This study examines the genotoxic effects of coumarin (2H-1-benzopyran-2-one) on Lens culinaris Medik cv Sultan in terms of DNA polymorphism and protein quantification. Effective concentration values were calculated according to a probit model, which is a type of regression where the dependent variable can take only two values, i.e., life or death, after 48 or 72 hours of treatment. Based on this analysis, the effective concentration value of EC50 identified as approximately 278 µM, and then adjusted to 300 µM. The bulb roots were treated with 300 µM (EC50), 600 µM (2X EC50) concentrations and the Hoagland was used in the control group.10 RAPD primers were used and as a result of the analysis, it was observed that 2 monomorphic and 8 polymorphic band profile primers for treatment of 600 μM and 300 μM, coumarin concentration according to the control group. Total of 39 polymorphic bands were detected from total of 97 bands and the percentage of polymorphism was detected as 48.75 %. SDS-PAGE analysis for total protein profile showed that there were differences between the treatment groups. In addition to PCR analysis, the root samples were examined by Fourier transform infrared (FT-IR) spectroscopy in order to determine the effects of coumarin on the quantity of biomolecules structure of L.culinaris One-way analysis of variance (ANOVA) was used in the calculation of statistical differences between the groups (P < 0.05).
Keywords: Lentil, Coumarin, RAPD-PCR, SDS-PAGE, FT-IR
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References
1. Ana, A.S., Penelope, G.A., Hugo, M., et al., The use of FTIR spectroscopy to monitor modifications in plant cell wall architecture caused by cellulose biosynthesis inhibitors, Plant Signal. Bah., 2011, vol. 6, pp. 1104–1110. https://doi.org/10.4161/psb.6.8.15793
2. Atienza, F.A., Conradie, M., Evenden, A.J., et al., Qualitative assessment of genotoxicity using random amplified polymorphic DNA: comparison of genomic template stability with key fitness parameters in Daphnia magna exposed to benzo[a]pyrene, Env. Tox. Chem., 1999, vol. 18, pp. 2275–2282. https://doi.org/10.1002/etc.5620181023
3. Atienzar, F.A., Venier, P., Jha, A.N., et al., Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations, Mut. Res./Gen Tox. Env. Mut., 2002, vol. 521, pp. 151–163. https://doi.org/10.1016/S1383-5718(02)00216-4
4. Atienzar, F.A. and Jha, A.N., The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review, Mut. Res. Rev., 2006, vol. 613, pp. 76–102. https://doi.org/10.1016/j.mrrev.2006.06.001
5. Barun, S.G., Bjorn, P.J., Tao, G., et al., Application of ATR-FTIR spectroscopy to compare the cell materials of wood decay fungi with wood mould fungi, Int. J. Spectrosc., 1999, vol. 2015, pp. 1–8. https://doi.org/10.1155/2015/521938
6. Borgaud, F., Hehn, A., Larbat, R., et al., Biosynthesis of coumarins in plants: a major pathway still to be unraveled for cytochrome P450 enzymes, Phytochem. Rev., 2006, vol. 5, pp. 293–308. https://doi.org/10.1007/s11101-006-9040-2
7. Damjanovich, S., Gáspár, R., and Panyi, G., An alternative to conventional immuno suppression: small-molecule inhibitors of Kv1. 3 channels, Mol. Int., 2004, vol. 4, pp. 250–254. https://doi.org/10.1124/mi.4.5.4
8. De Wolf, H., Blust, R., and Backeljau, T., The use of RAPD in ecotoxicology, Mut. Res./Rev. Mut. Res., 2004, vol. 566, pp. 249–262. https://doi.org/10.1016/j.mrrev.2003.10.003
9. Gnonlonfin, G.B., Sanni, A., and Brimer, L., Review scopoletin—a coumarin phytoalexin with medicinal properties, Crit. Rev. Plant Sci., 2012, vol. 31, pp. 47–56. https://doi.org/10.1080/07352689.2011.616039
10. Kawase, M., Sakagam, H., Motohash, N., et al., Coumarin derivatives with tumor-specific cytotoxicity and multi drug resistance reversal activity in vivo, IV. IIAR J., 2005, vol. 19, pp. 705–711.
11. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, vol. 227, pp. 680–685. https://doi.org/10.1038/227680a0
12. Liu, W., Yang, Y.S., Zhou, Q., et al., Impact assessment of cadmium contamination on rice (Oryza sativa L.) seedlings at molecular and population levels using multiple biomarkers, Chemosphere, 2007, vol. 67, pp. 1155–1163. https://doi.org/10.1016/j.chemosphere.2006.11.011
13. Malaiyandi, V., Sellers, E.M., and Tyndale, R.F., Implications of CYP2A6 genetic variation for smoking behaviors and nicotine dependence, Clin. Pharm. Ther., 2005, vol. 77, pp. 145–158. https://doi.org/10.1016/j.clpt.2004.10.011
14. Nei, M., Genetic distance between populations, Am. Nat., 1972, vol. 106, pp. 283–292. https://doi.org/10.1086/282771
15. Ozek, N., Bal, I., Sara, Y., et al., Structural and functional characterization of simvastatin-induced myotoxicity in different skeletal muscles, Biochim. Biophys. Acta, 2014, vol. 1840, pp. 406–415. https://doi.org/10.1016/j.bbagen.2013.09.010
16. Plumas, J., Drillat, P., Jacob, M., et al., Extra corporeal photochemotherapy for treatment of clonal T cell proliferations, Bul. Ducan., 2003, vol. 90, pp. 763–770.
17. Shinde, R.G., Khan, A.A., and Barik, A., Coumarin derivatives with antioxidant and anticancer potential: a review, Int. J. Med. Appl. Sci., 2014, vol. 3, pp. 165–184.
18. Turker, S., Dogan, F.T., and Severcan, F., The characterization and differentiation of higher plants by Fourier transform infrared spectroscopy, Appl. Spectrosc., 2007, vol. 61, pp. 300–308.
19. Venugopala, K.N., Rashmi, V., and Odhav, B., Review on natural coumarin lead compounds for their pharmacological activity, BioMed. Res. Int., 2013.
20. Williams, J.G., Kubelik, A.R., Livak, K.J., et al., DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res., 1990, vol. 18, pp. 6531–6535. https://doi.org/10.1093/nar/18.22.6531
21. Wulff, H., Rauer, H., Düring, T., et al., Alkoxypsoralens, novelnon peptide blockers of Shaker-type K+ channels: synthesis and photo reactivity, J. Med. Chem., 1998, vol. 41, pp. 4542–4549. https://doi.org/10.1021/jm981032o
22. Xu, R.X., Gao, S., Zhao, Y., Lou, H.X., and Cheng, A.X., Functional characterization of a Mg2+-dependent O‑methyltransferase with coumarin as preferred substrate from the liverwort Plagiochasma appendiculatum, Plant Physiol. Biochem., 2016, vol. 106, pp. 269–277.
23. Yuksel, B. and Aksoy, O., Cytological effects of coumann on the mitosis of Lens culinaris Medik, Fresemus Env. Bull., 2017, vol. 26, pp. 6400–6407.
24. Yuksel, B. and Aksoy, O., Analysis of the effects of coumarin on Lens culinaris Medik by some biochemical parameters using real-time polymerase chain reaction, Legume Res., 2019, vol. 42, pp. 640–645. https://doi.org/10.18805/LR-439