(T1030-02-08) Single Cell Gel Electrophoresis to Detect Genotoxicity of Iminostilbene, a Process Induced Impurity of Carbamazepine

Purpose: The current study aims to compare human lymphocytes for genotoxicity of carbamazepine (CBZ) and its process induced degradation product iminostilbene (IMB) by performing the single cell gel electrophoresis (SCGE) or The Comet assay (Singh et al., 1988; Anderson et al., 1997; Collins, 2004). CBZ is an anticonvulsant and analgesic drug used to treat epilepsy, terminal neuralgia, and other conditions. IMB is a process induced degradation product formed during processes like hot melt extrusion in formulation of CBZ (Bose, S. & Paradkar, A. & Kelly, A. & Magam, 2023).

Methods: Whole blood samples were collected from healthy non-smoker volunteers after completion of consent forms. Ethical approval was granted by the University of Bradford’s Sub-Committee for Ethics in Research involving Human Subjects (Reference no.: 0405/8). Control samples were also covered under IRAS/NRES application 12/YH/0464. Governance Office Bradford Teaching Hospital NHS Foundation granted the ReDA number: 1202. All viabilities measured by Trypan blue exclusion in freshly collected samples were above 90% to avoid artefacts due to toxicity. The cut-off point suggested 70%. (Henderson et al., 1997) The technique involves embedding the cells in agarose on slides, lysing them to expose supercoiled loops of DNA and subjecting them to electrophoresis under alkaline or neutral conditions. After neutralisation the slides are stained with DNA intercalating stains such as ethidium bromide, allowing the analysis of images that resemble a comet. These were analysed using fluorescence microscopy, a CCD camera and software Komet 6. The advantages of the technique include its ability to generate large amounts of data in a relatively short period of time; it is minimally invasive and requires a relatively small number of cells per sample (Dixon et al., 2002; Lee and Steinert, 2003). These have made it an effective tool in both in vitro and in vivo studies. The reason for using lymphocytes for this study include their availability in large numbers without using cell culture and although specialised, they reflect the overall state of the organism as they circulate through the whole body (Collins et al., 2008) In order to obtain a better comparison, each experiment involved incubating the blood sample with four different concentrations (1 µM , 10 µM , 25 µM and 50 µM ) of CBZ and IMB with a common negative control and positive control respectively. The stored blood samples were thawed at room temperature. 100 µL of blood was transferred to Eppendorf® tubes and incubated for 30 minutes at 37°C with 4 different concentrations (Table 1). Cell treatment was followed by cell lysis, unwinding and electrophoresis this was followed by neutralization and staining after this scoring was performed at 540nm using a Leica DMLB microscope (Leica Microsystems, Wetzlar GmbH) fitted with fluorescence attachment and CCD camera connected to a computer with a Komet 6.0. program (Kinetic Imaging, Liverpool, UK) and scored blind. All slides were coded before scoring and 50 cells were scored per slide.

Results: All the concentrations were compared to the negative control using ANOVA followed by Fisher LSD. The significance level of p < 0.05 was decided before the analysis. For ease of presentation, histograms have been produced of 3 repeat experiments showing means and standard errors for the % tail DNA. The mean % Tail DNA for the positive control of 26.51 was significantly higher compared to 14.39 for the negative control with a p value < 0.01. The mean % tail DNA of lymphocytes incubated with different concentrations of CBZ ranged between 14.68 and 16.01 was not significantly different compared to 14.39 of the negative control. On the other hand mean % tail DNA of lymphocytes incubated with different concentrations of IMB which ranged between 15.08 and 24.84 exhibited a steady increase with all concentrations (10 µM, 25 µM and 50 µM) except the lowest (1 µM) being significantly greater (p < 0.05) compared to the negative control.(Figure 1).

Conclusion: These results indicate that exposure to CBZ does not induce any significant genotoxic effect at the concentrations investigated in the current study. On the other hand, IMB appears to be capable of inducing a significantly higher level of damage on the genetic material of lymphocytes affecting their integrity compared to similar concentrations of CBZ.

References: 1. Anderson, D. et al. (1997) ‘Effects in the Comet assay of storage conditions on human blood.’, Teratogenesis, carcinogenesis, and mutagenesis, 17(3), pp. 115–125.
2. Bose, S. & Paradkar, A. & Kelly, A. & Magam, H. (2023) ‘Process induced degradation of carbamazepine during hot melt extrusion’, British Journal of Pharmacy 8(2), 8(2). Available at: https://www.bjpharm.org.uk/article/id/1392/.
3. Collins, A. R. (2004) ‘The comet assay for DNA damage and repair: principles, applications, and limitations.’, Molecular biotechnology, 26(3), pp. 249–261. doi: 10.1385/MB:26:3:249.
4. Collins, A. R. et al. (2008) ‘The comet assay: topical issues.’, Mutagenesis, 23(3), pp. 143–151. doi: 10.1093/mutage/gem051.
5. Dixon, D. R. et al. (2002) ‘Marine invertebrate eco-genotoxicology: a methodological overview.’, Mutagenesis, 17(6), pp. 495–507. doi: 10.1093/mutage/17.6.495.
6. Henderson, L. et al. (1997) ‘Industrial Genotoxicology Group collaborative trial to investigate cell cycle parameters in human lymphocyte cytogenetics studies.’, Mutagenesis, 12(3), pp. 163–167. doi: 10.1093/mutage/12.3.163.
7. Lee, R. F. and Steinert, S. (2003) ‘Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals.’, Mutation research, 544(1), pp. 43–64. doi: 10.1016/s1383-5742(03)00017-6.
8. Singh, N. P. et al. (1988) ‘A simple technique for quantitation of low levels of DNA damage in individual cells.’, Experimental cell research, 175(1), pp. 184–191. doi: 10.1016/0014-4827(88)90265-0.

Acknowledgements: I am grateful to University of Bradford, for providing adequate support and ethical approvals (Reference no.: 0405/8) to carry out the research. I extend my gratitude to HARKA Pharma for providing raw materials. I also acknowledge Bradford Teaching Hospital NHS Foundation for granting ReDA number: 1202. (REC reference: 12/YH/0464; IRAS project ID: 10484)

Table 1 - Concentration and volumes of the negative control, positive control, carbamazepine and iminostilbene used in the experiments

Table 2 - Dose response Experiments in terms of percentage Tail DNA for Carbamazepine and Iminostilbene using hydrogen peroxide as positive control.

Figure 1 - Mean Dose response in terms of percentage Tail DNA for carbamazepine and iminostilbene using H2O2 as positive control.