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Drug and Chemical Toxicology
ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: http://www.tandfonline.com/loi/idct20
In vitro cytogenetic evaluation of the particular
combination of flurbiprofen and roxithromycin
Taygun Timocin, Mehmet Tahir Husunet, Ebrahim Valipour, Mostafa
Norizadeh Tazehkand, Rima Celik, Mehmet Topaktas & Hasan B. Ila
To cite this article: Taygun Timocin, Mehmet Tahir Husunet, Ebrahim Valipour, Mostafa Norizadeh Tazehkand, Rima Celik, Mehmet Topaktas & Hasan B. Ila (2017) In vitro cytogenetic evaluation of the particular combination of flurbiprofen and roxithromycin, Drug and Chemical Toxicology, 40:3, 326-332, DOI: 10.1080/01480545.2016.1223097
To link to this article: http://dx.doi.org/10.1080/01480545.2016.1223097
Published online: 06 Sep 2016.
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ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2017; 40(3): 326–332
!2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/01480545.2016.1223097
RESEARCH A RTICL E
In vitro cytogenetic evaluation of the particular combination of
flurbiprofen and roxithromycin
Taygun Timocin1, Mehmet Tahir Husunet1, Ebrahim Valipour2, Mostafa Norizadeh Tazehkand3, Rima Celik1, Mehmet Topaktas4, and Hasan B. Ila4
1Department of Biology, Institute of Science, Cukurova University, Adana, Turkey,2Department of Molecular Biology and Genetics, Faculty of
Sciences and Arts, Bu¨lent Ecevit University, Turkey,3Department of Biotechnology, Institute of Science, Cukurova University, Adana, Turkey, and
4Department of Biology, Faculty of Science and Letters, Cukurova University, Adana, Turkey
Abstract
Flurbiprofen (FLB) (anti-inflammatory and analgesic drug) and roxithromycin (RXM) (antibiotic) were widely used in world wide. This study deals with investigation of genotoxicity, cytotoxicity, and oxidative stress effects of a particular combination of these drugs in human cultured lymphocytes. Also, DNA damaging-protective effects of combination of these drugs were analyzed on plasmid DNA. Human lymphocytes were treated with different concentra-tions (FLB + RXM; 10 mg/mL + 25 mg/mL, 15 mg/mL + 50 mg/mL, and 20 mg/mL + 100 mg/mL) of the drugs following by study of their genotoxic and cytotoxic effects by analysis of cytokinesis-block micronucleus test and nuclear division index, respectively. The effect of the combination in aspect of anti-oxidative and DNA damaging activity was evaluated on Pet-22b plasmid. According to our results, the combination of FLB and RXM did not show a notable genotoxic effect on cells. Although each of the substances had been shown as a cytotoxic agent by previous researchers, in this research, the combination of these drugs did not exhibit any adverse effect on cell division. FLB had DNA protection effect against H2O2 while in
combination with RXM had not the same effect on the plasmid.
Keywords
Flurbiprofen, Roxithromycin, micronucleus, genotoxicity, cytotoxicity, oxidative stress
History
Received 13 March 2016 Revised 29 June 2016 Accepted 30 July 2016
Published online 6 September 2016
Introduction
Flurbiprofen (FLB), a non-steroidal anti-inflammatory drug (NSAID), is commonly used for its analgesic, antipyretic, and anti-inflammatory effects (Tewari et al., 2010). FLB play its role by suppression of prostaglandin synthesis and inhibition of cyclooxygenase activity (Kawadkar et al., 2013). The drugs which include FLB as an active ingredient are used for pain killer, mouth hygiene (toothpaste and mouthwash), sore throat, and upper airway diseases (lozenge) and also for treatment of gout, osteoarthritis, rheumatoid arthritis, and sunburn (Battisti, 1994; Fang et al., 2003; Watson et al., 2000).
Roxithromycin (RXM) which belongs to the macrolide group of antibiotics is a drug approved by FDA (U.S. Food and Drug Administration) and it is used to treat chronic inflammatory airway diseases such as diffuse panbronchioli-tis, bronchitis and bronchial asthma (Ekici et al., 2002; Hodge et al., 2006; Labro, 1998; Rubin, 2004; Southern et al., 2012). Macrolides show their antibacterial effects by inhibiting protein synthesis (Young et al., 1989).
Lately, genotoxicity studies have become more important because of the increase in the use of chemical substances. According to Organization for Economic Co-operation and Development (OECD), chromosome mutations and related genetic changes are reason of many genetic diseases and it is demonstrated that those changes induced cancer in human and animals by effecting tumor suppressor genes or oncogenes (OECD, 1997). For this reason, drugs should be explored about their genotoxicity (Jaju et al., 1984). Also, as a result of oxidation, enzyme inhibition/denaturation and protein deg-radation and DNA damage can be resulted (Barzilai & Yamamoto, 2004; Stadtman, 1992). This event can cause mutations and cancer if DNA repair mechanisms cannot repair the damages (Nakabeppu et al., 2006; Valko et al., 2004).
In the literatures, there are insufficient studies about the genotoxicity of FLB and RXM. There is not any study about the genotoxic potential of combination form of these drugs. In one study, FLB did not have any genotoxic effect on peripheral blood cells of human who used FLB for two weeks (Kullich & Klein, 1986). There are some reports regarding cytotoxic and anti-proliferative effects of FLB (Kusuhara et al., 1999; Jurima-Romet et al., 1994; Masubuchi et al., 1998; Zielinska-Przyjemska et al., 2008) similarly our previous research showed that FLB had cytotoxic effect on
Address for correspondence: Taygun Timocin, Department of Biology, Institute of Science, Cukurova University, Adana, Turkey. E-mail: ttimocin@hotmail.com
cells and negative effect on mitotic index, nuclear division index, and proliferation index in vitro while it did not induce chromosome aberration, micronuclei formation, and sister chromatid exchange (Timocin & Ila, 2015; Timocin et al., 2016). We did not find any reports about the genotoxic potential of RXM, although in our unpublished study, RXM did not show any genotoxic effect in human lymphocytes in chromosome aberration, sister chromatid exchange, and micronucleus tests. When we talk about cytotoxicity of RXM, there are some studies showing the anti-proliferative effect of RXM (Dai et al., 2014; Yatsunami et al., 1999; Yoshimura et al., 1995). On the contrary, one study reported that RXM had anti-cytotoxic effect against sulfur mustard in human respiratory epithelial cells (Gao et al.’, 2007).
Nowadays, in some diseases, patients need to use an analgesic and an antibiotic at the same time. Generally, FLB and RXM are used by many patients. Also, the substances may have synergistic or antagonistic effects when they are together. As a result, the purpose of this study was to investigate genotoxic, cytotoxic, DNA damaging-protection and oxidative stress potential of the combination of FLB and RXM.
Materials and methods
Chemical substances
All chemical substances were purchased from a commercial brand. FLB was purchased from Sigma-Aldrich (CAS No: 5104–49-4; Steinheim, Germany), and its molecular structure is shown in Figure 1. Purity of FLB was 98.5%. RXM was purchased from Alfa Aesar (CAS No: 80214–83-1; Ward Hill, MA) and its molecular structure is shown in Figure 2. Purity of RXM was 97%. Dimethyl sulfoxide (DMSO) (CAS No: 67–68-5; Merck, Darmstadt, Germany) was used to resolve FLB and RXM. Mitomycin C (MMC) (CAS No: 50–07-7; Sigma-Aldrich, Steinheim, Germany) was used as positive
control. To culture human blood cells, PB-Max chromosome medium was used (Gibco, CAT No: 12557–013). Colchicine (CAS No: 64–86-8), bromodeoxyuridine (CAS No: 59–14-3), and cytochalasin D (CAS No: 22144–77-0) were purchased from Sigma-Aldrich (Steinheim, Germany).
In vitro cytokinesis-block micronucleus test
Yavuz-Kocaman et al. (2008)’s method was used with minor modifications. Human blood was provided from four healthy non-smokers volunteers (two males and two females, ages: 22) with the permission of the Cukurova University Clinical Research Ethics Committee. FLB and RXM concentrations in combination were selected according to the pre-assays. LD50
concentrations of FLB and RXM are 40 and 200 mg/mL, respectively. The half of these concentrations were selected as highest concentration. Therefore, the combination of the two drugs were prepared as follows: 10 mg/mL FLB+ 25 mg/mL RXM, 15 mg/mL FLB + 50 mg/mL RXM, and 20 mg/mL FLB + 100 mg/mL RXM. These combination concentrations were tested for two treatment periods of time (24 or 48 h). DMSO (10 mL for 2.5 mL medium) and MMC (0.15 mg/mL) were also used for two treatment periods (24 or 48 h).
At the beginning of experiment, 0.2 mL peripheral blood (1/10 heparinized) was added to 2.5 mL chromosome medium. The cells incubated at 37C for total of 68 h in polypropylene culture tubes. Forty-eight hours and 24 hours before the end of incubation, the cells treated with the drug combination for 48 h treatment period and 24 h treatment period, respectively. To block cytokinesis, 24 h before the end of incubation, 6 mg/mL cytochalasin D was added to the cells. After the 68 h incubation, the cells were centrifuged at 2000 rpm for 5 min and treated with 0.4% hypotonic solution KCl (37C). Without waiting, the cells were centrifuged at 1200 rpm for 10 min. After centrifugation, the cells were fixed with cold fixative (1/5/6: glacial acetic acid/methanol/0.9% NaCl isotonic solution) for 20 min. Then, they were fixed two times with another cold fixative (1/5: glacial acetic acid/ methanol) for 15 min. After every fixation process, the cells were centrifuged at 1200 rpm for 10 min. Later, the fixed cells were spread on cold glass slides, dried at room temperature and stained with Giemsa (5%). To determine the genotoxic effect, micronucleus (MN), nucleoplasmic bridge (NPB) and nuclear bud (NBUD) were scored in 1000 binucleated lymphocytes (4000 binucleated cells per every control and concentration). Also, micronucleated binucleated (MNBN) cells were scored (Fenech et al., 2011). For cytotoxic effect, 1000 cells (4000 binucleated cells per every control and concentration) were scored with 1, 2, 3, or 4 nuclei. Nuclear division index (NDI) was calculated by the following formula: NDI: [(1 1N) + (2 2N) + (3 3N) + (4 4N)]/1000. 1N– 4N was the number of cells with one to four nucleated and 1000 was the total number of cells scored (Fenech, 2000). DNA damaging and protective activity assay
DNA is a main locus of attack of cellular oxidants and is a molecule whose damage can lead to disease initiation and progression. A plasmid usually is a double stranded small DNA molecule that is physically separated from a chromo-somal DNA and can replicate independently. Plasmids may
Figure 2. Molecular structure of roxithromycin. Figure 1. Molecular structure of flurbiprofen.
appear in different forms that the prominent forms of monomeric plasmids are the covalently closed circular (CCC), the open circular (OC) form (also called ‘‘nicked form’’ or relaxed form and obtained by digestion of one of the strands of double strand), and the linear form (typically obtained by digestion of both strands of plasmid). In the CCC form, the plasmid is normally in supercoiled form and appears to migrate more rapidly than the other forms. These conformations, in order of electrophoretic mobility from slowest to fastest, are open-circular DNA, linear DNA, and supercoiled DNA (Fernandez et al., 2011; Simandan et al., 1998; Suksomtip & Pongsamart, 2008). DNA damaging and protection activities of drugs were evaluated on pET22b plasmid DNA. DNA damaging activities were investigated with three concentrations of drugs and DNA protection activities were investigated with three concentrations of drugs in the presence of %35 H2O2. Concentrations of drugs were
selected according to our previous studies (Table 1). In summary, the experiments were performed in a microfuge tube included 5 mL pET22b plasmid DNA (100 ng/mL), 2 mL FLB, RXM or FLB + RXM resolved in DMSO, and completed with dH2O to 10 mL. In addition, untreated
pET22b plasmid DNA as negative control and DMSO (2 mL) as a solvent control were used. For DNA damaging activity, plasmids were treated with substances for 30 min. Also the supercoiled DNA was treated with the combination along with 35% H2O2for 30 min at room temperature, for the
investigation of their anti-oxidative effect. H2O2modifies the
supercoiled structure into the circular one by a single-strand break. Further oxidative damage opens the circular structure to linear DNA and furthermore, small DNA fragments. After all treatments, 1 mL loading dye was added and the reactions were loaded 0.8% agarose gel. Then, electrophoresis was performed with 120 V for 80 min. The gels were stained with EtBr and photographed with Vilber Lourmat gel imaging system.
Total oxidant and anti-oxidant status
The measurements were performed by the Rel Assay Diagnostics Company, Turkey. Total oxidant status (TOS) and total antioxidant status (TAS) were measured in the supernatant of cells worked in the micronucleus test. About 0.2 mL peripheral blood (1/10 heparinized) was added to 2.5 mL chromosome medium. The cells incubated at 37C for total of 68 h in polypropylene culture tubes. 48 h and 24 h before the end of the incubation time, the cells treated with the drug combination. After the 68 h incubation, the cells were centrifuged at 2000 rpm for 5 min. Then supernatants were taken from tubes and stored at 80C until analysis. Calculation of TOS values was based on the oxidization of
divalent iron (ferrous ion¼ Fe+ 2) to trivalent iron (ferric
ion¼ Fe+ 3). After reaction, ferric ions form color complex in
acidic medium. This color density is commensurate with the amount of oxidizing molecules in samples and was measured spectrophotometrically (530 nm). Hydrogen peroxide was used to calibrate the test and results were presented as per liter of hydrogen peroxide micro molar (mmol H2O2Equiv./L)
(Erel, 2005)
Fe2+-o-dianisidine complex gives a Fenton-type reaction with the hydrogen peroxide to form the OH radical. This powerful reactive oxygen species reacts with the colorless o-dianisidine molecule at the reducting low pH and leads to the formation of yellow–brown dianisidyl radicals. Dianisidyl radicals attend in further oxidation reactions resulting in more color formation. But, in case of having antioxidants effect in samples, these oxidation reactions and color formation were inhibited. TAS values were measured spectrophotometrically (660 nm). Results were expressed as mmol Trolox equivalent/ L (Erel, 2004; Yumru et al., 2009)
Oxidative stress index (OSI) was determined by following formula: OSI¼ TOS/TAS (Harma et al., 2003; Kosecik et al., 2005; Yumru et al., 2009).
Statistical analysis
All values were presented as mean ± standard error (SE). In the Shapiro–Wilk test, all values had normal distribution. The data was analyzed with one-way analysis of variance (ANOVA) and post hoc Dunnett’s test in SPSS software (SPSS Inc., Chicago, IL). Concentration-dependent effect was evaluated by Pearson correlation. ‘‘p 0.05’’ was accepted as significant.
Results
Effects of the various concentration combination of FLB and RXM on genotoxicity
Genotoxic effects of the combination (FLB + RXM) were evaluated by cytokinesis-block micronucleus test. Consequently, the combination did not affect MN, MNBN, NPB, or NBUD formation at any concentrations for any treatment periods (24 or 48 h). MMC increased micronuclei as expected. So, comparisons between the combination and MMC were found statistically significant. The results are shown in Table 2.
Effects of the various concentration combination of FLB and RXM on cell division
In the study, when we compared with DMSO, there is not any statistically significant decrease or increase on NDI at any concentrations of the combination of any treatment periods. MMC decreased NDI as expected. So, comparisons between the combination and MMC were found statistically significant (Table 3).
DNA damaging and protection activity of FLB, RXM, and the various concentration combination of these drugs
By comparison of lanes 3, 4, and 5 with lanes 1 and 2, it can be found that the FLB has negligible effect on the plasmid,
Table 1. Concentrations of drugs used in DNA damaging and protection activity assay.
Flurbiprofen (mg/mL) Roxithromycin (mg/mL) Flurbiprofen + roxithromycin (mg/mL) 20 50 10 + 25 30 100 15 + 50 40 200 20 + 100
contrary to this, in lane 6, the bond regarding to the relaxed DNA was increased. The relaxed DNA (also, it is known as nicked DNA) was resulted from the breaking of super coiled plasmid DNA backbone by H2O2 which is an oxidative
chemical agent. In lanes 8 and 9, highest two concentrations (30 and 40 mg/mL) of FLB inhibited the breaking of DNA by the H2O2(Figure 3).
Three concentrations of RXM did not affect to plasmid (Figure 4; lines 3, 4, and 5) when compared with untreated and DMSO control (Figure 4; lines 1 and 2). In lines 7, 8, and 9, RXM could not avoid H2O2 effect on plasmid.
Consequently, RXM did not have any damaging or protection effect on the plasmid.
Looking for damaging and protection activity of combin-ation of FLB and RXM, the combincombin-ation did not have any damaging activity on the plasmid (Figure 5; lanes 3, 4, and 5). However, the combination did not have any protec-tion effect against H2O2on the plasmid (Figure 5; lanes 7, 8,
and 9).
Oxidative stress evaluation of the various concentration combination of FLB and RXM
The TOS values of sample groups treated with the combin-ation were not found statistically significant compared to the DMSO control group. The values for group treated with MMC for 24 h was found to be significantly higher than that of the groups treated with the all concentrations of the combination for 24 h. Also, the values for group treated with MMC for 48 h was found to be significantly higher than that of the sample group treated with highest concentration of the combination (20 + 100 mg/mL) for 48 h. The TAS values of the groups treated with combination were not found signifi-cant compared to the DMSO and MMC control groups. Because TAS and TOS values did not change, oxidative stress values did not change. 24 and 48 h MMC groups were found
Table 2. Effect of the combination of flurbiprofen and roxithromycin to micronucleus, micronucleated binucleated cells, nucleoplasmic bridge and nuclear bud in human cultured lymphocytes for 24 and 48 hours treatment periods.
Treatment
Test Substance Time (h) Conc.(mg/mL) MN ± SE (%) MNBN ± SE (%) NPB ± SE (%) NBUD ± SE (%) DMSO 24 10 mL 0.43 ± 0.063 0.43 ± 0.063 0.30 ± 0.041 0.18 ± 0.025 MMC 24 150 1.13 ± 0.125 1.03 ± 0.085 0.38 ± 0.063 0.25 ± 0.029 FLB + RXM 24 10 + 25 0.48 ± 0.063 b3 0.48 ± 0.063 b3 0.43 ± 0.063 0.10 ± 0.071 FLB + RXM 24 15 + 50 0.55 ± 0.065 b3 0.50 ± 0.041 b3 0.33 ± 0.063 0.13 ± 0.025 FLB + RXM 24 20 + 100 0.45 ± 0.065 b3 0.45 ± 0.065 b3 0.43 ± 0.025 0.15 ± 0.050 DMSO 48 10 mL 0.48 ± 0.048 0.45 ± 0.050 0.20 ± 0.041 0.10 ± 0.058 MMC 48 150 1.65 ± 0.096 1.48 ± 0.063 0.48 ± 0.085 0.25 ± 0.065 FLB + RXM 48 10 + 25 0.48 ± 0.063 b3 0.48 ± 0.063 b3 0.20 ± 0.041 b1 0.13 ± 0.063 FLB + RXM 48 15 + 50 0.63 ± 0.048 b3 0.58 ± 0.025 b3 0.23 ± 0.085 0.13 ± 0.048 FLB + RXM 48 20 + 100 0.53 ± 0.048 b3 0.53 ± 0.048 b3 0.33 ± 0.048 0.20 ± 0.041
MN, micronucleus; MNBN, micronucleated binucleated; NPB, nucleoplasmic bridge; NBUD, nuclear bud; SE, standard error; FLB, flurbiprofen; RXM, roxithromycin. b: significant from MMC (positive control); b1: p 0.05. b3: p 0.001.
Table 3. Effect of the combination of flurbiprofen and roxithromycin to nuclear division index in human cultured lymphocytes for 24 and 48 h treatment periods.
Treatment
Test substance Time (h) Conc. (mg/mL) NDI ± SE DMSO 24 10 mL 1.316 ± 0.0050 MMC 24 150 1.211 ± 0.0106 FLB + RXM 24 10 + 25 1.299 ± 0.0188 b3 FLB + RXM 24 15 + 50 1.302 ± 0.0076 b3 FLB + RXM 24 20 + 100 1.295 ± 0.0058 b3 DMSO 48 10 mL 1.314 ± 0.0076 MMC 48 150 1.199 ± 0.0073 FLB + RXM 48 10 + 25 1.311 ± 0.0042 b3 FLB + RXM 48 15 + 50 1.306 ± 0.0062 b3 FLB + RXM 48 20 + 100 1.303 ± 0.0108 b3
SE, standard error; FLB, flurbiprofen; RXM, roxithromycin; b: significant from MMC (positive control); b3: p 0.001.
Figure 3. Control (plasmid DNA); (2) 2 mL DMSO; (3) 20 mg/mL flurbiprofen; (4) 30 mg/mL flurbiprofen; (5) 40 mg/mL flurbiprofen; (6) 2 mL DMSO + 3 mL H2O2; (7) 20 mg/mL flurbiprofen + 3 mL H2O2; (8)
30 mg/mL flurbiprofen + 3 mL H2O2; (9) 40 mg/mL flurbiprofen + 3 mL
H2O2.
to be statistically significantly higher than that of the 24 and 48 h combination treatment groups (Table 4).
Discussion
To our knowledge, this is the first study that addresses the potential genotoxic and cytotoxic effects of a particular combination of the FLB and RXM in peripheral blood lymphocyte.
The combination did not have any genotoxic effect on cultured cells. In a previous study, FLB alone did not induce chromosome aberration and polymorphic bands (RAPD-PCR) in rat bone marrow cells (Timocin & Ila, 2015). There is not any study about the genotoxic potential of RXM. In our unpublished study, RXM did not have genotoxic effect. According to this, it is observed that FLB and RXM did not have any synergistic or antagonistic genotoxic effect in human cultured lymphocytes when they were used together.
The combination did not significantly reduce or increase nuclear division index in human cultured lymphocytes as compared with solvent control. In our previous study, FLB reduced mitotic index (cytotoxicity marker) (Carrano & Natarajan, 1988) for 12 and 24 h treatment periods in rat bone marrow cells. Also, this reduction was concentration dependent for 12 h (Timocin & Ila, 2015). In an another study, 200 mM FLB (48 h) lead to apoptosis in squamous cell carcinoma lines in vitro, inducing caspase-3 enzyme activity (Bock et al., 2007). Additionally, FLB was related to cytotoxicity by leading to LDH leakage in rat hepatocytes cultures (Jurima-Romet et al., 1994; Masubuchi et al., 1998).
Researchers found that FLB prevented the growth of A-431 human epidermoid carcinoma cells and cleaved transcription factor b-catenin (Nath et al., 2013). The cytotoxic activity of RXM was investigated in vivo and in vitro. RXM inhibited of tumor growth induced by cyclophosphamide, cis-diammine-dichloroplatinum (II), Adriamycin, and vindesine only in in vivo system (Yatsunami et al., 1999). RXM caused cell death by enhancing expression of FasL and caspase-3 in vitro T-lymphocytes cell system (Jun et al., 2003). In our unpub-lished study, RXM reduced nuclear division index. In addition, it was reported that RXM induced apoptosis in airway smooth muscle cells derived from a rat model of asthma in a dose-dependent manner by caspase-3- and caspase-9-dependent mitochondrial pathway (Dai et al., 2014). Generally, our and other researcher’s studies were revealed that FLB and RXM have cytotoxic effects separately. When we used the particular combination of these drugs, we did not find a statistically significant cytotoxic effect on cells. This suggests that FLB and RXM have antagonistic effect according our study. In the literature, we could not find any study which indicates the antagonistic effect of combination of an analgesic and an antibiotic. But, Paclitaxel and Carboplatin are chemotherapy drugs which have apoptotic effects on cells. There is a study which indicates that these drugs have an antagonistic effect when they are used together in vitro hormone-refractory prostate cancer system (Cabrespine et al., 2005). As seen, drugs can have such an effect against each other. Researchers reported that the combination of Rapamycin, an antibiotic from the macrolide group like RXM, and celecoxib, a NSAID drug like FLB, had a synergetic effect on chronic myelogenous leukemia cells. The combination had more antitumor effects than mono treatment of these drugs (Li et al., 2014). In another study, the combination of celecoxib and minocycline hydrochloride, a tetracycline antibiotic, had synergetic effect on breast cancer inhibiting in vitro. In the study, tumor weights and volumes were significantly lower and the tumor inhibition rate was significantly higher in the group treated with the combination than those of the control group and the group treated with celecoxib or minocycline hydrochloride alone (Niu et al., 2008).
FLB did not have DNA damaging effect on the plasmid. However, it had DNA protection effect against H2O2on the
plasmid. This is the first report mentioned about DNA protective effect of a NSAID. This results supports the findings about FLB’s genotoxicity (Timocin & Ila, 2015; Timocin et al., 2016). This first report of RXM’s DNA damaging and protection activity showed that RXM did not have damaging or protection activity on DNA. These result support finding of our previous research about RXM’s genotoxicity (unpublished data). Talking about the effects of the combination, it did not show damaging activity. But the image of plasmid treated by FLB and H2O2along with RXM
showed that RXM neutralized the antioxidant effect of FLB. In a study, RXM inhibited oxidative stress in a rat hepatocarcinogenetic model (Ueno et al., 2005). In our previous study, FLB did not change total oxidant and antioxidant status and hence oxidative stress in rat bone marrow cells (Timocin & Ila, 2015). According to research-ers, FLB did not have any effect on oxidative stress induced
Figure 5. Control (plasmid DNA); (2) 2 mL DMSO; (3) 20 mg/mL flurbiprofen + 50 mg/mL roxithromycin; (4) 30 mg/mL flurbiprofen + 100 mg/mL roxithromycin; (5) 40 mg/mL flurbiprofen + 200 mg/mL roxithromycin; (6) 2 mL DMSO + 3 mL H2O2; (7) 20 mg/mL flurbiprofen
+ 50 mg/mL roxithromycin + 3 mL H2O2; (8) 30 mg/mL flurbiprofen + 100 mg/mL roxithromycin + 3 mL H2O2; (9) 40 mg/mL flurbiprofen + 200 mg/mL roxithromycin + 3 mL H2O2.
Figure 4. Control (plasmid DNA); (2) 2 mL DMSO; (3) 50 mg/mL roxithromycin; (4) 100 mg/mL roxithromycin; (5) 200 mg/mL roxithro-mycin; (6) 2 mL DMSO + 3 mL H2O2; (7) 50 mg/mL roxithromycin +
3 mL H2O2; (8) 100 mg/mL roxithromycin + 3 mL H2O2; (9) 200 mg/mL
by aluminum in rats (Nivsarkar et al., 2008). On the contrary, it has been revealed that FLB had the scavenging effect on reactive oxygen and reactive nitrogen species (Costa et al., 2006). In our study, the particular combination of FLB and RXM did not change total oxidant, antioxidant and hence oxidative stress values. As seen, FLB and RXM did not have an antagonistic or synergetic effect together.
FLB and RXM which are set out as not a genotoxic agent in previous studies did not have a genotoxic effect together in our study. Also, plasmid assays showed that these drugs separately or together did not have DNA damaging effect. It may have occurred depending on the drugs made each other passive by interacting. It has been already revealed that FLB and RXM had a cytotoxic effect separately. In our study, surprisingly, the combination of these drugs did not have a cytotoxic effect in cultured cells. This shows that they have an antagonistic effect together. After all, it can lead us to develop different viewpoints.
Acknowledgements
This study was carried out by our own laboratory facilities.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Test substance Time (h) Conc. (mg/mL) Mean TOS values ± SE Mean TAS values ± SE Mean OSI ± SE DMSO 24 10 mL 3.234 ± 0.50 0.3217 ± 0.011 10.14 ± 1.70 MMC 24 150 1.752 ± 0.08 0.3228 ± 0.030 5.665 ± 0.88 FLB + RXM 24 10 + 25 3.960 ± 0.38 b3 0.3130 ± 0.009 12.59 ± 0.85 b3 FLB + RXM 24 15 + 50 3.958 ± 0.31 b3 0.3060 ± 0.034 13.17 ± 1.02 b3 FLB + RXM 24 20 + 100 4.094 ± 0.45 b3 0.3080 ± 0.013 13.27 ± 1.22 b3 DMSO 48 10 mL 1.976 ± 0.41 0.3292 ± 0.019 5.97 ± 1.07 MMC 48 150 1.828 ± 0.19 0.3453 ± 0.015 5.27 ± 0.40 FLB + RXM 48 10 + 25 3.005 ± 0.42 0.3090 ± 0.010 9.69 ± 1.19 b1 FLB + RXM 48 15 + 50 3.036 ± 0.34 0.3232 ± 0.013 9.54 ± 1.42 b1 FLB + RXM 48 20 + 100 3.234 ± 0.50 b1 0.3242 ± 0.012 9.96 ± 1.21 b1
SE, standard error; TOS, total oxidant status; TAS, total antioxidant status; OSI, oxidative stress index; FLB, flurbiprofen; RXM, roxithromycin. b: significant from MMC (positive control); b1: p 0.05; b3: p 0.001.
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