Toxicology Mechanisms and Methods
ISSN: 1537-6516 (Print) 1537-6524 (Online) Journal homepage: http://www.tandfonline.com/loi/itxm20
Oxcarbazepine-induced cytotoxicity and genotoxicity in human lymphocyte cultures with or without metabolic activation Zülal Atlı Şekeroğlu, Haluk Kefelioğlu, Seval Kontaş Yedier, Vedat Şekeroğlu & Berrin Delmecioğlu To cite this article: Zülal Atlı Şekeroğlu, Haluk Kefelioğlu, Seval Kontaş Yedier, Vedat Şekeroğlu & Berrin Delmecioğlu (2016): Oxcarbazepine-induced cytotoxicity and genotoxicity in human lymphocyte cultures with or without metabolic activation, Toxicology Mechanisms and Methods, DOI: 10.1080/15376516.2016.1273430 To link to this article: http://dx.doi.org/10.1080/15376516.2016.1273430
Accepted author version posted online: 20 Dec 2016.
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Date: 21 December 2016, At: 04:23
Oxcarbazepine-induced cytotoxicity and genotoxicity in human lymphocyte cultures with or without metabolic activation Zülal Atlı Şekeroğlu1*, Haluk Kefelioğlu2, Seval Kontaş Yedier1, Vedat Şekeroğlu1, Berrin Delmecioğlu2 1
Department of Biology, Faculty of Science and Letters, Ordu University, 52200 Ordu,
Turkey 2
Department of Biology, Faculty of Science and Letters, Ondokuz Mayıs University, 55139
Samsun, Turkey Correspondence to: Zülal Atlı Şekeroğlu, Department of Biology, Faculty of Science and
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E-mail:
[email protected] or
[email protected]
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Telephone: +90 4522345010/1667 Fax: +90 4522339149
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Letters, Ordu University, 52200 Ordu, Turkey.
Abstract
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There has been considerable debate about the relationship between epilepsy and cancer. Oxcarbazepine (OXC) is used for treating certain types of seizures in patients with epilepsy. There have been no detailed investigations about genotoxicity of OXC and its metabolites. Therefore, the aim of this study is to investigate the cytotoxic and genotoxic effects of OXC
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and its metabolites on cultured human lymphocytes. The cytotoxicity and genotoxicity of OXC on human peripheral blood lymphocytes were examined in vitro by sister chromatid
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exchange (SCE), chromosomal aberration (CA), and micronucleus (MN) tests. Cultures were treated with 125, 250 and 500 µg/ml of OXC in the presence (3 h treatment) and absence (24 h and 48 h treatment) of a metabolic activator (S9 mix). Dimethyl sulfoxide (DMSO) was used as a solvent control. OXC showed cytotoxic activities due to significant decreases in mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) in the absence of S9 mix when compared with solvent control. Metabolites of OXC also significantly reduced
MI and PI in cultures with S9 mix. OXC significantly increased the CAs, aberrant cells, SCE and MN values in the presence and absence of S9 mix. Our results indicated that both OXC and its metabolites have cytotoxic, cytostatic and genotoxic potential on human peripheral blood lymphocyte cultures under the experimental conditions. Further studies are necessary to elucidate the relationship between cytotoxic, cytostatic and genotoxic effects, and to make a possible risk assessment in patients receiving therapy with this drug. Keywords: oxcarbazepine; cytotoxicity; chromosome aberration; micronucleus; sister chromatid exchange Introduction
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Oxcarbazepine (OXC), an antiepileptic drug, currently widely registered worldwide (Sarikaya
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and Yüksel, 2008). It is an analogue of carbamazepine, with a comparable anticonvulsant
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efficacy. It exhibits similar chemical and therapeutic properties but fewer side effects such as low incidence of allergic reactions and enzyme induction. In case of combination therapy with
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other antiepileptic drugs, OXC is usually better tolerated than carbamazepine (Ambrosio et al., 2000; Sarikaya and Yüksel, 2008).
It has been stated that some antiepileptic drugs display some adverse effects which are a leading cause of treatment failure with these drugs. (Perucca and Gilliam, 2012). First and
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second generation antiepileptic drugs are associated with substantial risk of coordination difficulties. In a meta-analysis of randomized, placebo-controlled, adjunctive treatment trials
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of eight antiepileptic drugs including OXC, active treatment at any dose was associated with an almost three-fold increase in risk of coordination disturbances. Risk was particularly high for OXC, lamotrigine, topiramate and pregabalin (Perucca and Gilliam, 2012). Most of the evidence confirms that monotherapy with the most commonly used antiepileptic drugs is associated with a two to three times greater risk in birth defects (Perucca, 2005). Prenatal exposure to antiepileptic drugs, particularly in the first trimester, is associated with a higher
risk for major congenital fetal malformations and postnatal developmental anomalies than observed in the general population (Witczak et al., 2010; Perucca and Gilliam, 2012). There has also been considerable debate about the relationship between epilepsy and cancer, in particular whether the incidence of cancer is increased in people with epilepsy and whether antiepileptic drugs promote or protect against cancer. Although the potential for antiepileptic drugs to be carcinogenic was investigated in experimental, epidemiological and clinical research, particularly in the late 1960s, the issue has not attracted much attention (Singh et al., 2005). A literature survey has shown that genotoxicity and carcinogenicity data of OXC are
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very limited. Most of the data are belong to unpublished reports in the Physicians’ Desk
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Reference (PDR 2005). According to these reports, it yielded positive results in Ames and
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some in vitro cytogenetic tests. There were also positive results for carcinogenicity in both mice and rats (PDR 2005; Brambilla et al., 2009). Genotoxicity of OXC has not been
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investigated in human in detail. In addition, cytotoxic and genotoxic effects of both OXC and its metabolites using different genotoxicity tests at different treatment times have not been studied in cultured human peripheral blood lymphocytes with and without S9 mix. Taking into account the lack of information, we evaluated the cyto-genotoxic effects of this drug on
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human peripheral blood lymphocyte cultures in the presence and absence of S9 mix. For this purpose, the in vitro cytotoxic and genotoxic activities of OXC were evaluated by using by
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using chromosomal aberration (CA), sister chromatid exchange (SCE) and MN tests as genetic endpoints. The mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) were also calculated to evaluate cytotoxic/cytostatic effects of OXC.
Materials and methods Chemicals Oxcarbazepine (OXC) (trade names; Oxtellar XR, Trileptal, Oxcarpin, Epsile, Apilep) was obtained from Tokyo Chemical Industry Co., Ltd. (Portland, OR, USA) (>98%, CAS Number 28721-07-5). The chemical structure of OXC (10,11-dihydro- 10-oxo- 5H-dibenz(b,f)azepine5-carboxamide) is shown in Fig. 1. The test substance was dissolved in dimethyl sulfoxide (DMSO) supplied by Merck (Darmstadt, Germany, CAS 67-68-5). In the cultures without metabolic activation, Mitomycin-C (MMC) (Serva, Heidelberg, Germany, CAS 50-07-7) was used as positive control at 0.16 µg/ml for treatments. For cultures with metabolic activation,
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cyclophosphamide monohydrate (CP, Fisher Scientific, New Jersey, USA, CAS 6055-19-2)
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was used as a positive control at 45 µg/ml. They were dissolved in sterile distilled water.
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Cytochalasin-B (Cyt-B) was obtained from Sigma (Missouri, USA, CAS 14930-96-2) and colcemid was supplied by Biological Industries (Beit Haemek, Israel, Cat. No. 12-004-1).
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Human liver S9 pooled donors (20 mg/ml, product number 452961), NADPH Regenerating System Solution A (Product number 451220) and NADPH Regenerating System Solution B (Product number 51200) were supplied by Corning® Gentest™ (Massachusetts, USA). Other chemicals used for fixation and staining were obtained from Merck. All test solutions were
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freshly prepared prior to each experiment. Lymphocyte cultures and concentration selection
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Blood samples from four healthy and non-smoking donors (two males and two females) aged between 20 and 24 years were obtained by venipuncture and collected into heparinized tubes. All volunteers gave informed consent to participate in the study and signed consent forms. The study was approved by the Clinical Research Ethics Committee of University of Ondokuz Mayıs, Turkey (2013/492).
Heparinized whole blood (0.3 ml) was added to 2.5 ml of RPMI 1640 medium (Sigma), supplemented with 20% fetal bovine serum, 1% antibiotics (penicilin, streptomycin and amphotericin), 1.2% phytohemaglutinin and 2% L-glutamine. Negative control cultures were treated with DMSO at a final volume of 20 μl of DMSO. Positive control cultures were treated with MMC at a final concentration of 0.16 μg/ml (Kontaş and Atlı Şekeroğlu, 2015). In this study, the cytotoxic and genotoxic effects of OXC were tested at 125, 250 and 500 μg/ml concentrations. These concentrations were chosen on the basis of a preliminary cytotoxicity test using MI, an established standard assay for the detection of chemical compound genotoxicity on mammalian cell cultures in vitro. According to the preliminary
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cytotoxicity test procedure, we prepared a series of blood lymphocyte cultures of OXC at
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concentration range between 0 and 2000 μg/ml. MI frequency was scored in each OXC
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culture as well as in the solvent control culture. MI values decreased linearly as OXC concentration increased. Average 70, 65, 59, 51, 44, 39 and 34 metaphases in 2000 cells were
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found at concentrations of 0, 50, 100, 200, 300, 400 and 500, respectively. No metaphases were observed at 1000 and 2000 μg/ml concentrations. A concentration of 500 μg/ml that reduced the MI to about 50% (50% inhibitory concentration, IC50) was used as the highest concentration in the cytogenetic analysis. The concentrations of 250 μg/ml (half of IC50) and
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125 μg/ml (one-fourth of IC50) were taken as medium and low concentrations in this study, respectively. OXC concentrations were prepared by dissolving in DMSO, and they added a
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constant volume of 20 μl for each test concentration. MN, CA and SCE assay (without S9 mix) SCE and CA analyses were performed using the methods developed by Evans (1984) and Perry and Thompson (1984). Briefly, lymphocyte cultures were set up by adding 0.3 ml of whole blood from each of four healthy donors to 2.5 ml of medium. The blood cultures were incubated for 72 h at 37°C. The cultures were treated with OXC at three concentrations (125,
250 and 500 µg/ml) for 24 h and 48 h. 5-bromodeoxyuridine (10 µg/ml) was added to medium at the beginning of the SCE cultures. Colcemid at a final concentration of 0.06 μg/ml was added 1 h before harvesting to the all cultures. The cells were harvested by replacing the culture medium with a hypotonic solution (0.4% KCI) in which cells were incubated for 15 min at 37°C. Until the supernatant is clear and the pellet become white, the cells were fixed at least three times with methanol and acetic acid (3:1 v/v). To prepare the slides, the fixed cell suspension were dropped on clean and cold slides and air-dried. The slides were stained with 5% Giemsa in Sorenson's buffer (pH 6.8) for 15 min for CA analysis, and with a modified Fluorescence plus Giemsa method (FPG) for SCE analysis (Speit and Haupter 1985). After
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Giemsa in Sorenson's buffer (pH 6.8) for 20 min.
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irradiation, the slides were incubated in 1X SSC at 60 °C for 60 min and stained with 5%
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The cytokinesis-block micronucleus (CBMN) assay was carried out using technique of Fenech (2000). Peripheral lymphocyte cultures were incubated at 37°C for 68 h. The cultures
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were treated with OXC (125, 250 and 500 µg/ml) for 24 h and 48 h. Cyt-B, an inhibitor of actin polymerization, (final concentration of 8 μg/ml) is added 44 h after initiation of the cultures in order to obtain binucleated (BN) cells. The cells were harvested by centrifugation and the pellet was re-suspended in hypotonic solution for 5 min. After the cells were fixed in
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a cold fixative, slides were prepared and air-dried. The slides were stained with 5% Giemsa stain solution (diluted with Sorenson's buffer, pH 6.8) for 15 min.
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MN, CA and SCE assays (with S9 mix) Human liver S9 microsomal fraction and NADPH generation system (NADPH regenerating system solution A and solution B) were used for metabolic activation. The S9 mixture was prepared in 1X Tris buffer, pH 7.6. The final concentration of S9 mixture in the culture medium was 2%. The cultures were incubated for a total of 72 h at 37˚C. All test chemicals (DMSO, CP or three different concentrations of OXC) and S9 mix were added 48 h after
initiating the cultures. While the cultures without S9 were treated with OXC for 24 and 48 h, the cultures with S9 mix were exposed to the test compound only for 3 h. After the incubation period, the cultures were centrifuged at 2000 rpm for 4 min. After the supernatant was aspirated, the pellet was washed twice with 2.5 ml fresh medium. 5-bromodeoxyuridine (10 µg/ml) for SCE cultures and Cyt-B (8 μg/ml) for MN cultures were also added to the cells which were re-suspended in fresh medium. All cultures were incubated for an additional 24 h prior to harvesting. Colcemid was added to CA and SCE cultures 1 h before harvesting. A solvent control (DMSO) and a positive control (cyclophosphamide monohydrate, 45 µg/ml) were also included with each experiment. In the presence of S9 mix, CP was used as a
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positive control because CP required a metabolic activator for the presence of its mutagenic
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effect. The cultures were harvested in the same way as in cultures without S9 mix.
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Scoring
Slides were examined using a Leica DM2500 light microscope at 1000X magnification. All
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slides were coded and scored blind. The MI was calculated as the number of metaphases in 2000 cells (a total of 8000 cells) analyzed per culture, for each treatment and donor. For the analysis of CAs, 100 well-spread and intact metaphases were analyzed per culture, for each treatment and donor (a total of 400 metaphases). Twenty-five second-division metaphase cells
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were analyzed per culture, for each treatment and donor for SCE scoring. A total of 400 cells (100 cells per donor) were scored to determine the PI, which was calculated using the
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formula: PI= 1 X M1 + 2 X M2 + 3 X M3 / 100, where M1, M2, and M3 represent the number of cells undergoing first, second and third mitosis, respectively. For the MN analysis, the number of MN in 2000 BN cells was scored per culture, for each treatment and donor giving a total of 8000 BN cells. For the determining NDI, the number of cells containing 1-4 nuclei in 1000 cells was scored per culture, for each treatment and
donor.
The
NDI
was
calculated
using
the
following
formula:
NDI=1×M1+2×M2+3×M3+4×M4/1000; where M1 through M4 represent the number of cells with one to four nuclei (Eastmond and Tucker 1989). Statistical analysis All of the subjects (i.e., the four donors; n=4) were used as the experimental unit (n) for all statistical analyses. Statistical analysis was performed using one-way analysis of variance (ANOVA). The experimental values were expressed as the mean ± standard error (SE). Comparisons of parameters between the each treatment group and solvent control were evaluated with Student’s t test. P
