The Assessment of Cytotoxicity and Genotoxicity of Tetracycline Antibiotic in Human Blood Lymphocytes Using CBMN and SCE Analysis, in Vitro

Tetracycline antibiotic, a widely used antimicrobial drug was tested for nuclear DNA damage in cultured peripheral blood lymphocytes in terms of chromosome alterations. The extent of cytogenetic damage, expressed as chromosome breakage and chromosome loss, was evaluated employing the Cytokinesis Block Micronucleus method (CBMN), the mutagenic effect of these antimicrobial drugs was investigated using Sister Chromatid Exchange (SCE) assay in cultured peripheral blood lymphocytes. Cytotoxicity was measured by evaluating mitotic activity (MA) and cytostatic effect was designated by measuring Proliferation Index and Nuclear Division index (NDI) in cultures. In this study,five different concentrations were used for tetracycline antibiotic. All the concentrations led to decrease in mitotic activity (p<0.05). SCE frequency was significantly different from negative control, (p<0.05) except at the concentration of 0.1μg/ml. All the concentrations induces the increase in MN frequency, but there is no significant difference between negative control and all the tested concentrations of tetracycline for micronucleus frequency (p>0.05). Tetracycline antibiotic has a moderate genotoxic from the point of view MN induction. Our results indicate that tetracycline has a positive response and is potentially more toxic than being genotoxic in the present study. continued medication (National Ambulatory Care Survey Data base 2007). Moreover, the drug has gained wide acceptance in the third world countries because of its easy accessibility, availability and cost effectiveness. As such, the drug can be abused due to self-prescription, which is rife in this part of the world. Among a wide variety of pharmaceutical compounds, antibiotics are of special concern due to their extensive use in human and veterinary medicine. Because of the increasing amount in the environment of antibiotics, their biological impacts have been widely discussed (Lindsey et al. 2001; Kolpin et al. 2002; Giger et al. 2003; Gao and Pedersen 2005). The presence of traces of this kind of pharma-ceutical in the environment can induce the development of antibiotic-resistant pathogens, causing serious problems for human health (Hirsch et al. 1999; Bautitz and Nogueira 2007). There are many antibiotics showing similar effects as TC. It is shown that these antibiotics also have toxic effects on non-target organisms. It is determined that erythromycin, lincomycin, clarithromycimn antibiotics lead the toxic effects on non-target organisms such as aquatic organisms (Isidori et al. 2005). Genotoxicity testing of pharmaceuticals prior to commercialization is obligated by regulatory agencies worldwide. For the most part, a three or AYLA ÇELIK AND DILEK EKE four-test battery including bacterial mutagenesis, in vitro mammalian mutagenesis, in vitro chromosome aberration analysis and in vivo chromosome stability assay are required (Synder and Green 2001). Therefore, it is important to determine the genotoxic potential of a drug that will be used in therapy, particularly in native human cells. In general, CAs, SCEs and micronuclei are considered to be essential markers of genotoxicity in in vitro studies. Thus, the aim of present study was to evaluate the genotoxic and cytotoxic effects of tetracycline, an antibiotic, on peripheral blood lymphocytes using the CBMN, SCE test and measuring MA and PRI or/and NDI respectively in vitro. 2. MATERIALS AND METHODS 2.1. The Collection of Blood Samples The study was carried out using human peripheral lymphocytes from fresh blood samples. Blood samples were obtained from five young non-smoking healthy donors. All donors were healthy and non-smokers. They were not exposed to X-ray or to treatment with any drugs. Approximately, 10 ml of blood was collected, by venipuncture, into syringes containing sodium heparin as anticoagulant. Lymphocyte cultures were used for the CBMN test and SCE assay. 2.2. The Preparation of Antibiotic Solutions Tetracycline hydrochloride (minimum purity 95 %, from SIGMA, 64-75-5 ) plasma concentration was considered for determination of doses of tested antibiotic. It is reported that the free blood concentrations of this drug is 1 μg/ml after usual therapeutic dose (Sakellari et al. 2000). In this study, dose range is 0,1-2 μg/ml. 2.3. The Preparation of Lymphocytes Cultures for SCE and CBMN Test 1. Sister Chromatid Exchange (SCE) 2. Cytokinesis Block Micronucleus Method (CBMN). 1. Sister Chromatid Exchange (SCE) Analysis: The study was carried out by using blood samples from five healthy non-smoking female donors, aged 22, 24, 24, 26 and 24 years. In the five donors, results of clinical routine laboratory analyses were in normal range, and the absence of exposure to known genotoxicants was considered. Briefly, lymphocyte cultures were set up by adding 0.5 ml of heparinized whole blood to 4.5 ml of RPMI 1640 chromosome medium supplemented with 15% heat-inactivated fetal calf serum, antibiotics (penicillin and streptomycin) and L-glutamine (all obtained from Gibco, Eragny, France). Lymphocytes were stimulated by 1% phytohaemagglutinin (PHA, Gibco). Test chemical (TC) and 5-Bromo-2-deoxyuridine (9 mg/mL, BrdU, Sigma) was added to cultures at 24 h after phytohaemagglutinin stimulation the at 37°C for SCE analysis. Lymphocytes were cultured in the dark for 72 h, and metaphases were blocked during the last 1.5 h with Colcemid at final concentration of 0.2 mg /mL. The cells were harvested by replacing the culture medium with KCl (0.075 M) in which cells were incubated for 20 min. at 37°C. The cells were fixed in Carnoy’s fixative (methanol: acetic acid, 3:1 v:v) five times, and slides were kept at room temperature overnight. Air-dried slides were stained according to Fluoresence-Plus Giemsa method by Perry and Wolf (1974) with slight modification. Two slides were prepared for each concentration. The number of SCEs was counted in 50 second-metaphase cells from each of the cultures on coded slides. Thus, 100 cells were scored in blind per dose for SCEs. Mitomycin C (2 mg /mL) was used as positive control. 2. Cytokinesis Block Micronucleus Method (CBMN): The CBMN assay was carried out using the Standard technique proposed by Fenech (1993), with slight modifications. Cytochalasin-B (Cyt-B, Sigma), at a final concentration of 3μg/ml was added at 44 h after the cultures were established, to arrest cytokinesis of dividing cells. Binucleated lymphocytes were harvested 72 h after culture setting. The test chemical (tetracycline) was added at 24 h after phytohaemmaglutinin stimulation. The cells were collected by centrifugation and washed with hypotonic solution (0.075 M KCl) at room temperature using vortex for two minutes. The induction of MN was evaluated by scoring a total of 1000 BN cell with well-preserved cytoplasm at 1000 x magnification. Only BN cells were included in the microscopic analysis. 2.4. Cell Proliferation Kinetic (CPK) and Mitotic Activity The mitotic activity (MA) was calculated as proportion of metaphases among the total cell 24 THE ASSESSMENT OF CYTOTOXICITY AND GENOTOXICITY OF TETRACYCLINE ANTIBIOTIC population by counting a total of 1000 cells. The cell proliferation kinetics was defined as the proportion of the relative frequency of first division metaphases (M1, identifiable by uniform staining of both the sister chromatids), seconddivision metaphases (M2, identifiable by differential staining of the sister chromatids), and third and subsequent division metaphases (M3, identifiable by non-uniform pattern of staining). Replication index or proliferation index (RI) was calculated according to Ivett and Tice (1982). RI is the average number of replications completed by metaphase cells and is calculated as follows: PRI = 1x(first division metaphases) + 2x(% second division metaphases) + 3x(% subsequent division metaphases)/500 In the MN study, toxicity was evaluated by classifying 500 cells according to the number of nuclei. The well-known cytotoxicity index was used: an index for measuring the cell proliferation kinetics, called the Nuclear Division Index (NDI) which was calculated following the expression: where MI–MIV represent the numbers of cells with one to four nuclei, respectively, and MIII and MIV are equally considered to be in their third cell cycle. As previously demonstrated, the NDI is an accurate and biologically relevant index in detecting cellular toxicity or cell-cycle delay (Fenech 2007). NDI = MI +2 MII +3(MIII, MIV)/500 2.5 Statistical Analysis Data were compared by one-way analysis of variance. Statistical analysis was performed using the SPSS for Windows 9.05 package program. Multiple comparisons were performed by Student Newman-Keuls (SNK) test. Statistical decisions were based on a significance level of 0.05.