Effects of Grayanotoxin-III on different cell lines: in vitro ischemia model

Abstract

Grayanotoxins (GTXs) are natural products and are mostly found in plants of the Ericaceae family, especially in the Rhododendron. With their ability to bind to voltage-gated sodium channels, they keep these channels constantly active and cause tissue damage. However, despite this feature, the use of Rhododendron leaves or its secondary products as an alternative product is especially common in Turkey. This study aims to evaluate the possible dose-related effects of GTX-III in ischemia-induced in vitro cell models. Within the scope of the study, an ischemia model was established in two different cell lines (H9c2 and Cos-7) and treated with various concentrations of GTX-III. In this context, cell viability, cytotoxicity, apoptosis and necrosis were examined. In the results of MTT, a significant decrease (+p < 0.05) in cell viability was observed in all GTX-III concentrations in H9c2 cells compared to the control, while a significant difference (+p < 0.05) was observed in Cos-7 cells, especially at the 24th hour. LDH cytotoxicity was increased in a dose-dependent manner in both cell models. It was concluded that GTX-III caused apoptosis, and reduced cell viability in ischemia models; however, promoted cell proliferation in healthy cells. Based on the literature review, this study is the first to document the cytotoxic properties and apoptotic potential of GTX-III in an in vitro cell culture ischemia model. Our findings support the usage of GTX-III, however it should be remembered that the dose needs to be verified before being used medically.

Keywords

• Cardiomyocyte
• Cells culture
• Grayanotoxin-III
• Renal cell line
• Cytotoxicity

References

1. Abdel Wahab, S.I., Abdul, A.B., Alzubairi, A.S., Mohamed Elhassan, M., Mohan, S. (2009). In vitro ultramorphological assessment of apoptosis induced by zerumbone on (HeLa). Journal of Biomedicine and Biotechnology, 2009. https://doi.org/10.1155/2009/769568
2. Aliyev, F., Türkoğlu, C., Çeliker, C., Firatli, I., Alici, G., & Uzunhasan, I. (2009). Chronic mad honey intoxication syndrome: a new form of an old disease?. Europace, 11(7), 954-956. https://doi.org/10.1093/europace/eup126
3. Argun, M., Üzüm, K., Sönmez, M.F., Özyurt, A., Karabulut, D., Soyersarıca, Z., ... & Narin, N. (2016). Cardioprotective effect of metformin against doxorubicin cardiotoxicity in rats. Anatolian Journal of Cardiology, 16(4), 234. https://doi.org/10.5152/akd.2015.6185
4. Bilir, E.K., Tutun, H., Sevin, S., Kısmalı, G., & Yarsan, E. (2018). Cytotoxic effects of Rhododendron ponticum L. extract on prostate carcinoma and adenocarcinoma cell line (DU145, PC3). Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 24(3). https://doi.org/10.9775/kvfd.2017.19219
5. Brown, B.S., Akera, T., & Brody, T.M. (1981). Mechanism of grayanotoxin III-induced afterpotentials in feline cardiac Purkinje fibers. European Journal hf Pharmacology, 75(4), 271-281. https://doi.org/10.1016/0014-2999(81)90554-9
6. Choo, Y.K., Kang, H.Y., & Lim, S.H. (2008). Cardiac problems in mad-honey intoxication. Circulation Journal, 72(7), 1210-1211. https://doi.org/10.1253/circj.72.1210
7. Condreay, J.P., Witherspoon, S.M., Clay, W.C., & Kost, T.A. (1999). Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proceedings of the National Academy of Sciences, 96(1), 127 132. https://doi.org/10.1073/pnas.96.1.127
8. Doğanyiğit, Z., Silici, S., Demirtaş, A., Kaya, E., & Kaymak, E. (2019). Determination of histological, immunohistochemical and biochemical effects of acute and chronic grayanotoxin III administration in different doses in rats. Environmental Science and Pollution Research, 26(2), 1323-1335. https://doi.org/10.1007/s11356-018-3700-9

9. Doǧanyiǧit, Z., Kaymak, E.M.İ.N., & Silici, S.İ.B.E.L. (2020). The cardiotoxic effects of acute and chronic grayanotoxin-III in rats. Human & Experimental Toxicology, 39(3), 374-383. https://doi.org/10.1177/0960327119889668
10. Dubey, L., Maskey, A., & Regmi, S. (2009). Bradycardia and severe hypotension caused by wild honey poisoning. Hellenic Journal of Cardiology, 50(5), 426-428.
11. Durdagi, S., Scozzafava, G., Vullo, D., Sahin, H., Kolayli, S., & Supuran, C.T. (2014). Inhibition of mammalian carbonic anhydrases I-XIV with grayanotoxin III: solution and in silico studies. Journal of Enzyme Inhibition and Medicinal Chemistry, 29(4), 469-475. https://doi.org/10.3109/14756366.2013.804072
12. Elahi, M.M., & Matata, B.M. (2011). Endothelial Dysfunction during Cardiac Development: A Heart to Heart Discussion of the Significance of the Nitrosative-Oxidative Disequilibrium Hypothesis. An Overview of Association with Oxidative Stress, 58-71.
13. Incalza, M. A., D'Oria, R., Natalicchio, A., Perrini, S., Laviola, L., & Giorgino, F. (2018). Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases. Vascular Pharmacology, 100, 1 19. https://doi.org/10.1016/j.vph.2017.05.005
14. Kuznetsov, A.V., Javadov, S., Sickinger, S., Frotschnig, S., & Grimm, M. (2015). H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1853(2), 276 284. https://doi.org/10.1016/j.bbamcr.2014.11.015
15. Kühn, B., Del Monte, F., Hajjar, R.J., Chang, Y.S., Lebeche, D., Arab, S., & Keating, M.T. (2007). Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair. Nature Medicine, 13(8), 962-969.
16. Lim, M.Y., Phua, D.H., & Ooi, C.K. (2016). Heart stopping honey—not just Turkish honey. The American Journal of Emergency Medicine, 34(9), 1915 e1. https://doi.org/10.1016/j.ajem.2016.02.014
17. Maejima, H., Kinoshita, E., Seyama, I., & Yamaoka, K. (2003). Distinct sites regulating grayanotoxin binding and unbinding to D4S6 of Nav1. 4 sodium channel as revealed by improved estimation of toxin sensitivity. Journal of Biological Chemistry, 278(11), 9464-9471. https://doi.org/10.1074/jbc.M212133200
18. MacKay, M., & Gardiner, S.E. (2017). A model for determining ex situ conservation priorities in big genera is provided by analysis of the subgenera of Rhododendron (Ericaceae). Biodiversity and Conservation, 26(1), 189 208. https://doi.org/10.1007/s10531-016-1237-0
19. Mladěnka, P., Applová, L., Patočka, J., Costa, V.M., Remiao, F., Pourová, J., ... & TOX‐OER and CARDIOTOX Hradec Králové Researchers and Collaborators. (2018). Comprehensive review of cardiovascular toxicity of drugs and related agents. Medicinal Research Reviews, 38(4), 1332-1403. https://doi.org/10.1002/med.21476
20. Okuyan, E., Uslu, A., & Ozan Levent, M. (2010). Cardiac effects of “mad honey”: a case series. Clinical Toxicology, 48(6), 528 532. https://doi.org/10.3109/15563650.2010.497150
21. Poon, W.T., Ho, C.H., Yip, K.L., Lai, C.K., Cheung, K.L., Sung, R.Y., ... & Mak, T.W. (2008). Grayanotoxin poisoning from Rhododendron simsii in an infant. Hong Kong Medical Journal, 14(5), 405-407.
22. Popescu, R., & Kopp, B. (2013). The genus Rhododendron: an ethnopharmacological and toxicological review. Journal of Ethnopharmacology, 147(1), 42 62. https://doi.org/10.1016/j.jep.2013.02.022
23. Ritter, O., & Neyses, L. (2003). The molecular basis of myocardial hypertrophy and heart failure. Trends in Molecular Medicine, 9(7), 313-321. https://doi.org/10.1016/S1471-4914(03)00114-X
24. Small, D.M., Coombes, J.S., Bennett, N., Johnson, D.W., & Gobe, G.C. (2012). Oxidative stress, anti‐oxidant therapies and chronic kidney disease. Nephrology, 17(4), 311-321. https://doi.org/10.1111/j.1440-1797.2012.01572.x
25. Türck, P., Nemec-Bakk, A., Talwar, T., Suntres, Z., Belló-Klein, A., da Rosa Araujo, A.S., & Khaper, N. (2022). Blueberry extract attenuates norepinephrine-induced oxidative stress and apoptosis in H9c2 cardiac cells. Molecular and Cellular Biochemistry, 1-10. https://doi.org/10.1007/s11010-021-04313-z
26. Qiang, Y., Zhou, B., & Gao, K. (2011). Chemical constituents of plants from the genus Rhododendron. Chemistry&Biodiversity, 8(5), 792 815. https://doi.org/10.1002/cbdv.201000046
27. Xu, J., Tang, Y., Bei, Y., Ding, S., Che, L., Yao, J., ... & Xiao, J. (2016). miR-19b attenuates H2O2 induced apoptosis in rat H9c2 cardiomyocytes via targeting PTEN. Oncotarget, 7(10), 10870.
28. Wang, Z., Su, G., Zhang, Z., Dong, H., Wang, Y., Zhao, H., ... & Sun, Q. (2018). 25-Hydroxyl-protopanaxatriol protects against H2O2-induced H9c2 cardiomyocytes injury via PI3K/Akt pathway and apoptotic protein down-regulation. Biomedicine & Pharmacotherapy, 99, 33-42. https://doi.org/10.1016/j.biopha.2018.01.039
29. Weeke-Klimp, A., Bax, N.A., Bellu, A.R., Winter, E.M., Vrolijk, J., Plantinga, J., ... & Lie-Venema, H. (2010). Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes. Journal of Molecular and Cellular Cardiology, 49(4), 606-616. https://doi.org/10.1016/j.yjmcc.2010.07.007