis. Commercial TOS
and TAS kits (Relassay, Turkey) were used to determine total oxi-dant and total antioxioxi-dant status (TOS and TAS).
Oxidants, convert divalent iron (ferrous ion¼ Feþ2) to triva-lent iron (ferric ion¼ Feþ3). In acidic medium, ferric ions form the colored complex. This color density allows measurement of oxidized molecules spectrophotometrically (530 nm). Hydrogen peroxide was used to calibrate the test and the results were expressed as per liter of hydrogen peroxide micromolar (mmol H2O2Equiv/L) (Erel 2005). Determination of TAS values is
deter-mined by bleaching of ABTS (2,20-azino-bis [3-ethylbenzothiazol-6- sulfonic acid]) radical cation with antioxidants. TAS values were measured spectrophotometrically at 660 nm. Results were expressed as mmol Trolox equivalent/L (Erel2004).
Oxidative stress index (OSI) was calculated by using the following formula: OSI¼ TOS/TAS (Harma et al. 2003, Kosecik et al.2005, Yumru et al.2009).
2.9. Statistical analysis
All values were expressed as means ± standard error (SE). All data were analyzed with One-Way Analysis of Variance (ANOVA) and Dunnett’s post hoc test using SPSS software (Armonk, NY). The concentration-response effects were deter-mined using the Pearson Correlation. In group comparisons, the standard level of significance was p¼ 0.05.
2.10. Results
2.10.1. DNA damaging and protective effect of R. coriaria
The DNA protection assay is a simple and quick test for the in vitro characterization of the protective properties of pro-teins or chemicals. Two hundred fifty, 500, or 750mg/mL con-centrations of RCE did not have DNA damaging effect on pET22-b(þ) plasmid. However, none of the concentrations of RCE showed a protective effect against DNA damaging effect of FeSO4þH2O2(Figure 1).
Figure 1. Plasmid gel electrophoresis image. 1: 3ml plasmid þ 3 ml 250 mg/mL RCE; 2: 3ml plasmid þ 3 ml 500 mg/mL RCE; 3: 3 ml plasmid þ 3 ml 750 mg/mL RCE; 4: Control 3 ml plasmid; 5: Positive Control 3 ml plazmid þ 3 ml FESO4þ1 mL H2O2; 6: 3 ml plasmid þ 3 ml 250 mg/mL RCEþ 3 ml FESO4þ1 mL H2O2; 7: 3ml plasmid þ 3ml 500 mg/mL RCEþ 3 ml FESO4þ1 mL H2O2; 8: 3ml plasmid þ 3 ml 750 mg/mL RCEþ 3 ml FESO4þ1 mL H2O2.
2.10.2. Genotoxic-antigenotoxic effects of R. coriaria in vitro
In human lymphocytes, 250, 500, or 750mg/mL concentra-tions of RCE did not induce micronuclei at any treatment periods (24 or 48 h). The mixture of RCE and MMC induced the micronuclei when compared to negative control (Table 1). This finding was interpreted as the lack of antigenotoxic effect of RCE in vitro.
2.10.3. Genotoxic-antigenotoxic effects of R. coriaria in vivo
Five hundred, 1000, and 2000 mg/kg concentrations of RCE were administrated to rats for two treatment periods (12 or 24 h). The results of the study showed that RCE alone did not induce chromosome aberrations in rat bone marrow cells. Moreover, the highest concentration of RCE (2000 mg/kg)
showed a significant antigenotoxic effect against the geno-toxic effect of urethane for both treatment periods (12 or 24 h) (Table 2).
2.10.4. Cytotoxic effect of R. coriaria in vitro
In human lymphocytes, RCE showed cytotoxic effect revealed by the statistically significant decreases in the NDI. Moreover, for both treatment periods, RCE showed a synergistic effect and increased cytotoxic effect of MMC when they were administrated to lymphocytes together (Figure 2).
2.10.5. Cytotoxic effect of R. coriaria in vivo
In rat bone marrow cells, 500, 1000, and 2000 mg/kg concen-trations of RCE were not cytotoxic as shown by the MI. As expected, urethane reduced the mitotic index for both treat-ment periods (12 or 24 h). The highest concentration of RCE
Table 1. Percentage of micronuclei and percentage of micronucleated binuclear cells in human per-ipheral blood treated with different concentrations ofR. coriaria extract and MMC for 24 or 48 h treatment period.
Test substance Treatment per hour Cons.mg/mL % MN ± SE % MNBNþ SE Control – – 0.050 ± 0.029 0.050 ± 0.029 MMC 24 0.25 1.375 ± 0.138a3 1.275 ± 0.138a3 R. coriaria E. 24 250 0.075 ± 0.048b3 0.075 ± 0.048b3 500 0.075 ± 0.048b3 0.075 ± 0.048b3 750 0.025 ± 0.025b3 0.025 ± 0.025b3 MMC 48 0.25 2.325 ± 0.202a3 2.225 ± 0.189a3 R. coriaria E. 48 250 0.050 ± 0.029b3 0.050 ± 0.029b3 500 0.050 ± 0.029b3 0.050 ± 0.029b3 750 0.075 ± 0.048b3 0.075 ± 0.048b3 R. coriaria E.þ MMC 24 250þ 0.25 1.275 ± 0.175a3 1.250 ± 0.185a3 500þ 0.25 1.325 ± 0.085a3 1.275 ± 0.063a3 750þ 0.25 1.075 ± 0.171a3 1.000 ± 0.071a3 R. coriaria E. þ MMC 48 250þ 0.25 1.975 ± 0.138a3 1.925 ± 0.175a3 500þ 0.25 1.925 ± 0.131a3 1.825 ± 0.131a3 750þ 0.25 2.025 ± 0.138a3 1.775 ± 0.131a3 a: Significant from control; b: significant from positive control.a3b3p 0.001.
Table 2. Chromosome Abnormalities Types, Percentage of Abnormal Cell, CA/Cell ratio in bone marrow cells of rat treated with different concentrations ofR. coriaria extract and Urethane for 12 or 24 h treatment period.
Test substance Treatment per hour Cons. mg/kg b.w.
Abnormalities
Abnormal cell (%)±SE CA/Cell ± SE B’ B’’ F CC Control – – 5 2 – – 1.17 ± 1.17 1.17 ± 1.17 Urethane 12 400 16 6 3 2 4.00 ± 0.52a3 4.50 ± 0.67a3 R. coriaria E. 12 500 6 3 1 – 1.68 ± 0.50b2 1.67 ± 0.49b2 1000 5 0 – – 0.83 ± 0.31b3 0.83 ± 0.31b3 2000 6 3 – – 1.33 ± 0.33b3 1.50 ± 0.43b2 Urethane 24 400 15 8 3 5 4.68 ± 0.67a3 5.17 ± 0.83a3 R. coriaria E. 24 500 5 3 – – 1.33 ± 0.21b3 1.33 ± 0.21b3 1000 5 0 – – 0.83 ± 0.31b3 0.83 ± 0.31b3 2000 4 4 1 – 1.50 ± 0.34b3 1.50 ± 0.34b3 R. coriaria E.þ Urethane 12 500 13 5 2 1 3.00 ± 0.37a1 3.50 ± 0.43a2 1000 19 3 – – 3.33 ± 0.49a2 3.67 ± 0.67a2 2000 12 1 – – 2.00 ± 0.37b1 2.17 ± 0.48b1 R. coriaria E. þ Urethane 24 500 19 4 3 2 4.33 ± 0.56a3 4.67 ± 0.67a3 1000 14 5 4 – 3.67 ± 0.42a3 3.67 ± 0.42a2 2000 9 2 2 1 2.18 ± 0.48b2 2.17 ± 0.48b2 a: Significant from control; b: significant from positive control.a1b1p 0.05;a2b2p 0.01;a3b3p 0.001; B’: chromatid brake; B’’:
chromosome brake; F: fragment; CC: chromatid change. 4 T. TIMOCIN ET AL.
(2000 mg/kg) inhibited the cytotoxic effect of urethane (Figure 3) for both 12 and 24 h treatment periods (p< 0.05).
2.10.6. Oxidative stress effect of R. coriaria
The highest concentration of RCE (2000 mg/kg) significantly reduced the total oxidant level for 12 h treatment. Interestingly, the lowest total oxidant level was found in the blood of rats treated with the lowest concentration RCE (500 mg/kg) and urethane together. Thousand and 2000 mg/ kg concentrations of RCE decreased the total antioxidant lev-els of rat blood for 24 h treatment period (Table 3). Oxidative stress levels of various RCE concentrations and negative con-trol were found similar when compared with each other. When the highest concentration of RCE (2000 mg/kg) and urethane were administrated together for 24 h treatment period, the oxidative stress level was higher than control. But this increase was not statistically significant.
3. Discussion
In this study, genotoxic, cytotoxic, and oxidant-antioxidant, DNA damaging-protective effects of RCE were investigated in human peripheral lymphocytes, and rat bone marrow and blood cells. Researchers have commonly used these tests for these pur-poses (Ahn et al. 2007, Timocin and Ila 2015, Norizadeh Tazehkand et al.2016, Akyil et al.2017, Ali et al.2018).
In the literature, this is the first study to reveal the geno-toxic effects of RC extract using these tests. According to our results, neither in vitro micronucleus test nor in vivo chromo-some aberration test showed a genotoxic effect for RCE. In rats, even the highest concentration we could apply accord-ing to OECD, 2000 mg/kg RCE, showed no genotoxic effect in the bone marrow cells. In the micronucleus test, RCE concen-trations of 250, 500, and 750mg/mL did not induce micronu-cleus formation. Similarly, methanol and water extracts of RC and quercetin, flavonol extracted from RC, did not demon-strate mutagenic potential in Salmonella strains (Seino et al.
1978, Al-Bataina et al. 2003). In one study, albino rats were fed with food comprising a 1/3 ratio RC seed for a period of 9–28 weeks. At the end of feeding, RC did not show carcino-genic effect (Pamukcu et al. 1996). In our study, when 250, 500, and 750mg/mL concentrations of RCE administered, pet-22b (þ) plasmid remained intact. Likewise, there was no change in DNA migration of the RC ethanolic extract treated lymphocytes in comet test (Chakraborty et al. 2009). When the studies in the literature and our findings are evaluated together, it can be concluded that RC is not genotoxic.
Mitomycin-C is a commonly used agent in genotoxicity and anti-genotoxicity studies as positive control (Takai et al.
2015, Kocaman and Guzelkokar 2018). When RCE and mito-mycin-C were applied to human lymphocytes in combination, the genotoxic effect of mitomycin-c was not altered. That is, RCE did not show an antigenotoxic effect in vitro. Urethane is
known as a genotoxic agent in rats (Schlatter and Lutz1990, Topaktas¸ et al.1996, Kayraldiz and Topaktas¸2007, Azirak and Rencuzogullari 2008, Bemis et al.2015). The results obtained in our study indicate that the highest concentration of RCE (2000 mg/kg) treatment significantly reduced the frequency of structural CAs induced by urethane in bone marrow cells for both 12 and 24 h treatment periods. The antigenotoxic
effect observed in this study is thought to be caused by the acids in the RC. Parallel to our study, Toxicodendron vernicifl-uum (former Rhus verniciflua), which belongs to Anacardiaceae family like RC, has been investigated for its anti-mutagenic effect. The Ames test showed that RC metha-nol extract potentially inhibited the mutagenic effect caused by aflatoxin B1 (Park et al. 2004). In addition, RC inhibited
Figure 3. Mitotic index in bone marrow cells of rat administrated with different concentrations ofRhus coriaria extract and urethane for 12 or 24 h treatment period.
Table 3. Total Oxidant Status, Total Antioxidant Status and Oxidative Stress Index in peripheral blood of rats treated with different concentrations ofR. coriaria extract and Urethane for 12 or 24 h treatment period.
Test substance Treatment per hour
Cons. mg/kg
b.w. TOS ± SE TAS ± SE OSI ± SE Control – – 24.58 ± 0.90 1.759 ± 0.050 14.05 ± 0.72 Urethane 12 400 27.18 ± 3.13 1.784 ± 0.050 15.12 ± 1.45 R. coriaria E. 12 500 23.11 ± 1.10 1.691 ± 0.036 13.70 ± 0.71 1000 23.74 ± 2.19 1.858 ± 0.055 12.67 ± 0.84 2000 19.00 ± 0.87b1 1.576 ± 0.047 12.09 ± 0.55 Urethane 24 400 24.23 ± 1.77 1.772 ± 0.101 13.76 ± 1.03 R. coriaria E. 24 500 26.48 ± 3.31 1.767 ± 0.061 15.13 ± 2.00 1000 20.59 ± 1.87 1.505 ± 0.032a1b2 13.76 ± 1.42 2000 22.61 ± 2.02 1.557 ± 0.031b1 14.47 ± 1.07 R. coriaria E. þ Urethane 12 500þ 400 18.16 ± 1.59b2 1.537 ± 0.137 11.93 ± 0.57 1000þ 400 31.13 ± 1.85 1.792 ± 0.070 17.38 ± 0.84 2000þ 400 20.55 ± 1.09 1.604 ± 0.039 12.88 ± 0.88 R. coriaria E. þ Urethane 24 500þ 400 25.16 ± 1.59 1.671 ± 0.028 15.08 ± 0.96 1000þ 400 19.65 ± 1.09 1.509 ± 0.025a1b2 13.02 ± 0.67 2000þ 400 25.24 ± 1.66 1.573 ± 0.039b1 16.07 ± 1.01 a: Significant from control; b: significant from positive control.a1b1p 0.05;b2p 0.01; TOS: total oxidant status;
TAS: total antioxidant status; OSI: oxidative stress index; SE: standard error. 6 T. TIMOCIN ET AL.
DNA damage induced by H2O2 and
(±)-anti-benzo[a]pyrene-7,8-dihydro-diol-9,10-epoxide (BPDE) in the comet assay (Chakraborty et al. 2009). Kizil and Turk (2010) investigated the fatty acid compositions of RC in the southeastern Anatolia region of Turkey. The most abundant fatty acids in the extract were found to be linoleic acid, oleic acid, and pal-mitic acid. €Unver (2006) has studied the biochemical properties of the RC plants fruit obtained from a location close to the RC plant we used in our study. According to the study, RC was very rich in palmitic acid and oleic acid. These acids are known to have antimutagenic/anticlastogenic properties. In addition, palmitic acid showed antimutagenic activity in rats (Harada et al. 2002). In the study by Kritchevsky (2000), a linoleic acid form reduced dimethylbenz(a)anthracene(DMBA)-induced mammary tumors and adenocarcinomas in rats. The antigeno-toxic effect of RCE detected in our study, may be due to the palmitic and oleic acid contents of the RC.
The cytotoxic effect of RCE was evaluated by mitotic index (Yuet Ping et al. 2012, Morais et al. 2016, Topaktas et al.
2017) and nuclear division index (Alimba et al. 2016, Kocaman and Bucak 2016, Kocaman and Guven 2016). The statistically significant decrease of the NDI showed that RCE was cytotoxic at all concentrations (p< 0.001). Even, when lymphocytes were treated with RCE in combination with positive control MMC, the cytotoxic effect further increased for both treatment periods (24 and 48 h). This result shows that RCE synergistically increases the cytotoxic effect of MMC in vitro conditions. In a similar study, researchers have investi-gated the cytotoxic effect of RC ethanolic extract on breast cancer cell lines (El Hasasna et al.2015). RC ethanolic extract inhibited the proliferation of these cell lines in a concentra-tion-dependent manner by arresting the cell cycle in G1
phase via upregulation of p21. We are in the opinion that the reduction in cell division in our study may have depended on this pathway. Kizilsahin et al. (2015) demon-strated that oleic acid and a linoleic acid form, the compo-nents of RC, had significant cytotoxicity on human prostate cancer (PC3) cell line. There are many studies in the literature showing that RC is cytotoxic in vitro conditions (Sokmen
2001, Munusamy and Eid2013, El Hasasna et al.2015, Mirian et al.2015).
The relationship between cell-cycle progression and inhib-ition of cell proliferation was examined by mitotic index in vivo. In in vivo conditions, no concentrations of RCE exerted cytotoxic effects when the mitotic index was evaluated. Contrarily, mitotic index, which decreased with urethane treatment, were higher when RCE and urethane were admin-istered to rats in combination. However, the only significant difference was observed in the group treated with the high-est concentration of RCE for both 12 and 24 h treatment peri-ods (p< 0.05). The cells undergo apoptosis after DNA damage give rise to the accumulation of cells in the sub-G1
phase (Plesca et al. 2008, Franco et al. 2009, Kim and Ryu
2013). Urethane-induced genotoxicity may have caused cells to enter apoptosis, following which, a cytotoxic effect was observed. The RCE may have ameliorated the genotoxic effect caused by the urethane and prevented the cells from entering the apoptosis. El Hasasna et al. (2016) tested the effects of RCE on tumor growth in vivo using the chick
embryo. They found that RCE significantly inhibited tumor growth compared with controls, even at a low concentration (50mg/mL).
According to our results, 2000 mg/kg RCE concentration statistically significantly reduced the total oxidant level after 12 h of treatment. This effect was not observed in the 24 h treatment. There are many studies in the literature showing that RCE reduces oxidative stress. In the study performed by Saglam et al. (2015), 20 mg/kg of RCE administered to rats for 11 days, and RCE was found to have reduced the total oxi-dant status and oxidative stress at the end of treatment period. RCE had an antioxidant effect on lymphocytes which was potentiated by the enhanced GST activity (Chakraborty et al. 2009). Interestingly, concentrations of RCE of 1000 and 2000 mg/kg decreased the total antioxidant levels of rat blood after 24 h treatment period.
When all our results are evaluated together, we conclude that RC is not genotoxic in vivo or in vitro. On the other hand, RC had an antigenotoxic effect at the highest concen-tration in vivo. The cytotoxic effect obtained in vitro was dis-appeared in vivo conditions. The highest concentration of RCE (2000 mg/kg) prevented a urethane-induced cytotoxic effect, and thus, a genotoxic effect in bone marrow cells. Cancer drugs (Wang et al. 1998, Sung and Shuler 2009) and antimutagenic substances (Horn and Vargas 2003) often show their effects through cytotoxicity. In our study, the anti-genotoxic effect in in vivo conditions occurred without cyto-toxic effect. These results reveal the importance of this study and the fact that this plant should be investigated with dif-ferent genotoxicity/mutagenicity test systems.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was funded by Unit of Scientific Research Projects, Cukurova University, Turkey (Project No: FBA-2015–3645). Also, we are grateful to The Scientific and Technological Research Council of Turkey (TUBITAK) for providing scholarship support throughout the project (Scholarship No: 1649B031500496).
ORCID
Taygun Timocin http://orcid.org/0000-0002-5519-5744
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