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Drug and Chemical Toxicology
ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: http://www.tandfonline.com/loi/idct20
Assessment of in vitro genotoxic and cytotoxic
effects of flurbiprofen on human cultured
lymphocytes
Taygun Timocin, Hasan Basri Ila, Tuba Dordu, Mehmet Tahir Husunet,
Mostafa Norizadeh Tazehkand, Ebrahim Valipour & Mehmet Topaktas
To cite this article: Taygun Timocin, Hasan Basri Ila, Tuba Dordu, Mehmet Tahir Husunet, Mostafa Norizadeh Tazehkand, Ebrahim Valipour & Mehmet Topaktas (2016) Assessment of in vitro genotoxic and cytotoxic effects of flurbiprofen on human cultured lymphocytes, Drug and Chemical Toxicology, 39:3, 338-343, DOI: 10.3109/01480545.2015.1121276To link to this article: http://dx.doi.org/10.3109/01480545.2015.1121276
Published online: 07 Jan 2016.
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ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2016; 39(3): 338–343
!2016 Taylor & Francis. DOI: 10.3109/01480545.2015.1121276
RESEARCH A RTICL E
Assessment of in vitro genotoxic and cytotoxic effects of flurbiprofen on
human cultured lymphocytes
Taygun Timocin1, Hasan Basri Ila2, Tuba Dordu3, Mehmet Tahir Husunet1, Mostafa Norizadeh Tazehkand3, Ebrahim Valipour3, and Mehmet Topaktas2
1Department of Biology, Institute of Science, Cukurova University, Adana, Turkey,2Department of Biology, Faculty of Science and Letters,
Cukurova University, Adana, Turkey, and3Department of Biotechnology, Institute of Science, Cukurova University, Adana, Turkey
Abstract
Flurbiprofen is non-steroidal anti-inflammatory drug which is commonly used for its analgesic, antipyretic, and anti-inflammatory effects. The purpose of the study was to explore the genotoxic and cytotoxic effects of flurbiprofen in human cultured lymphocytes by sister chromatid exchange, chromosome aberration, and cytokinesis-blocked micronucleus tests. 10, 20, 30, and 40 mg/mL concentrations of flurbiprofen (solvent is DMSO) were used to treatment of human cultured lymphocytes at two different treatment periods (24 and 48 h). Flurbiprofen had no significant genotoxic effect in any of these tests. But exposing to flurbiprofen for 24 and 48 h led to significant decrease on proliferation index, mitotic index, and nuclear division index (NDI). Also, all decreases were concentration-dependent (except NDI at 24 h treatment period). Consequently, the findings of this research showed that flurbiprofen had cytotoxic effects in human blood lymphocytes.
Keywords
Flurbiprofen, human cultured lymphocytes, SCE, CA, MN, cytotoxicity
History
Received 18 June 2015 Revised 21 September 2015 Accepted 13 November 2015 Published online 8 January 2016
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are com-monly sold in the worldwide (Brambilla & Martelli, 2009). They are used for their analgesic (pain-killing), antipyretic (fever-reducing), and in higher doses, anti-inflammatory effects (Tewari et al., 2010). Flurbiprofen, one of the NSAIDs, is a phenylalkanoic acid derivative and used in the treatment of gout, osteoarthritis, rheumatoid arthritis, and sunburn (Fang et al., 2003) and also for mouth hygiene as a toothpaste and mouthwash (Battisti, 1994). In addition, flurbiprofen lozenge is efficient for sore throat and upper airway diseases (Watson et al., 2000). It shows its anti-inflammatory, anti-pyretic, and analgesic effect by inhibits the activity of cyclooxygenase, leading to the suppression of prostaglandin synthesis (Kawadkar et al., 2013).
Chromosome mutations and related genetic changes are cause of many human genetic diseases and there is much evidence that the changes induce cancer in human and experimental animals by effecting oncogenes or tumor suppressor genes (OECD, 1997, TG 473). Therefore, drugs must be investigated about their genotoxic potential (Jaju et al., 1984). In the literatures, studies on the genotoxic and cytotoxic effects of flurbiprofen are limited. In one study, it is demonstrated that flurbiprofen did not have any genotoxic
effect in peripheral blood cells in vivo (Kullich & Klein, 1986). Meanwhile, some NSAID drugs, which belongs profens group (flurbiprofen is member of this group according to World Health Organization), did not have genotoxic effect (Falfushynska et al., 2014; Koseoglu et al., 2008; Oldham et al., 1986; Williams et al., 1989). But, they had cytotoxic and apoptotic effects on cells (Jurima-Romet et al., 1994; Kim et al, 2004; Koga et al., 2011; Rakesh et al., 2014). Flurbiprofen caused lactate dehydrogenase (LDH) release related with cytotoxicity in rat hepatocytes culture (Jurima-Romet et al., 1994; Masubuchi et al., 1998). In a study of human polymorphonuclear neutrophils, flurbiprofen induced apoptosis (Zielinska-Przyjemska et al., 2008). In addition, flurbiprofen increased apoptotic DNA fragmentation and mRNA expression involved COX-2 in human gastric epithelial cells (Kusuhara et al., 1999). R-flurbiprofen, a single enantiomer of R-flurbiprofen, was led to apoptosis in human colon carcinoma cells (Grosch et al., 2005) and in gastric cancer cells (Jin et al, 2010). Also, it reduced proliferation in pituitary adenoma cell culture (Liu et al., 2012).
As a result, the purpose of this study was to evaluate the genotoxic and cytotoxic potential of flurbiprofen. Genotoxic potential of flurbiprofen was determined by sister chromatid exchange (SCE), chromosome aberration (CA), and cytokin-esis-blocked micronucleus (CBMN) tests in human cultured lymphocytes. Also, effect of flurbiprofen on cell proliferation was determined by proliferation index (PI), mitotic index
Address for correspondence: Taygun Timocin, Department of Biology, Institute of Science, Cukurova University, Adana, Turkey. E-mail: ttimocin@hotmail.com
(MI), and nuclear division index (NDI) in human cultured lymphocytes.
Materials and methods
Chemical substances
Flurbiprofen was purchased from Sigma-Aldrich (CAS 5104-49-4; Steinheim, Germany), and its molecular structure is shown in Figure 1. Purity of flurbiprofen was 98.5%. Dimethyl sulfoxide (DMSO) (CAS No: 67-68-5; Merck, Darmstadt, Germany) was used as solvent control. PB-Max chromosome medium was used to culture cells (Gibco, CAT No: 12557-013). Colchicine (CAS No: 64-86-8), bromodeox-yuridine (CAS No: 59-14-3), and cytochalasin B (CAS No: 14930-96-2) was purchased from Sigma-Aldrich.
Experimental design
This study was performed with permission of Cukurova University Ethics Committee (36/2). Human blood was provided from four healthy volunteers (non-smokers two males and two females, ages: 22–25) and treated with four concentrations of flurbiprofen (10, 20, 30, and 40 mg/mL; determined by LD50) at two different treatment periods (24 and 48 h). Also, solvent control (DMSO; 3.7 mL for 1 mL medium) was also used for two treatment periods.
In vitro chromosome aberration and sister chromatid exchange tests
This study was conducted according to IPCS (International Programme on Chemical Safety) guidelines (Albertini et al., 2000). 0.2 mL whole blood was added to 2.5 mL chromosome medium. At the same time, 10 mg/mL bromodeoxyuridine was added to medium. Blood cultures were incubated at 37C for 72 h. 10, 20, 30, and 40 mg/mL concentrations of flurbiprofen were added to culture 48 h after initiating the culture (24 h treatment period) and added 24 h after initiating the culture (48 h treatment period). The cells were treated with 0.06 mg/ mL colchicine 2 h before harvesting time to arrest cells in metaphase. At the end of 72 h incubation, the cells were centrifuged at 2000 rpm for 5 min. Then, the cells were treated with 0.4% KCl (hypotonic solution), incubated at 37C for 8 min and centrifuged at 1200 rpm for 10 min. After centri-fugation process, the cells were exposed to cold fixative (1/3 acetic acid/methanol), incubated at room temperature (15 min) and then centrifuged at 1200 rpm for 10 min. This fixative process was repeated three times in total. After that, the cells were spread on cold glass slides. Then, slides were stained with 5% Giemsa (prepared in Sorenson buffer) for CA and stained with fluorescence plus Giemsa method for SCE (Speit & Haupter, 1985). The cells were irradiated with 30 W, 254 nm UV lamp at 15 cm distance in Sorensen buffer for
30 min, then incubated in 1 X SSC (standard saline citrate) at 60C for 60 min and stained with 5% Giemsa prepared with Sorensen buffer. Chromosome aberrations and SCEs were used to investigate the genotoxicity. Chromosome aberrations were explored by examination of metaphases of 100 well-spread samples for each donor (400 metaphases for each group). Structural chromosome aberrations were used to determine genotoxicity. Structural CAs scoring was performed with following classification: chromatid-type aberrations (fragments, breaks, and exchanges) and chromo-some-type aberrations (fragments, breaks, sister chromatid union, rings, and dicentrics). Percentage of abnormal cell (cell with CA) and CA/Cell ratio were calculated. The formation of SCE was explored by the examination of 25 s metaphases (100 s metaphases for each group). PI and MI were used to investigate cytotoxic effect of flurbiprofen. PI was calculated by the following formula: PI¼ [(1 M1) + (2 M2) + (3 M3)]/total score cells (100 cells per donor). M1, M2, and M3 are cells within the first, second, and third mitosis during the incubation time of cell culture. MI was determined by analyzing 3000 cells from each donor and scoring the cells that were in metaphase or not.
In vitro cytokinesis-blocked micronucleus test
The CBMN test was performed as described by Rothfuss et al. (2000) with some modifications. 0.2 mL peripheral blood was added to 2.5 mL PB-Max chromosome medium and incubated at 37C for 68 h. Flurbiprofen concentrations were added 44 h after initiating the culture (24 h treatment period) and added 20 h after initiating the culture (48 h treatment period). To block cytokinesis, 44 h after the start of incubation, 6 mg/mL cytochalasin B was added to the culture. At the end of incubation time (68 h), the cells were centrifuged at 2000 rpm for 5 min and exposed to 37C 0.4% KCl (hypotonic solution). The cells were centrifuged at 1200 rpm for 10 min. Then, the cells were fixed one time with cold fixative (1/5/6 glacial acetic acid/methanol/NaCl isotonic solution) for 20 min and then 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. The cells were spread on cold glass slides, dried and stained with 5% Giemsa. 1000 binucleated lymphocytes were examined from each donor (total 4000 binucleated cells for each group) to determine the percentage of MN and percentage of micronucleated binuclear (MNBN) cell fre-quency, which is indicator of genotoxic effect. To evaluate cytotoxic effect, 1000 cells for each donor (4000 cells for each group) were scored as 1, 2, 3, or 4 nuclei and used following formula to calculate NDI: [(1 M1) + (2 M2) + (3 M3) + (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 (Fenech, 2000).
Statistical analysis
All values were presented as mean ± standard error (SE). The data had a normal distribution (Shapiro–Wilk) and were analyzed with one-way analysis of variance (ANOVA) and post hoc Dunnett’s test, using SPSS software (Chicago,
Figure 1. Molecular structure of flurbiprofen (C15H13FO2).
IL). Concentration-dependent effect was determined by Pearson Correlation. In comparisons, ‘‘0.05’’ was accepted as significant.
Results
Genotoxicity of flurbiprofen
Comparisons showed that flurbiprofen did not induce SCE frequency at any concentrations for any treatment periods (Table 1). When abnormal cell and CA/Cell ratio values were compared with DMSO groups, no significant differences were found at any different treatment periods (24 and 48 h) (Table 2). Similar to SCE and CA results, there were not significant differences on MN and MNBN values when compared with DMSO control groups at any treatment periods (Table 3). Effects of flurbiprofen on cell division
Effects of flurbiprofen on cell division were determined by PI, MI, and NDI data (Table 4). Considering the results of PI, flurbiprofen was cytotoxic. It reduced PI at 30 and 40 mg/mL concentrations for 24 h treatment period and all concentra-tions for 48 h treatment period. Additionally, reduction of PI was concentration-dependent manner for both treatment periods (Figure 2). As compared to DMSO treatments, flurbiprofen reduced MI at all concentrations for 24 and 48 h treatment. Also, reduction of MI was concentration-dependent manner for both treatment periods (Figure 2).
When compared with DMSO treatments, flurbiprofen reduced NDI at two highest concentrations (30 and 40 mg/mL) for 24 h treatment and at three concentrations (20, 30, and 40 mg/mL) for 48 h treatment. Also, reduction of NDI for 48 h treatment period was concentration-dependent manner (Figure 2).
Discussion
Flurbiprofen did not statistically induce SCE, chromosome aberration or micronuclei. According to these results, flurbiprofen is not a genotoxic agent. In one study, SCE rates were measured before and after flurbiprofen application (two weeks) in human lymphocytes in vivo. Eventually, flurbiprofen did not induce SCE. In the same study; NSAIDs such as diclofenac, ibuprofen, indomethacin, isoxicam, ketoprofen, piroxicam, pirprofen, and tiaprofenic acid did not induce SCE, too (Kullich & Klein, 1986). Ibuprofen, naproxen, and ketoprofen did not lead to reverse mutation on Salmonella TA97, TA100, and TA102 strains. But consider-ing the frequency of SCE, they had weak genotoxic effects on mouse bone marrow cells (Philipose et al., 1997). This finding is opposite to our findings in this study. In salmonella plate test with TA98, TA100, TA1535, TA1537, and TA1538 strains, ibuprofen did not reveal any genotoxic effect (Oldham et al., 1986). Different doses of ibuprofen (10, 20, 40, and 60 mg/kg body weight) was administered to mice and, unlike previous studies, it was reported that ibuprofen was genotoxic in higher concentrations (40 and 60 mg/kg) (Tripathi et al., 2012). As a result, most genotoxicity studies of flurbiprofen and other NSAIDs in the literature support our report about genotoxic effect of flurbiprofen.
In this study, flurbiprofen showed cytotoxic activity in three tests. In an earlier study, similar to our results, 48 h treatments of flurbiprofen, ketoprofen, and naproxen (200 mM) leaded to apoptosis in squamous cell carcinoma lines in vitro, inducing caspase-3 enzyme activity (Bock et al., 2007). In addition, it has been shown that flurbiprofen leaded to LDH leakage, which is related with cytotoxicity, in rat hepatocytes cultures (Jurima-Romet et al., 1994; Masubuchi et al., 1998). Researchers have reported that flurbiprofen inhibited the growth of A-431 human epidermoid carcinoma cells and cleaved transcription factor b-catenin (Nath et al., 2013). On the contrary, in one study, flurbiprofen protected hepatocytes from apoptosis-inducing hepatic ischemia/reper-fusion (Fu et al., 2012). In addition, flurbiprofen, ibuprofen,
Table 2. Chromosome aberrations types, abnormal cell percentage, and CA/Cell ratio in human cultured lymphocytes treated with flurbiprofen. Treatment Structural CA
Test substance Time (hour) Conc. (mg/mL) B0type B00type
Abnormal cell % ± SE CA/Cell ± SE DMSO 24 3.7 mL 16 1 4.25 ± 0.750 0.0425 ± 0.00750 FLB 24 10 15 2 4.25 ± 0.479 0.0425 ± 0.00479 20 18 2 4.75 ± 0.629 0.0500 ± 0.00707 30 17 4 5.25 ± 0.750 0.0525 ± 0.00750 40 15 2 3.75 ± 0.479 0.0425 ± 0.00629 DMSO 48 3.7 mL 20 3 5.00 ± 0.577 0.0575 ± 0.00479 FLB 48 10 15 2 4.00 ± 0.707 0.0425 ± 0.00629 20 20 3 5.75 ± 0.479 0.0575 ± 0.00479 30 24 0 5.75 ± 0.750 0.0600 ± 0.00707 40 19 2 5.00 ± 0.408 0.0525 ± 0.00479
FLB, flurbiprofen; B0, chromatid type CA; B00, chromosome type CA. Table 1. Minimum–maximum SCE and mean SCE values in human cultured lymphocytes treated with flurbiprofen.
Treatment Test substance Time (hour) Conc. (mg/mL) Min–Max SCE SCE/Cell ± SE DMSO 24 3.7 mL 1–15 6.28 ± 0.16 FLB 24 10 1–16 6.25 ± 0.29 20 1–14 6.72 ± 0.23a 30 1–17 6.30 ± 0.23 40 1–15 5.74 ± 0.44 DMSO 48 3.7 mL 1–17 6.52 ± 0.41 FLB 48 10 0–16 5.96 ± 0.39 20 1–16 6.33 ± 0.20 30 1–19 6.95 ± 0.31 40 1–15 6.24 ± 0.33 FLB, flurbiprofen.
and diclofenac did not induce apoptosis in mouse fibrosar-coma cells (Hoferova et al., 2004). Fratelli et al. (2003) reported that nitric oxide-releasing NSAIDs were safer than their parents. They demonstrated that flurbiprofen nitroxybu-tyl ester inhibited activity of transcription nuclear factor (NF)-kappaB and cell growth in L929 (mouse fibroblast) cells at a concentration of 100 mM. In colon (SW480) and breast (MCF-7) cancer cell lines, phospo-flurbiprofen inhibited prolifer-ations and when compared to chemotherapeutic drugs, it induced apoptosis more effectively (Sun et al., 2011). An enantiomer of flurbiprofen, r-flurbiprofen (or tarenflurbil) leaded to significantly lower incidence of metastasis and a reduction in the primary tumor incidence (Wechter et al., 2000). Tarenflurbil inhibited transcription factor NF-kappaB (Tegeder et al., 2001), which is key regulator molecule especially organizes anti-apoptotic genes such as TRAF1 and TRAF2, so defects of these molecules may cause apoptosis and increased cell death (Sheikh & Huang, 2003). R-flurbiprofen and ibuprofen induced expression of p75 tumor suppressor gene and thereby reduced the cell growth in human prostate cancer lines (Andrews et al., 2008). According to Escarcega et al. (2007), inhibiting of activity of NF-kappaB leaded to block proliferation of tumor cells, eliminate cells or
make tumor cells to be more sensitive for antitumor agents. R-flurbiprofen had apoptotic activity on prostate tumors, inducing expression of p75 protein, which is related with tumor suppressor activity (Quann et al., 2007). R-flurbiprofen prevented metastasis of gastric cancer and decreased tumor size in vitro. It induced expression of tumor suppressor receptor p75 via the p38 signaling pathway (Jin et al., 2010). In another study, it was revealed that r-flurbiprofen decreased proliferation and induced apoptosis in GH4C1 pituitary adenoma cells in vitro (Liu et al., 2012). 30 mg/kg concen-tration of r-flurbiprofen and 10 mg/kg concenconcen-tration of racemic flurbiprofen enhanced apoptosis on Azoxymethane-induced rat colorectal cancer (Martin et al., 2013). Unlike to previous studies mentioned above, in one study, r-flurbiprofen prevented to cytotoxicity of amyloid beta or H2O2on cultured
human neuroblastoma cell lines and primary neurons (Zhao et al., 2008). In a study, ibuprofen inhibited the growth of cancer cell lines, inducing expression of p75 neurotrophin receptor (Khwaja et al., 2004). Tripathi et al. (2012) has demonstrated that ibuprofen reduced mitotic index in mouse at 40 and 60 mg/kg/day concentrations for two weeks. Heat shock protein 70 (Hsp70) is usually overexpressed in tumor cells. Researchers found that ibuprofen reduced the regulation
Table 4. Mean values of proliferation index (PI), mitotic index (MI), and nuclear division index (NDI) in human cultured lymphocytes treated with flurbiprofen.
Treatment Test substance Time (hour) Conc. (mg/mL) PI ± SE MI ± SE NDI ± SE DMSO 24 3.7 mL 2.120 ± 0.021 4.30 ± 0.05 1.294 ± 0.0089 FLB 24 10 2.050 ± 0.053 3.93 ± 0.08 a1 1.281 ± 0.0138 20 2.010 ± 0.042 2.99 ± 0.06 a3 1.247 ± 0.0084 30 1.928 ± 0.025 a2 2.66 ± 0.11 a3 1.202 ± 0.0041 a3 40 1.788 ± 0.041 a3 2.18 ± 0.07 a3 1.203 ± 0.0040 a3 DMSO 48 3.7 mL 2.120 ± 0.033 3.83 ± 0.07 1.271 ± 0.0010 FLB 48 10 1.923 ± 0.019 a1 3.33 ± 0.06 a2 1.242 ± 0.0088 b3 20 1.793 ± 0.040 a3 2.63 ± 0.13 a3 1.227 ± 0.0146 a1 30 1.713 ± 0.064 a3 2.24 ± 0.06 a3 1.172 ± 0.0049 a3 40 1.545 ± 0.033 a3 1.92 ± 0.06 a3 1.132 ± 0.0039 a3
FLB, flurbiprofen; a, significant from DMSO (solvent control); a1: p 0.05;
a2: p 0.01;
a3: p 0.001.
Table 3. Percentage of micronucleated binuclear (MNBN) cells and percentage of micronucleus (MN) in human cultured lymphocytes treated with flurbiprofen.
Treatment
Test substance Time (hour) Conc. (mg/mL) MNBN ± SE (%) MN ± SE (%)
DMSO 24 3.7 mL 0.63 ± 0.048 0.65 ± 0.065 FLB 24 10 0.70 ± 0.071 0.70 ± 0.071 20 0.58 ± 0.048 0.63 ± 0.063 30 0.70 ± 0.071 0.70 ± 0.071 40 0.75 ± 0.065 0.75 ± 0.065 DMSO 48 3.7 mL 0.65 ± 0.087 0.65 ± 0.087 FLB 48 10 0.88 ± 0.095 0.90 ± 0.108 20 0.68 ± 0.085 0.68 ± 0.085 30 0.75 ± 0.065 0.75 ± 0.065 40 0.78 ± 0.075 0.80 ± 0.091 FLB, flurbiprofen.
of Hsp70 in lung cancer cells in vitro and thereby it enhanced the antitumor activity of chemotherapy drug cisplatin in lung cancer cells (Endo et al., 2014). In the literature, flurbiprofen inhibited cell proliferation by inhibiting kappaB or transcrip-tion factor, inducing caspase activity, leading LDH leakage, or inducing tumor suppressor gene activity. As mentioned above, most of studies about flurbiprofen’s cytotoxicity support our result. Probably, flurbiprofen inhibited cell proliferation by one of these ways or more.
Conclusion
According to the findings of this research, flurbiprofen did not have genotoxic effect on human cultured lymphocytes. These results suggest that flurbiprofen is not a genotoxic agent. Nevertheless, flurbiprofen had significantly cytotoxic effects on human peripheral blood cells in vitro. Further, these cytotoxic effects were mostly concentration-dependent manner. In light of this information, it is demonstrate that flurbiprofen can be used as potential antiproliferative agent especially in cancer cases.
Declaration of interest
The authors report no financial conflicts of interest. This research was funded by Unit of Scientific Research Projects, Cukurova University (Project no: FBA-2015–3645).
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